CN104013974A - Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain - Google Patents
Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain Download PDFInfo
- Publication number
- CN104013974A CN104013974A CN201410258665.2A CN201410258665A CN104013974A CN 104013974 A CN104013974 A CN 104013974A CN 201410258665 A CN201410258665 A CN 201410258665A CN 104013974 A CN104013974 A CN 104013974A
- Authority
- CN
- China
- Prior art keywords
- pain
- growth factor
- hepatocyte growth
- injury
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses an application of a hepatocyte growth factor gene in preparation of a medicament for preventing and/or treating pain. The paint caused by skin damages, the pain caused by muscle damages or neuropathic pain caused by nerve injuries can be significantly relieved by the application of the medicament. The application disclosed by the invention has an important application prospect and practical significance.
Description
Technical field
The present invention relates to biomedicine field, in particular to a kind of liver cell growth factor gene in the application for the preparation of preventing and/or treating in pain medication.
Background technology
Pain is one of complication of the most general disease, and present most of hospitals are using pain as the 5th vital sign.Pain mainly comprises acute pain and chronic pain, and wherein chronic pain comprises inflammatory and neuropathic pain and cancer pain.Inflammatory pain and tissue injury and to infect the immunoreation causing relevant, caused by various diseases, as the chronic Pelvic Pain Syndrome of rheumatoid or arthritis, pancreatitis, inflammatory bowel, interstitial cystitis or bladder etc.; The pain that neuropathic pain is caused by primary lesion or nervous system dysfunction, is produced by neural tissue injury.Postoperative pain is the Physiological Psychology reaction of a kind of complexity of human body to tissue injury and repair process, as post surgery treatment not in time, can there is the complication such as respiratory insufficiency, myocardial infarction, severe patient can cause shock even dead.
At present, although existing opiates, morphines analgesics and nonsteroidal anti-inflammatory analgetic clinically, the selectivity that these medicines provide for patient or fewer, therefore, still needs to develop the medicine of new treatment pain.
Summary of the invention
The present invention aims to provide a kind of liver cell growth factor gene in the application for the preparation of preventing and/or treating in the medicine of pain, for liver cell growth factor gene provides a kind of new application.
For more medicament selection is provided to pain patients, according to an aspect of the present invention, provide a kind of hepatocyte growth factor in the application for the preparation of preventing and/or treating in the medicine of pain.
Further, above-mentioned pain comprises the neuropathic pain that the pain that caused by skin injury or muscle injury and nerve injury cause.
Further, the above-mentioned pain being caused by skin injury or muscle injury comprises traumatic acute pain and Chronic Traumatic pain.Further, traumatic acute pain comprise skin or muscle shell wound, stab, acute pain and postoperative acute pain that lacerated wound causes.Preferred above-mentioned postoperative acute pain comprises the postoperative acute pain such as operation on neck, operation on breast, stomach wall operation, abdominal operation, stomach/operation on duodenum, operation on intestine, colonic operation, operation on liver, pancreatic surgery and vascular surgery.
Further, Chronic Traumatic pain comprises chronic pain and the postoperative chronic pain that burn and scald, radiation injury, cold injury cause, preferably above-mentioned postoperative chronic pain comprises the postoperative chronic pains such as amputation, breast operation, open chest surgery, indirect inguinal herniorrhaphy, coronary artery bypass and cesarean section.
Further, the neuropathic pain that above-mentioned nerve injury causes comprises sciatica or the caused pain of diabetic neuropathy.
Further, above-mentioned liver cell growth factor gene is human hepatocyte growth factor gene.
Further, above-mentioned human hepatocyte growth factor gene is carried by carrier, and described carrier can be expressed human hepatocyte growth factor albumen in mammalian cell.
Further, preferred above-mentioned carrier is eukaryon expression plasmid, viral vector, gene expression frame or minicircle dna.
Further, in the time that above-mentioned carrier is eukaryon expression plasmid, eukaryon expression plasmid is super spirial plasmid DNA, comprising: eukaryotic cell promoter, human hepatocyte growth factor gene, polyadenylic acid tail, antibacterial replication sequence and antibiotics resistance gene; In the time that above-mentioned carrier is viral vector, viral vector is retroviral vector, adenovirus vector, gland relevant viral vector, herpesvirus vector or vaccinia virus vector; In the time that above-mentioned carrier is gene expression frame, gene expression frame comprises: eukaryotic cell promoter, human hepatocyte growth factor gene and polyadenylic acid tail; In the time that above-mentioned carrier is minicircle dna, minicircle dna is superhelix closed-circular DNA, comprising: the recognition sequence of eukaryotic promoter, human hepatocyte growth factor gene, poly-A tail Palestine and Israel and part recombinase.
Further, in the time that said medicine is injection, the method that described medicine shifts by local intramuscular injection, particle gun or conduit mediation is transferred to human hepatocyte growth factor gene in mammalian cell, to give expression to human hepatocyte growth factor albumen.
Apply the application of technical scheme human hepatocyte growth factor gene of the present invention in the medicine for the preparation for the treatment of pain.The present invention first means using human hepatocyte growth factor gene as a kind of gene therapy is applied in the preparation of pain disease medicine, not only can reach analgesic object, and the method for this gene therapy do not have the side effect such as dependency, addiction, therefore have broad application prospects.
Brief description of the drawings
The Figure of description that forms the application's a part is used to provide a further understanding of the present invention, and schematic description and description of the present invention is used for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows in embodiments of the invention 2 liver cell growth factor gene to SD In The Rat Sole otch machinery pain contracting pawl threshold value temporal evolution curve;
Fig. 2 shows in embodiments of the invention 2 liver cell growth factor gene to the hot pain contracting of SD In The Rat Sole otch pawl threshold value temporal evolution curve;
Fig. 3 shows in embodiments of the invention 3 human hepatocyte growth factor gene to SD rat skin/muscle otch tractive pain machinery contracting pawl threshold value temporal evolution curve; And
Fig. 4 shows in embodiments of the invention 4 hepatocyte growth factor to C57 mice SNI neuropathic pain machinery contracting pawl threshold value curve over time.
Detailed description of the invention
It should be noted that, in the situation that not conflicting, the feature in embodiment and embodiment in the application can combine mutually.Describe below with reference to the accompanying drawings and in conjunction with the embodiments the present invention in detail.
Hepatocyte growth factor (hepatocyte growth factor, HGF), at first as a kind of hepatocyte mitogen, separates and obtains the serum of the rat removing from hepatietomy.HGF is the multi-functional somatomedin of one extensively distributing in body, has the effect that the each organ morphology of cell migration, propagation and promotion period of embryo occurs, and fetal development, tissue and organ regeneration, wound healing and angiogenesis are played to important regulating action.It is by being combined and bringing into play multifarious biological action with its specific membrane c-met's expression, be the important information molecule of interstitial and the interphase interaction of epithelium/endotheliocyte, fetal development, tissue and organ regeneration, wound healing and blood vessel are played to important regulating action.Research discovery simultaneously, HGF is a kind of neurotrophic factor, and nerve is had to nutrition and protective effect.
Once accidental chance, inventor finds that this hepatocyte growth factor has obvious inhibitory action for pain, and the means that proposed audaciously first HGF gene as a kind of gene therapy are applied in the preparation of pain disease medicine.By above-mentioned application of the present invention, not only can reach analgesic object by topical application liver cell growth factor gene, and well-known, hepatocyte growth factor albumen does not act on opiate receptor, compare the medicine of existing treatment pain, there is no dependence, without addicted advantage, there is important application prospect and practical significance.
Comprise various common various pain clinically in the applicable types of pain of said medicine of the present invention, especially in the pain that is applied to skin injury or muscle injury and causes, or the neuropathic pain being caused by nerve injury.No matter be the Chronic Traumatic pain that the traumatic acute pain that causes of skin injury or muscle injury or skin injury or muscle injury cause, or the neuropathic pain that causes of nerve injury, slow pain effect is all very obvious.
One of the present invention preferred embodiment in, the traumatic acute pain acute pain and postoperative acute pain that said medicine is applicable to including the shell wound of skin or muscle, stabs, lacerated wound causes; Wherein, postoperative acute pain comprises any postoperative acute pain in operation on neck, operation on breast, stomach wall operation, abdominal operation, stomach/operation on duodenum, operation on intestine, colonic operation, operation on liver, pancreatic surgery and vascular surgery.Application said medicine can be alleviated the pain of the wound of above-mentioned wound more significantly.May be the expression that HGF can suppress site of injury inflammatory factor IL-1 β, IL-6, TNF-α, PGE2 to the analgesic mechanism of the above-mentioned postoperative acute pain causing, reduce the transmission of peripheral nerve impulsion, thereby reduce the activation of maincenter glial cell.
In the present invention, above-mentioned Chronic Traumatic pain comprises chronic pain and the postoperative chronic pain that burn and scald, cold injury, radiation injury cause, medicine of the present invention makes HGF gene express at site of injury by the mode of smearing or injecting, and all can effectively alleviate above-mentioned chronic pain.The postoperative chronic pain that medicine of the present invention is also suitable for comprises amputation, breast operation, open chest surgery, indirect inguinal herniorrhaphy, coronary artery bypass and cesarean section etc.Refer to that at above-mentioned postoperative chronic pain of the present invention wound heals, but pain still continues.Therefore, before wound healing or/and apply afterwards said medicine not only can lenitive severe degree, can also shorten corresponding pain duration.Above-mentioned is that HGF reaches central nervous system against aixs cylinder transport to the possible mechanism of action of the inhibition of postoperative chronic pain, by suppressing with neurogliocyte c-met receptors bind the expression that p38MAPK, NF-KB signal path suppress inflammatory factor, reduce neurogliocyte and activate.HGF plays analgesic activity by anti-inflammatory activity from surrounding tissue to central nervous system.
In the application of the neuropathic pain causing in the applicable nerve injury of said medicine of the present invention, the neuropathic pain that nerve injury causes comprises sciatica or the caused pain of diabetic neuropathy.In the typical embodiment of one of the present invention, the neuropathic pain that above-mentioned nerve injury causes can be effectively alleviated in above-mentioned application.The analgesic mechanism of the neuropathic pain that HGF causes nerve injury may be to arrive central nervous system by local muscle multiple injection HGF gene against aixs cylinder transport, suppresses nociception P
2x, P
2the expression of Y receptor and inflammatory factor IL-6 has repair to injured nerve around simultaneously, thereby reaches analgesic effect.
In above-mentioned application of the present invention, be that the form that HGF gene is carried with carrier is applied.The effect of carrier is transported to HGF gene in cell, and goes out human hepatocyte growth factor albumen at cells, thereby makes HGF albumen can in cell, bring into play lenitive effect.
In above-mentioned application of the present invention, above-mentioned carrier there is no particular/special requirement, conventional eukaryon expression plasmid, viral vector, gene expression frame or minicircle dna are all applicable to the present invention, as long as can HGF gene is transported in cell and give expression to HGF albumen.
In a kind of preferred embodiment of the present invention, in the time that above-mentioned carrier is eukaryon expression plasmid, eukaryon expression plasmid is super spirial plasmid DNA, comprising: eukaryotic cell promoter, human hepatocyte growth factor gene, polyadenylic acid tail, antibacterial replication sequence and antibiotics resistance gene.In the time that above-mentioned carrier is viral vector, the applicable carrier of the present invention comprises retroviral vector, adenovirus vector, gland relevant viral vector, herpesvirus vector or vaccinia virus vector.In the time that above-mentioned carrier is gene expression frame, gene expression frame comprises: eukaryotic cell promoter, human hepatocyte growth factor and polyadenylic acid tail.In the time that above-mentioned carrier is minicircle dna, minicircle dna is superhelix closed-circular DNA, comprising: the recognition sequence of eukaryotic cell promoter, human hepatocyte growth factor, poly-A tail Palestine and Israel and part recombinase.
Above-mentioned carrier of the present invention not only all can be transported to HGF gene in cell and at cell inner expression and go out HGF albumen, plays lenitive curative effect; But also there is high, the easy purification of stability, with short production cycle and cost is low, thereby be conducive to suitability for industrialized production, further preferably use eukaryon expression plasmid, the safety of this carrier is higher, curative effect is also better.
The carrier of above-mentioned carrier's liver cell growth factor gene of the present invention can be separately or be made the various dosage forms that use clinically with other pharmaceutically useful excipient, as long as can be safely and steadly at cell inner expression HGF albumen by the above-mentioned carrier that makes to carry HGF gene.The present invention preferably makes local injection agent, spray, liniment or biodegradable embedding medium.Injection, spray and liniment can be according to the conventional method preparations of pharmaceutical field, and finished product is in 4 DEG C of preservations.And spray, liniment directly smear in wound, easy to use.Embedding medium is because using the biology material of can demoting, and embedded material can directly be absorbed by the body, without subsequent treatment.
The preparation of above-mentioned various dosage forms comprises the carrier that carries HGF gene of the effective dose that is enough to treatment or the treatment easing the pain or prevent irritation." treatment effective dose " refers to, in necessary dosage and time limit, effectively realize desirable therapeutic outcome, as the amount of pain relief.In a specific embodiment, can be by regulating dosage to provide optimal treatment to reply dosage, the treatment effective dose that reduces the carrier that carries HGF gene can become according to following factor: individual morbid state, age, sex, body weight and preparation cause desirable ability of replying in individuality.Treatment effective dose is still treated the amount that beneficial effect exceedes its toxicity or harmful effect." prevention effective dose " refers to, in necessary dosage and time limit, effectively realize desirable prevention result, as the amount of prevention or inhibition of pain outbreak.Can determine prevention effective dose according to the above-mentioned description to treatment effective dose.For any concrete experimenter, can regulate in time given dose according to individual need and the professional judgment of using people.
In the time that the dosage form of said medicine of the present invention is injection, the method that preferably said medicine shifts by local intramuscular injection, particle gun or conduit mediation is transferred to human hepatocyte growth factor in people's muscle cell and is given expression to human hepatocyte growth factor albumen.
To beneficial effect of the present invention be described further combined with embodiment below.
It should be noted that the experimental technique in the following example if no special instructions, is conventional method.
Embodiment 1
The acquisition of the recombinant plasmid vector of carrier's liver cell growth factor gene
One, thalline preparation
The plasmid of the carrier's liver cell growth factor gene building voluntarily, the linear expression cassette that contains CMV promoter, human hepatocyte growth factor gene, ployA tail.Host Strains is bacillus coli DH 5 alpha, ferments according to conventional method, centrifugal rear acquisition thalline, and-20 DEG C save backup.
Two, alkaline lysis smudge cells extracts plasmid DNA
Be that (Granted publication day is on August 28th, 2013 to ZL201110089982.2 according to the patent No., patent name is alkaline lysis system and the combined system of preparation plasmid DNA) in the disclosed cell lysis method and apparatus that extracts plasmid DNA carry out the extraction of plasmid DNA, improvement step is wherein as follows:
1. the ultrafiltration post that the clarification alkaline lysis liquid obtaining by continuous alkaline lysis process is 300KDa by molecular cut off carries out ultrafiltration and concentration.
2. the ultrafiltration and concentration liquid obtaining separates the plasmid DNA of carrier's stem cell factor gene through Sepharose 6 Fast Flow fillers (GE company, article No.: 17-0159-01) with RNA.
3. the plasmid DNA solution obtaining is separated superhelix through Plasmidselect Xtra filler (GE company, article No.: 28-4024-02) with the plasmid DNA of open loop, obtain super spirial plasmid DNA.
The super spirial plasmid DNA obtaining is further refining through Source 15Q filler (GE company, article No.: 17-0947-05) 4., obtain the higher plasmid DNA of purity.
5. the plasmid DNA high purity of acquisition is precipitated through ethanol, filtrated air is dry, by frozen solid plasmid DNA in-20 DEG C.
Three, the configuration of different dosage form
By 2 or 3mg/ml be configured to the dosage forms such as injection, spray, liniment or biodegradable embedding medium.The formula of injection contains: Na
2hPO
4, NaH
2pO
4the buffer of composition, and the protective agent such as EDTA, NaCl.Liniment is on injection basis, to add carbomer to make paste.Spray is to add benzalkonium chloride on injection basis.Degradable embedding medium is to add poly lactic-co-glycolic acid etc. on injection basis.The dosage form configuring is stored in 4 DEG C, for subsequent use.
Embodiment 2
Human hepatocyte growth factor gene causes the analgesic activity of acute pain to SD rat skin muscle otch
One, animal model
SPF level male SD rat 210～230g, lumbar injection pentobarbital sodium (45mg/kg) anesthesia, by left back rat foot sterilization, use No. 11 knife blades to be about the longitudinal cut of 1.0cm to toe from near-end 0.5cm at the bottom of pawl, cut after skin, provoke muscle stringer blunt separation at the bottom of pawl with ophthalmic tweezers, but keep muscle start-stop and adhere to complete.After pressing haemostatic, with 5-0 nylon line suture skin totally two pins.The requirement of sewing up be otch skin can not be overlapping, in turn over, split.Postoperative injection 0.1ml gentamycin.
This pattern die can cause that spontaneous pain appears in rat, mechanicalness and thermal hyperalgesia and tactile Provocative pain, these pain behavior performances continue 4 to 7 days, these pain behaviors and persistent period and clinically acute injury pain status have similarity, this model can be used in the shell wound of simulated skin or muscle, stab, the acute pain that lacerated wound causes and operation on neck, operation on breast, stomach wall hands art, abdominal operation, stomach/operation on duodenum, operation on intestine, colonic operation, operation on liver, the peripheral neuralgia that in pancreatic surgery and vascular surgery, any postoperative acute wound causes is quick.
Two, administering mode
It is 4mg/ml that the recombiant plasmid of carrier's liver cell growth factor gene is configured to concentration, respectively 6 postoperative rats is carried out to administration with microsyringe, and the administration volume of every rat is 50 μ l.Injection site is muscle at the bottom of the pawls of 6 rats; The administration number of times of every rat is once.Matched group gives the plasmid empty carrier of same volume same concentrations.
Three, the mensuration of pain
Pain determining adopts 50% machinery contracting pawl threshold value (g) to embody, and wherein, 50% machinery contracting pawl threshold value (g) is calculated as follows:
50% machinery contracting pawl threshold value (g)=(10[X
f+ k δ])/10000, wherein, X
ffor last Von Frey intensity level; K is tabular value (list of references: Chaplan, S.R., F.W.Bach, J.W.Pogrel, et al., Quantitative assessment of tactile allodynia in the rat paw.J Neurosci Methods, 1994,53 (1): 55-63.); δ is the mean deviation (0.224) stimulating.The contracting pawl threshold value that this formula calculates represents can cause 50% contracting pawl reaction at the bottom of the von Frey filament stimulation in rats pawl by this intensity.In experiment, the size of this value reflects the severe degree to pain, and this value is stronger than the lower explanation pain of basic value Shaoxing opera.
The mensuration of 3.1 mechanical pain thresholds: respectively at for three days on end preoperative and postoperative 6h, 1d, 2d, 3d, 5d, 7d, in room temperature, in quiet environment, is placed in metal grill by rat, makes rat adaptation, quiet 15～30min; With in the middle of the left back pawl of a set of Von Frey filament stimulation in rats with differently curved pressure palm bottom near the region of otch or preoperative identical region with it.From 4.0g, in the time that bending 90 degree of Von Frey Hair, time are greater than 8S rat and still react without contracting pawl, change the Von Frey Hair of adjacent slightly large pressure; If there is contracting pawl reaction (or add pawl or hind leg unsettled etc.), select the Von Frey Hair of adjacent slightly little pressure, carry out so continuously, until while there is for the first time sun (the moon) property response value, then METHOD FOR CONTINUOUS DETERMINATION four times.Press interval at every turn and be greater than 15S, for preventing tissue injury, high threshold is made as 26g.
The mensuration of 3.2 burning pain contracting pawl threshold values: respectively at for three days on end preoperative and postoperative 6h, 1d, 2d, 3d, 5d, 7d is in room temperature, in quiet environment, rat is placed in to the transparent organic glass case on testing jig glass plate, treat that it quiets down, application of stimulus instrument alignment light, according to getting In The Rat Sole fixed position (in the middle of otch), is changed stimulation high light, record starts, to the reflex time (S) that occurs that contracting pawl is escaped, to be contracting pawl threshold value from strong illumination; If exceed 20S still without the reaction of contracting pawl, stop irradiating, in order to avoid cause vola to organize heat loss.Replication 5 times, every minor tick 5min.
Four, effect assessment
The effect of machinery pain threshold is shown in Fig. 1, and when in figure, * represents P < 0.05, human hepatocyte growth factor group is compared empty carrier group and had significant difference; When * represents P < 0.01, hepatocyte growth factor group is compared empty carrier group and is had utmost point significant difference.Fig. 1 result shows, the postoperative plasmid DNA group 6h to the after transgenic that gives 200 μ g carrier liver cell growth factor genes all significantly improves than the tactile machinery contracting pawl threshold value utmost point that brings out that gives empty plasmid vehicle group for 5 days.After administration, 6h just has analgesic activity, obviously early than repair, therefore, illustrates that HGF can play analgesic effect, instead of reaches analgesic effect because of repair.
The effect of burning pain contracting pawl threshold value is shown in Fig. 2, and when in figure, * represents P < 0.05, human hepatocyte growth factor group is compared empty carrier group and had significant difference; When * represents P < 0.01, hepatocyte growth factor group is compared empty carrier group and is had utmost point significant difference.Fig. 2 result shows, the postoperative plasmid DNA group that gives 200 μ g carrier liver cell growth factor genes all significantly improves than the hot pain contracting pawl threshold value that gives empty plasmid vehicle group for the 2nd day to the 5th day after transgenic, and there is significant difference, the 2nd, 0.01, the 5 day P < 0.05 of 3 days P <.
Above result shows, the skin that human hepatocyte growth factor gene causes acute injury or the shell wound of muscle, stab, acute pain that lacerated wound causes and peripheral neuralgia is quick comprises that in operation on neck, operation on breast, stomach wall operation, abdominal operation, stomach/operation on duodenum, operation on intestine, colonic operation, operation on liver, pancreatic surgery and vascular surgery, any postoperative acute pain has analgesic activity.
Embodiment 3
The analgesic activity of the irritated pain of maincenter that human hepatocyte growth factor gene causes SD rat skin/muscle otch tractive
One, animal model
SPF level male SD rat 210～230g, lumbar injection pentobarbital sodium (45mg/kg) anesthesia, dorsal position is fixed, inside the large midleg saphena of right hind, the skin incision of a long 1.5～2cm is done at 4mm place, legs exposed muscle, then does long 7～10mm otch, blunt separation shallow-layer muscle in shallow-layer muscle (gracilis), see after white adductor muscles tendon fascia, in otch, insert micro-retractor.Micro-retractor tip is inserted to shallow-layer muscle below, pull open integumentary musculature to 2cm, expose adductor muscles fascia, continue tractive 1h.In traction process, saphenous nerve is stretched and is shifted by retractor, but because it is not subject to hard objects as the compressing of bone etc. in muscle top layer.During tractive, cover the moisturizing of rat incision, insulation with physiological saline solution gauze, skin suture muscle after tractive 1h, postoperative routine gives 0.1ml gentamycin anti-infective therapy.
This model just can be observed mechanical hyperalgesia for the 3rd day after surgery, and the 10th to 13 days after surgery especially remarkable, at least lasts till postoperative 22 days, and until disappears completely for postoperative 32 days.Its abnormal pain behavior reaction main manifestations is hyperpathia, touches and bring out pain.This model comprises that for the Chronic Traumatic pain of simulating people the nervus centralis pain that chronic pain that burn and scald, cold injury, radiation injury cause and postoperative constant pain, difficult healing wounds cause is quick, comprises the chronic pain that amputation, breast operation, open chest surgery, indirect inguinal herniorrhaphy, coronary artery bypass and cesarean section etc. cause.
Two, administering mode
It is 2mg/ml that the recombiant plasmid of carrier's liver cell growth factor gene is configured to concentration, and administration volume is 100 μ l, directly injects recombiant plasmid 200 μ g, every 40 μ g local 5 of 6 SD rat cutting part muscle by microsyringe.Matched group gives the plasmid empty carrier of same volume same concentrations.
Three, the mensuration of pain
The mensuration of machinery pain threshold (computational methods are with embodiment 2): respectively at for three days on end preoperative, postoperative 1d, 4d, 7d, 11d, 12d, 18d, 25d, 28d, 32d, in room temperature, in quiet environment, is placed in metal grill by rat, makes rat adaptation, quiet 15～30min; With in the middle of the right back pawl of a set of von Frey filament stimulation in rats with differently curved pressure palm bottom near the region of otch or preoperative identical region with it.From 4.0g, be greater than 8S rat still when lifting foot and react when bending 90 degree of Von Frey Hair, time, change the Von Frey Hair of adjacent slightly large pressure; If there is contracting pawl reaction (or add pawl or hind leg unsettled etc.), select the Von Frey Hair of adjacent slightly little pressure, carry out so continuously, until while there is for the first time sun (the moon) property response value, then METHOD FOR CONTINUOUS DETERMINATION four times.Press interval at every turn and be greater than 15S, for preventing tissue injury, high threshold is made as 26g.
Four, effect assessment
Effect is shown in Fig. 3, and when * represents P < 0.05, hepatocyte growth factor group is compared empty carrier group and had significant difference; When * represents P < 0.01, hepatocyte growth factor group is compared empty carrier group and is had utmost point significant difference.Fig. 3 result shows, postoperative the 3rd day, rat starts to occur that machinery pain is quick, and quick the most serious at 11,12 days central pains, after the recombiant plasmid at the local muscle multi-point injection of wound carrier hepatocyte growth factor, since the 7th day, rat machinery contracting pawl threshold value significantly improved (P < 0.01) compared with injecting unloaded group.
From the observation to rat, healing completely of site of injury skin in the 7th day after surgery, but now occur that central pain is quick, the effect of not playing pain relieving of repairing is described.And it is quick to give to suppress central pain after human hepatocyte growth factor.Visible, the effect of above-mentioned inhibition of pain is not because the reparation of wound causes, but HGF causes owing to giving.Therefore, HGF has analgesic activity to the Chronic Traumatic pain such as chronic pain and amputation, breast operation, open chest surgery, indirect inguinal herniorrhaphy, coronary artery bypass and cesarean section that has the quick scald of above-mentioned central pain, cold injury, radiation injury and cause.
Embodiment 4
The analgesic activity of human hepatocyte growth factor gene to C57BL/6J mice sciatic nerve branching selection damage (SNI) model
One, animal model
SPF level male C 57 BL/6 J mouse 16～18g, lumbar injection pentobarbital sodium (50mg/kg) anesthesia.Mice lateral position is fixed on operating board, after iodophor disinfection by mice right hind upper limb incision of skin, separating muscle, expose sciatic trunk and 3 branches (tibial nerve, common peroneal nerve and sural nerve), ligation cut off common peroneal nerve and sural nerve respectively, retain tibial nerve and avoid tractive, suture muscles and skin successively; Sham operated rats mice only exposes the sciatic nerve of operation side, not ligation, successively suture muscles and skin.
This model can damage the pain causing by analog neuron clinically, as the treatment of sciatica, the caused pain disease of diabetic neuropathy.
Two, the mensuration of pain
Get 8 mices, the front mechanical stimulus basis threshold value of measuring mice of operation, after operation, the mechanical stimulus pain sensation of 1d, 3d, 5d mensuration mice is super quick.After administration, 1d, 3d, 5d, 7d, 8d, 10d, 12d, 14d, 17d, 21d, 24d, 28d, 32d measure the mechanical pain threshold (computational methods are with embodiment 2) of mice.In room temperature, in quiet environment, rat is placed in to metal grill, make rat adaptation, quiet 15-30min; With the left back vola of a set of stimulation of the Von Frey filament with differently curved pressure mice outside.From 0.04g, be greater than 8S mice still when lifting foot and react when bending 90 degree of Von Frey Hair, time, change the Von Frey Hair of adjacent slightly large pressure; If there is contracting pawl reaction (or add pawl or hind leg unsettled etc.), select the Von Frey Hair of adjacent slightly little pressure, carry out so continuously, until while there is for the first time sun (the moon) property response value, then METHOD FOR CONTINUOUS DETERMINATION four times.Press interval at every turn and be greater than 15S, for preventing tissue injury, high threshold is made as 2.0g.
Three, administering mode
In the time that mice forms allodynia (postoperative 5d gives pUDK-HGF treatment).1. mice is divided into the local intramuscular injection group of pUDK-HGF wound, and (100 μ g); 2. (10 μ g) for pUDK-HGF intrathecal drug delivery group; 3. the local intramuscular injection group of empty carrier wound.
Four, effect assessment
Experimental result is shown in Fig. 4.In Fig. 4, when * represents P < 0.05, liver cell growth factor gene intrathecal injection group is compared empty carrier group, and there were significant differences; When * represents P < 0.01, liver cell growth factor gene intrathecal injection group is compared empty carrier group utmost point significant difference; When # represents P < 0.05, liver cell growth factor gene intramuscular injection group is compared empty carrier group and is had significant difference; When ## represents P < 0.01, liver cell growth factor gene intramuscular injection group is compared empty carrier group and is had utmost point significant difference.
As can be seen from Figure 4, SNI mice starts to occur the super quick phenomenon of mechanical pain for the 3rd day after surgery, administration for the first time in the 5th day after surgery, after administration, find that mice machinery contracting pawl threshold value increases and administration after the 2nd day machinery contracting pawl threshold value HGF organize and there is significant difference (P < 0.05) than giving normal saline group, administration for the second time after 1 week, the analgesic effect of finding intrathecal drug delivery after administration is obviously better than injured nerve topical, and this advantage is continued until that experiment finishes.
Above-mentioned intrathecal drug delivery group directly proves that HGF has analgesic activity to pain, and irrelevant with reparation.Therefore the neuropathic pain that, pUDK-HGF causes mice nerve injury has analgesic activity.May be the inhibitory action that HGF activates glial cell by intrathecal injection pUDK-HGF, thereby play analgesic activity, it is quick that peripheral neuralgia is alleviated in the transmission that the release that may suppress the local inflammation factor by the local intramuscular injection pUDK-HGF of injured nerve suppresses neural impulse, (Tsuchihara according to the literature, T., S.Ogata, K.Nemoto, et al.Nonviral retrograde gene transfer of human hepatocyte growth factor improves neuropathic pain-related phenomena in rats.Mol Ther, 2009, 17 (1): 42-50.), the recombiant plasmid of local intramuscular injection can be entered in spinal cord by contrary aixs cylinder means of transportation by neuron, thereby pUDK-HGF is transported to central nervous system by contrary aixs cylinder glial cell is activated and plays inhibitory action, play analgesic activity.
As can be seen from the above description, in the rat pawl undercut pain model of the above embodiment of the present invention 2, give the recombiant plasmid of carrier's hepatocyte growth factor after 6h just there is analgesic activity and lasted till that rat returns to preoperative level.Visible, human hepatocyte growth factor has obvious analgesic activity to postoperative acute pain, and the reparation of this analgesic effect and wound is irrelevant.Simultaneously, rat skin/muscle otch tractive art at embodiment 3 causes in the quick pain model of central pain, quick by suppressing central pain at the above-mentioned plasmid of the local muscle multi-point injection of wound, and the rat that gives empty plasmid occurs that central pain is quick, and central pain was quick the most serious in postoperative the 10th to 13 days, within the 7th day after surgery, heal by gross examination of skeletal muscle rat skin, this is with that pain generation time-histories occurs is inconsistent, and it doesn't matter also to show analgesic effect that HGF of the present invention has and the reparation pain relieving of wound.Can find out from the above results, human hepatocyte growth factor has analgesic effect to postoperative chronic pain.In the mice sciatic nerve branch damage pain model of embodiment 4, can play obvious analgesic effect by the above-mentioned plasmid of intrathecal injection, the neuropathic pain that human hepatocyte growth factor causes nerve injury as can be seen here has analgesic activity.From the various embodiments described above result, by utilizing the method for HGF gene therapy of the present invention, can significantly alleviate the acute peripheral nervous pain that skin injury or muscle injury cause, the chronic neuropathic pain that nervus centralis pain is quick and nerve injury causes.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. liver cell growth factor gene is in the application for the preparation of preventing and/or treating in the medicine of pain.
2. application according to claim 1, is characterized in that, described pain comprise the pain that caused by skin injury, pain that muscle injury causes or the neuropathic pain that caused by nerve injury at least one.
3. application according to claim 2, is characterized in that, the described pain being caused by skin injury or muscle injury comprises traumatic acute pain and Chronic Traumatic pain.
4. application according to claim 3, is characterized in that, described traumatic acute pain comprise skin or muscle shell wound, stab, acute pain and postoperative acute pain that lacerated wound causes; Preferred described postoperative acute pain comprises any postoperative acute pain in operation on neck, operation on breast, stomach wall operation, abdominal operation, stomach/operation on duodenum, operation on intestine, colonic operation, operation on liver, pancreatic surgery or vascular surgery.
5. application according to claim 3, is characterized in that, described Chronic Traumatic pain comprises chronic pain and the postoperative chronic pain that burn and scald, cold injury, radiation injury cause; Preferably described postoperative chronic pain comprises any postoperative chronic pain in amputation, breast operation, open chest surgery, indirect inguinal herniorrhaphy, coronary artery bypass or cesarean section.
6. application according to claim 2, is characterized in that, the neuropathic pain that described nerve injury causes comprises sciatica or the caused pain of diabetic neuropathy.
7. according to the application described in any one in claim 1 to 6, it is characterized in that, described liver cell growth factor gene is human hepatocyte growth factor gene.
8. application according to claim 7, it is characterized in that, described human hepatocyte growth factor gene is carried by carrier, and described carrier can be expressed human hepatocyte growth factor albumen in mammalian cell, preferred described carrier is eukaryon expression plasmid, viral vector, gene expression frame or minicircle dna.
9. application according to claim 8, is characterized in that,
Described eukaryon expression plasmid is super spirial plasmid DNA, and described super spirial plasmid DNA comprises: eukaryotic cell promoter, human hepatocyte growth factor gene, polyadenylic acid tail, antibacterial replication sequence and antibiotics resistance gene;
Described viral vector is retroviral vector, adenovirus vector, gland relevant viral vector, herpesvirus vector or vaccinia virus vector;
Described gene expression frame comprises: eukaryotic cell promoter, human hepatocyte growth factor gene and polyadenylic acid tail;
Described minicircle dna is superhelix closed-circular DNA, and described superhelix closed-circular DNA comprises: the recognition sequence of eukaryotic promoter, human hepatocyte growth factor gene, poly-A tail Palestine and Israel and part recombinase.
10. application according to claim 9, it is characterized in that, described medicine is local injection agent, spray, liniment or biodegradable embedding medium, preferably in the time that described medicine is injection, the method that described medicine shifts by local intramuscular injection, particle gun or conduit mediation is transferred to human hepatocyte growth factor gene in mammalian cell, to give expression to human hepatocyte growth factor albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410258665.2A CN104013974A (en) | 2014-06-11 | 2014-06-11 | Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410258665.2A CN104013974A (en) | 2014-06-11 | 2014-06-11 | Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104013974A true CN104013974A (en) | 2014-09-03 |
Family
ID=51431152
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410258665.2A Pending CN104013974A (en) | 2014-06-11 | 2014-06-11 | Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104013974A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497975A (en) * | 2016-10-21 | 2017-03-15 | 浙江生创精准医疗科技有限公司 | The preparation method of genetic recombination stem cell medicine and its application in skin injury reparation, scar suppress |
| CN108611367A (en) * | 2018-04-24 | 2018-10-02 | 北京诺思兰德生物技术股份有限公司 | The gene therapy recombinant vector that one kind is mediated by plasmid vector |
| CN113521309A (en) * | 2020-04-16 | 2021-10-22 | 中国人民解放军军事科学院军事医学研究院 | Application of human hepatocyte growth factor gene in eczema treatment and micro-needle medical instrument |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1367703A (en) * | 1999-05-31 | 2002-09-04 | 三菱化学株式会社 | Freeze dried HGF preparations |
| CN1453039A (en) * | 2002-04-26 | 2003-11-05 | 中国人民解放军军事医学科学院放射医学研究所 | Method of speeding wound repair and preventing complications |
-
2014
- 2014-06-11 CN CN201410258665.2A patent/CN104013974A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1367703A (en) * | 1999-05-31 | 2002-09-04 | 三菱化学株式会社 | Freeze dried HGF preparations |
| CN1453039A (en) * | 2002-04-26 | 2003-11-05 | 中国人民解放军军事医学科学院放射医学研究所 | Method of speeding wound repair and preventing complications |
Non-Patent Citations (5)
| Title |
|---|
| SENDA AJROUD-DRISS: "Phase 1/2 Open-label Dose-escalation Study of Plasmid DNA Expressing Two Isoforms of Hepatocyte Growth Factor in Patients With Painful Diabetic Peripheral Neuropathy", 《MOLECULAR THERAPY》, vol. 21, no. 6, 31 December 2013 (2013-12-31) * |
| TOYOKAZU TSUCHIHARA, ET AL: "Noniviral Retrograde Gene Transfer of Human Hepatocyte Growth Factor Improves Neuropathic Pain-related Phenomena in Rats", 《MOLECULAR THERAPY》, vol. 17, no. 1, 31 January 2009 (2009-01-31) * |
| ZHANG J, MIDDLETON KK, FU FH, ET AL: "HGF mediates the anti-inflammatory effects of PRP on injured tendons", 《PLOS ONE》, 31 December 2013 (2013-12-31) * |
| 胡春生 等: "重组质粒pUDK-HGF 的中试纯化工艺", 《生物工程学报》, vol. 27, no. 2, 31 December 2011 (2011-12-31) * |
| 陈杏 等: "携带人肝细胞生长因子重组质粒pUDK-HGF对大鼠肾纤维化的防治作用", 《生物技术通讯》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106497975A (en) * | 2016-10-21 | 2017-03-15 | 浙江生创精准医疗科技有限公司 | The preparation method of genetic recombination stem cell medicine and its application in skin injury reparation, scar suppress |
| CN108611367A (en) * | 2018-04-24 | 2018-10-02 | 北京诺思兰德生物技术股份有限公司 | The gene therapy recombinant vector that one kind is mediated by plasmid vector |
| CN113521309A (en) * | 2020-04-16 | 2021-10-22 | 中国人民解放军军事科学院军事医学研究院 | Application of human hepatocyte growth factor gene in eczema treatment and micro-needle medical instrument |
| CN113521309B (en) * | 2020-04-16 | 2023-07-07 | 中国人民解放军军事科学院军事医学研究院 | Application of human hepatocyte growth factor gene in treatment of eczema and microneedle medicine instrument |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20100266532A1 (en) | Methods for inhibiting scarring | |
| DE69807710T2 (en) | Use of latency-associated peptides in the manufacture of a medicament to improve wound healing. | |
| CN107050423A (en) | Low frequency glatiramer acetate therapy | |
| CN104013974A (en) | Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain | |
| EP3154551B1 (en) | The use of dextran sulfate having an average molecular weight below 10000 da for inducing angiogenisis in a subject | |
| MX2014003256A (en) | Use of indolyl and indolinyl hydroxamates for treating heart failure of neuronal injury. | |
| CN112843229A (en) | Application of medicine for promoting skin injury repair in plastic surgery repair | |
| CN103705910A (en) | Ziconotide injection hypodermic implant and preparation method thereof | |
| US20090136587A1 (en) | Pharmaceutical composition for the treatment of nerve damage comprising blood plasma or serum | |
| CN101057823B (en) | Composite biological preparation with antisenility and beautifying function | |
| CN110372800B (en) | Fusion polypeptide with multifunctional activity and application thereof | |
| DE60110247T2 (en) | Angiogenic tri- or tetrapeptides derived from AcSDKP | |
| CN112569222A (en) | Application of trifluroicaritin in preparation of medicine for improving pain, swelling and motor function | |
| Chen et al. | Combining reverse end-to-side neurorrhaphy with rapamycin treatment on chronically denervated muscle in rats | |
| CN113855676B (en) | Application of AD16 in preparation of medicine for relieving chronic inflammatory pain | |
| CN112675294B (en) | Preparation of fibroblast and application of fibroblast in plastic surgery repair | |
| JP2011506413A (en) | Methods for inhibiting scarring | |
| CN101411700B (en) | Use of 5,7,4'-trihydroxy flavanone or derivative thereof for analgesia | |
| CN116173134A (en) | Application of traditional Chinese medicine composition in preparation of medicine for treating organ fibrosis | |
| US20110152189A1 (en) | Methods for the inhibition of scarring | |
| CN101152559A (en) | Pharmaceutical composition having function of protecting and treating nerve damnification | |
| Maan et al. | Germ Layer Restricted Wnt and Hedgehog Signaling Regulate Digit Tip Regeneration | |
| RU2486605C1 (en) | Method for simulating hinged epigastric venous flap with arterialisation of deep veins of lower extremity | |
| CN100484565C (en) | Compound skins lintement of Chinese medicine | |
| RU2406505C2 (en) | Method of treating inflammatory disease of metacarpus and metatarsus in dogs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140903 |
|
| RJ01 | Rejection of invention patent application after publication |