CN113425742A - Application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of drugs for treating pulmonary fibrosis - Google Patents
Application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of drugs for treating pulmonary fibrosis Download PDFInfo
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- CN113425742A CN113425742A CN202110771657.8A CN202110771657A CN113425742A CN 113425742 A CN113425742 A CN 113425742A CN 202110771657 A CN202110771657 A CN 202110771657A CN 113425742 A CN113425742 A CN 113425742A
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Abstract
The invention provides application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of a drug for treating pulmonary fibrosis, belonging to the technical field of biological drugs. The endometrial stem cells (CP EMSCs) over-expressing CTLA-4Ig/PD-L1 can remarkably enhance the effect of the endometrial stem cells (EMSCs) on treating pulmonary fibrosis, and comprise the functions of more effectively reducing the expression level of alpha-SMA protein of lung tissues and the expression levels of mRNA of TGF-beta, IL-6, IL-1 beta and MCP-1, so that the fibrosis and inflammation levels after lung injury are more effectively reduced, therefore, the invention provides the application of the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 or cell preparations prepared from the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in the preparation of medicaments for preventing and/or treating pulmonary fibrosis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of a medicine for treating pulmonary fibrosis.
Background
Pulmonary fibrosis is a permanent lung diseaseSexual injury is a serious disease characterized by high mortality, usually permanent damage to the lungs due to severe external trauma, spontaneous immune response, or adverse drug side effects. Studies have shown that the progression of pulmonary fibrosis is roughly divided into the following 3 stages: in the first stage, when the lung is damaged or otherwise harmfully stimulated, extracellular matrix (ECM) -producing cells of fibroblasts are activated; in the second stage, the activated ECM-producing cells are changed in structure and phenotype to produce a large amount of ECM, intracellular mitogen-activated protein kinase and nuclear transcription factor pathways are activated, and T cells are activated to produce various cytokines; in the third stage, the injury factor exists continuously, fibroblasts are continuously activated to generate more ECM, the cytokine continuously causes tissue inflammation and collagen over-expression, ECM is continuously deposited, pulmonary fibrosis is gradually formed, and finally the lung function of a special part is lost[1]。
The pulmonary fibrosis pathogenesis process is a complex pathological process, and a plurality of cells and cytokines are involved. TNF-alpha and TGF-beta are important indexes for clinical examination of pulmonary fibrosis, and research finds that mRNA levels of TNF-alpha and TGF-beta in fibrotic lung tissues are obviously higher than those of normal lung tissues[2]Inhibiting the synthesis and function of TNF-alpha and TGF-beta can reduce or regulate the progress of pulmonary fibrosis and delay the acute stage of pulmonary fibrosis. It is also shown by research that IL-6 can promote the occurrence of pulmonary fibrosis, and the increase of the expression of IL-6 can cause severe inflammatory reaction, so that the structure of certain areas of lung tissues is damaged to cause severe damage, ECM is continuously deposited, and pulmonary fibrosis is gradually formed.
In recent years, although research on pulmonary fibrosis mechanisms and therapeutic drugs has progressed with the advancement of technical means, the pathogenesis has not been fully elucidated and an effective therapeutic method is lacking. The authors state that dysregulation of any one or more of these processes during the injury-inflammation-repair cycle can lead to the development of pulmonary fibrosis, suggesting that we can prevent the formation of fibrosis by interfering with these processes[3]. Currently, the main therapeutic strategies for lung maintenance are: anti-inflammatory, anti-fibrosis and anti-oxidation, and can be used for treating handThe main treatment is medication and operation. However, drug therapy has major side effects, and lung transplantation, which is the most effective means for treating pulmonary fibrosis at present, has limited its application due to the defects of shortage of donated organ resources, rejection, infection, complications, high cost, and the like. Recently, the treatment of stem cells such as umbilical cord mesenchymal stem cells, embryonic stem cells, umbilical cord blood mesenchymal stem cells and the like is reported to improve the symptoms of pulmonary fibrosis induced by bleomycin, but the treatment of the stem cells usually has the function of clearing immune cells of an organism, so that the dosage of the stem cells is increased, and the treatment effect of the stem cells is reduced.
Reference to the literature
[1] Penguilu, huangwenhai, shengzheng, etc. development mechanism and treatment research progress of pulmonary fibrosis [ J ]. china journal of pharmacy 2012, 47 (23): 1873-76.
[2]Higgins DF，Kimura K，Bernhardt WM，et al.Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition[J].J Clin Invest.2007，117(12)：3810-20.
[3]Wilson MS，Wynn TA.Pulmonary fibrosis:pathogenesis，etiology andregulation.[J].Mucosal Immunol.2009，2(2)：103-21.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of endometrial stem cells (CP EMSCs) over-expressing CTLA-4Ig/PD-L1 in the preparation of a drug for treating and/or preventing pulmonary fibrosis, wherein the cells can be used for not only effectively treating and/or preventing pulmonary fibrosis, but also reducing the usage amount of EMSCs.
The invention provides application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of a medicament for preventing and/or treating pulmonary fibrosis.
Preferably, the method for constructing the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 comprises the following steps:
1) extracting RNA of the spleen of the animal, and obtaining cDNA through reverse transcription;
2) respectively amplifying CTLA-4Ig gene and PD-L1 gene by using the cDNA as a template to obtain CTLA-4Ig gene segment and PD-L1 gene segment;
3) cloning the CTLA-4Ig gene fragment and the PD-L1 gene fragment to a pLenti CMV GFP Puro (658-5) vector to obtain a recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro;
4) mixing the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, PMD2 and PSPAX2, treating eukaryotic cells under the action of a transfection reagent, culturing and collecting supernatant;
5) infecting the supernatant into endometrial stem cells, and performing cell culture to obtain the endometrial stem cells over-expressing CTLA-4 Ig/PD-L1.
Preferably, the primer for amplifying the CTLA-4Ig gene in the step 2) has a nucleotide sequence shown in SEQ ID NO:1 and the nucleotide sequence of the forward primer shown in SEQ ID NO:2, and a reverse primer;
the primer for amplifying the PD-L1 gene has a nucleotide sequence shown as SEQ ID NO:3 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:4, or a reverse primer as shown in figure 4.
Preferably, the mass ratio of the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, PMD2 and PSPAX2 in the step 4) is 3: 1.5-2.5: 2.5 to 3.5.
Preferably, the endometrial stem cells in step 5) are selected from any generation of primary to tenth generation of endometrial stem cells.
Preferably, the infection in step 5) is performed under the action of a transfection-enhancing agent; the transfection enhancing agent comprises polybrene reagent.
Preferably, the pulmonary fibrosis comprises pulmonary fibrosis caused by drug induction;
the drug comprises bleomycin and/or lipopolysaccharide.
Preferably, the medicament comprises an injection; the injection is a sodium chloride solution containing endometrial stem cells over-expressing CTLA-4 Ig/PD-L1;
the mass volume percentage of the sodium chloride solution is 0.8-1.0%.
The invention provides a cell preparation for preventing and/or treating pulmonary fibrosis, which comprises a sodium chloride solution containing endometrial stem cells over-expressing CTLA-4 Ig/PD-L1;
the mass volume percentage of the sodium chloride solution is 0.8-1.0%.
Preferably, the density of the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 is independently 2.0 × 105～4.0×105one/mL.
The invention provides application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of a medicament for preventing and/or treating pulmonary fibrosis. Experiments show that the pulmonary fibrosis phenomenon of mice can be obviously improved and the expression level of the alpha-SMA protein in lung tissues can be adjusted down after the tail veins of the mice with the pulmonary fibrosis induced by bleomycin are injected with endometrial stem cells (EMSCs) or CP EMSCs, but the treatment effect of the CP EMSCs is better than that of the endometrial stem cells with equal dosage. Meanwhile, CP EMSCs can effectively reduce inflammatory reaction in pulmonary fibrosis. The invention takes CP EMSCs as cell components, and compared with the endometrium stem cells, the expression of mouse lung tissues IL-6, IL-1 beta, MCP-1 and the like induced by bleomycin is more effectively inhibited. Therefore, CP EMSCs can effectively reduce the fibrosis and inflammation level after lung injury, and a new idea is provided for clinical treatment of pulmonary fibrosis.
Drawings
FIG. 1 shows the results of detection of cell surface markers in an example of the present invention;
FIG. 2 shows the results of real-time fluorescent quantitative PCR detection of CTLA-4Ig and PD-L1 gene and protein expression in CP EMSCs according to the present invention;
FIG. 3 is a graph of CP EMSCs in accordance with an embodiment of the present invention for treating fibrosis in lung tissue;
FIG. 4 shows the result of detecting the expression level of the lung tissue fibrosis marker alpha-SMA protein in the model group of mice in the example of the present invention;
FIG. 5 shows the result of detecting the expression level of genes related to lung fibrosis by EMSCs in the present invention.
Detailed Description
The invention provides application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of a medicament for preventing and/or treating pulmonary fibrosis.
In the present invention, the pulmonary fibrosis includes, but is not limited to, permanent damage to the lung caused by external trauma, spontaneous immune response, or adverse side effects of drugs. In the embodiment of the invention, the animal model of bleomycin induced pulmonary fibrosis is a classic pulmonary fibrosis animal model. After the mouse tail vein injection of the bleomycin induced pulmonary fibrosis CP EMSCs, the mouse pulmonary fibrosis phenomenon can be obviously improved, the lung tissue alpha-SMA protein expression level is simultaneously regulated, and the treatment effect is superior to that of the EMSCs with equal dosage.
In the present invention, the pulmonary fibrosis also preferably includes pulmonary fibrosis inflammatory response. In the inflammatory reaction of pulmonary fibrosis, the CP EMSC can more effectively inhibit the expression of inflammatory factors such as IL-6, IL-1 beta, MCP-1 and the like of mouse lung tissues induced by bleomycin than EMSC, and the CP EMSC can more effectively reduce the inflammatory reaction of pulmonary fibrosis than EMSC.
In the invention, the construction method of the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 preferably comprises the following steps:
1) extracting RNA of the spleen of the animal, and obtaining cDNA through reverse transcription;
2) respectively amplifying a mouse-derived CTLA-4Ig gene and a PD-L1 gene by using the cDNA as a template to obtain a CTLA-4Ig gene segment and a PD-L1 gene segment;
3) cloning the CTLA-4Ig gene fragment and the PD-L1 gene fragment to a pLenti CMV GFP Puro (658-5) vector to obtain a recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro;
4) mixing the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, PMD2 and PSPAX2, treating eukaryotic cells under the action of a transfection reagent, culturing and collecting supernatant;
5) infecting the supernatant into endometrial stem cells, and performing cell culture to obtain the endometrial stem cells over-expressing CTLA-4 Ig/PD-L1.
The invention extracts the RNA of animal spleen, and obtains cDNA through reverse transcription.
The method for extracting the RNA from the spleen of the animal is not particularly limited, and the RNA extraction protocol well known in the art can be adopted, for example, an animal RNA extraction kit can be adopted. The method of the reverse transcription is not particularly limited in the present invention, and a reverse transcription method well known in the art may be used. In the present example, since mice were used as the subjects, overexpression cells were prepared using mouse spleen RNA as the material.
After obtaining the cDNA, the invention respectively amplifies CTLA-4Ig gene and PD-L1 gene by using the cDNA as a template to obtain CTLA-4Ig gene segment and PD-L1 gene segment.
In the embodiment of the invention, a mouse is taken as an experimental object, so that the amplified CTLA-4Ig gene and the amplified PD-L1 gene are both mouse-derived CTLA-4Ig gene and PD-L1 gene. The preferable primer for amplifying the murine CTLA-4Ig gene has a nucleotide sequence shown as SEQ ID NO:1 (5'-ACTCTCCTGGTAAATGATTAATTAACCCCTCTCCCTCCCCCCCCC-3') and the nucleotide sequence is shown in SEQ ID NO:2 (5'-CGCCGGATCCGGCGCGCCTGTTAACTTATCATCGTGTTTTTCAAA-3'). The primer for amplifying the murine PD-L1 gene has a nucleotide sequence shown as SEQ ID NO:3 (5'-TTGAAAAACACGATGATAAGGATCCATGAGGATATTTGCTGGCAT-3') and the nucleotide sequence is shown in SEQ ID NO:4 (5'-TACCGATAAGCTTGATATCGAATTCTTACGTCTCCTCGAATTGTGTAT-3'). The reaction procedure of the amplification is preferably 95 ℃ for 5 min; at 95 ℃ for 10s, at 61 ℃ for 45s, at 70 ℃ for 1min, for 39 cycles; 5min at 70 ℃; storing at 16 ℃. The reaction system for amplification is preferably KOD DNA polymerase (TOYOBO) reaction system, the volume of the system is 50 μ L, and the system mainly comprises 1 μ L of template, 5 μ L of KOD buffer, 5 μ L of 2mM dNTP, 1.5 μ L of 10 μ M forward and reverse primer, and 1.5 μ L of KOD MgSO43. mu.L, KOD polymerase 1. mu.L and 33.5. mu.L of purified water.
After obtaining the CTLA-4Ig gene segment and the PD-L1 gene segment, the CTLA-4Ig gene segment and the PD-L1 gene segment are cloned to a pLenti CMV GFP Puro (658-5) vector to obtain a recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro.
In the present invention, the cloning method is not particularly limited, and a cloning method well known in the art may be used. The cloning sites of the pLenti CMV GFP Puro (658-5) vector are preferably SalI and BamHI.
After cloning, the resulting recombinant vector was identified. The identification method is not particularly limited in the present invention, and can be performed by methods known in the art, for example, introducing the recombinant vector into escherichia coli, culturing, detecting a target gene by a colony, obtaining a positive transformant, and extracting a plasmid to obtain the recombinant vector.
After obtaining the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, the invention mixes the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, PMD2 and PSPAX2, treats eukaryotic cells under the action of transfection reagent, and collects the supernatant after culturing.
In the invention, the mass ratio of the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, PMD2 and PSPAX2 is preferably 3: 1.5-2.5: 2.5 to 3.5, and more preferably 3:2: 3. The source of the plasmids of PMD2 and PSPAX2 is not limited in any way, and the plasmids of PMD2 and PSPAX2 which are well known in the art can be used. The transfection reagent preferably comprises a PEI transfection reagent. In embodiments of the invention, the eukaryotic cells are 293FT cells. The culture was carried out for 8h and then fresh culture medium was replaced. The fresh medium is a medium commonly used in the art for culturing 293FT cells. And (4) after replacing the fresh culture solution, continuously culturing for 72h, collecting supernatant, and concentrating to obtain the recombinant lentivirus.
After obtaining the supernatant, the invention infects the endometrial stem cells with the supernatant, and then obtains the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 through cell culture.
In the present invention, the endometrial stem cells are preferably selected from any generation of primary to tenth generation of endometrial stem cells, more preferably 6 th generation or 7 th generation. The infection is preferably carried out under the action of a transfection-enhancing agent; the transfection enhancing agent preferably comprises polybrene reagent. The polybrene reagent is preferably used at a concentration of 10. mu.g/mL. The cell culture was carried out for 24h and then the fresh culture medium was replaced. Cells were collected 48h after the exchange. The collected cells were identified. The identification preferably includes identification of cell surface markers and identification of the expression level of the foreign gene and the protein expression thereof. The identification of the expression level of the foreign gene is carried out by real-time fluorescent quantitative PCR, preferably, the expression level of CTLA4-IgG gene is detected by adopting a forward primer of a nucleotide sequence SEQ ID NO. 5 and a reverse primer of a nucleotide sequence SEQ ID NO. 6, and the expression level of PD-L1 gene is detected by adopting a forward primer of a nucleotide sequence SEQ ID NO. 7 and a reverse primer of a nucleotide sequence SEQ ID NO. 8.
In the present invention, the drug preferably includes an injection. The injection is preferably sodium chloride solution containing endometrium stem cells or endometrium stem cells over-expressing CTLA-4 Ig/PD-L1. The mass volume percentage of the sodium chloride solution is preferably 0.8-1.0%, and more preferably 0.9%. The density of the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 is preferably 2.0 x 105～4.0×105one/mL, more preferably 3.0X 105one/mL.
The invention provides a cell preparation for preventing and/or treating pulmonary fibrosis, which comprises a sodium chloride solution containing endometrial stem cells or endometrial stem cells over-expressing CTLA-4 Ig/PD-L1; the mass volume percentage of the sodium chloride solution is 0.8-1.0%. The density of the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 is preferably 2.0 x 105～4.0×105one/mL.
In the present invention, the method for preparing the cell preparation preferably comprises the following steps:
culturing the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 prepared by the technical scheme until the cell confluency reaches 90-95%, digesting with trypsin, and collecting the cells;
and (3) suspending the collected cells in a sodium chloride solution containing 0.8-1.0% to obtain the cell preparation.
In the invention, the preparation process of the cell preparation needs to be carried out under aseptic conditions, and the environment temperature and/or the temperature of the sodium chloride solution are preferably 20-26 ℃. The administration mode of the cell preparation is intravenous injection and/or intraperitoneal injection. The intravenous and/or intraperitoneal dose is 500. mu.L of cell suspension. The cell preparation can effectively improve pulmonary fibrosis and reduce inflammatory reaction in the pulmonary fibrosis process.
The following examples are provided to illustrate the application of the endometrial stem cells or the endometrial stem cells over-expressing CTLA-4Ig/PD-L1, but they should not be construed as limiting the scope of the present invention.
Source of experimental material and description of primer sequences involved
1 experimental animal: c57BL/6 mice, SPF grade, 6-8 weeks old, male; purchased in the experimental animal center of Guangdong province, and the certification number: no. 44007200042765.
2. Primary reagent
Bleomycin (Bleomycin, pharmaceutical industry ltd, national drug group); EMSCs medium (seiko (guangzhou) biotechnology limited, HUXUB-90011); sodium chloride (Sigma, usa); phosphate buffered saline (PBS, Gibco, USA); dimethyl sulfoxide (Gibco, USA); 0.25% pancreatin (Thermo Fisher, usa); BCA protein concentration assay kit (Beyotime Biotechnology); anti-a-SMA antibodies (Abcam, uk); anti-beta-actin antibodies (CST, usa); anti-PD-L1 antibody (Abcam, USA), and secondary antibody (goat anti-rabbit, CST, USA); PrimerScriptTMRT reagent Kit with gDNA Eraser reverse transcription Kit (Dalianbao biology Co., Ltd.);
Premix Ex TaqTMII (Dalibao BioLimited); other reagents are all domestic analytical purifiers.
3 Main Instrument
Clean bench, CO2Incubator (Thermo corporation, usa); inverted microscopes (Chongqing Otta optical instruments, Inc.); low speed centrifuges, high speed centrifuges (eppendorf, germany); ChemiDocTMTouch imaging System, electrophoresis apparatus (Bio-Rad, USA); fluorescent quantitative PCR instrument (applied biosystems, USA).
4. The primer sequences involved in the examples of the present invention are shown in Table 1.
TABLE 1 primer sequences for amplification or detection of respective genes
Example 1
Construction method of recombinant lentivirus for expressing murine CTLA-4Ig gene and PD-L1 gene
1. Construction method of recombinant vector for over-expressing murine CTLA-4Ig gene and PD-L1 gene
Taking spleen of normal mouse, grinding, extracting RNA, and then reverse transcribing into cDNA library. The cDNA library was used as a PCR template to design the amplification primers of murine CTLA-4Ig gene (SEQ ID NO:1 and SEQ ID NO:2) and the amplification primers of PD-L1 gene overexpression PCR (SEQ ID NO:3 and SEQ ID NO:4, the specific sequences are shown in Table 1). Amplifying the murine CTLA-4Ig gene and PD-L1 gene sequences by PCR, wherein the reaction program is 95 ℃ for 5 min; at 95 ℃ for 10s, at 61 ℃ for 45s, at 70 ℃ for 1min, for 39 cycles; storing at 70 deg.C for 5min 7, 16 deg.C. The reaction system is a KOD DNA polymerase (TOYOBO) reaction system, the volume of the system is 50 mu L, and the reaction system mainly comprises 1 mu L of template, 5 mu L of KOD buffer, 5 mu L of 2mM dNTP, 1.5 mu L of 10 mu M forward and reverse primers, and 1.5 mu L of KOD MgSO43. mu.L, KOD polymerase 1. mu.L and 33.5. mu.L of purified water. Then, two sequences are cloned on a pLenti CMV GFP Puro (658-5) (Addgene, Plasmid #17448) vector (using restriction enzymes SalI and BamHI for enzyme digestion) by using a molecular cloning gene recombination method to obtain a pLenti CMV CTLA-4Ig IRES PD-L1 Puro recombinant vector.
2. Murine CTLA-4Ig gene and PD-L1 gene overexpression lentivirus preparation and cell infection
Extracting the recombinant vector prepared by the method by a plasmid extraction kit, and packaging the lentivirus. The method comprises the following specific steps: culturing 293FT in a 15cm culture dish, and changing into a fresh culture medium for culturing in an incubator 2h before packaging lentivirus when the density reaches 80-90%. When packaging the virus, 1mL of serum-free medium opti-MEM (thermo fisher) was placed in a 1.5EP tube, 10. mu.g of PMD2, 15. mu.g of PSPAX2 and 15. mu.g of pLenti CMV CTLA-4Ig IRES PD-L1 Puro or pLenti CMV IRES Puro plasmid (control plasmid) were added, and the mixture was inverted and mixed. Then 58. mu.L of 1mg/mL PEI transfection reagent was added, shaken rapidly on a shaker for 10s, and left to stand at room temperature for 15min in the dark. Then, the cells were added to 293FT cells and replaced with fresh medium after 8 hours. Collecting supernatant after 72h of liquid change, adding virus concentrated solution after centrifugation, concentrating overnight at 4 ℃, freezing and storing at-80 ℃ after centrifugation and collection of precipitate for later use, and obtaining the recombinant lentivirus of over-expressed CTLA-4Ig and PD-L1 genes.
Example 2
Cell preparation containing EMSCs or CP EMSCs 1
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (sixth Generation), 2.0 × 105Per mL; the concentration of the sodium chloride solution is 0.9 percent.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 1 for use.
The EMSCs cell preparation of the control group is prepared by the steps of after cells grow to 90-95% of density, digesting with trypsin, resuspending the cells by using sterilized sodium chloride with the mass volume percentage of 0.9%, and subpackaging the cells into a 1mL syringe to obtain the EMSCs cell preparation 1 for later use.
Example 3
Cell preparation containing EMSCs or CP EMSCs 2
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (sixth Generation), 4.0 × 105Per mL; sodium chloride, 0.9%.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 2 for use.
After the cells of the control EMSCs grow to 90-95% density, and are digested with 0.25% trypsin for 3min, the cells are respectively resuspended in sterilized sodium chloride with the mass volume percentage of 0.9%, and then are subpackaged into 1mL syringes, so that the cell preparation 2 of the EMSCs is obtained for later use.
Example 4
Cell preparation containing EMSCs or CP EMSCs 3
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (seventh generation), 2.0 × 105Per mL; sodium chloride, 0.9%.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 3 for use.
After the cells of the control group of EMSCs grow to 90-95% density, digesting the cells for 3min by using 0.25% trypsin, respectively resuspending the cells by using 0.9% sterilized sodium chloride in percentage by mass and volume, and subpackaging the cells into a 1mL syringe to obtain a cell preparation 3 of the EMSCs for later use.
Example 5
Cell preparation containing EMSCs or CP EMSCs 4
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (seventh Generation), 4.0 × 105Per mL; sodium chloride, 0.9%.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 4 for use.
After the cells of the control group of EMSCs grow to 90-95% density, digesting the cells for 3min by using 0.25% trypsin, respectively resuspending the cells by using 0.9% sterilized sodium chloride in percentage by mass and volume, and subpackaging the cells into a 1mL syringe to obtain a cell preparation 4 of the EMSCs for later use.
Example 6
Cell preparation containing EMSCs or CP EMSCs 5
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (eighth generation), 2.0 × 105Per mL; sodium chloride, 0.9%.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 5 for use.
After the cells of the control group of EMSCs grow to 90-95% density, digesting the cells for 3min by using 0.25% trypsin, respectively resuspending the cells by using 0.9% sterilized sodium chloride in percentage by mass and volume, and subpackaging the cells into a 1mL syringe to obtain a cell preparation 5 of the EMSCs for later use.
Example 7
Cell preparation containing EMSCs or CP EMSCs 6
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (eighth generation), 4.0 × 105Per mL; sodium chloride, 0.9%.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 6 for use.
After the cells of the control group of EMSCs grow to 90-95% density, digesting the cells for 3min by using 0.25% trypsin, respectively resuspending the cells by using 0.9% sterilized sodium chloride in percentage by mass and volume, and subpackaging the cells into a 1mL syringe to obtain a cell preparation 6 of the EMSCs for later use.
Example 8
Cell preparation containing EMSCs or CP EMSCs 7
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (ninth generation), 2.0 × 105Per mL; sodium chloride, 0.9%.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 7 for use.
After the cells of the control group of EMSCs grow to 90-95% density, digesting the cells for 3min by using 0.25% trypsin, respectively resuspending the cells by using 0.9% sterilized sodium chloride in percentage by mass and volume, and subpackaging the cells into a 1mL syringe to obtain a cell preparation 7 of the EMSCs for later use.
Example 9
Cell preparation containing EMSCs or CP EMSCs 8
(1) The proportion of the ingredients of the final product is as follows: EMSCs or CP EMSCs (ninth generation), 4.0 × 105Per mL; sodium chloride, 0.9%.
(2) The preparation method comprises the following steps: inoculating EMSCs into 10cm cell culture dish, culturing with EMSCs medium at 37 deg.C and CO2Culturing in a cell culture box with the concentration of 5%. After the cells were grown to 60% to 70% density, the cells were infected with the lentivirus overexpressing CTLA-4Ig and PD-L1 genes prepared in example 1 and digested with 0.25% trypsin for 3min at 48 hours. The digested cells were resuspended in sterile 0.9% by weight sodium chloride and dispensed into 1mL syringes to obtain CP EMSCs cell preparation 8 for use.
After the cells of the control group of EMSCs grow to 90-95% density, digesting the cells for 3min by using 0.25% trypsin, respectively resuspending the cells by using 0.9% sterilized sodium chloride in percentage by mass and volume, and subpackaging the cells into a 1mL syringe to obtain a cell preparation 8 of the EMSCs for later use.
Example 10
Identification of recombinant cells prepared in examples 2 to 9
Cell surface markers in the recombinant cells and the EMSCs cells prepared in examples 2-9 were detected by flow cytometry. The cells meet the following requirements in marker detection: markers positive for CD29, CD44, CD90, and CD105 expression; markers CD34, CD45, HLA-DR expression negative, see figure 1.
RNA from the recombinant cells prepared in examples 2 to 9 was extracted and reverse-transcribed to obtain cDNA. The cDNA is taken as a template, CTLA-4Ig gene primers (SEQ ID NO:5 and SEQ ID NO:6) and PD-L1 gene primers (SEQ ID NO:7 and SEQ ID NO:8) are adopted to carry out fluorescence quantitative PCR detection, and the beta-Actin gene is taken as an internal reference gene. In addition, the total protein of the recombinant cells prepared in examples 2 to 9 was extracted and subjected to western blot detection by a conventional method.
Real-time fluorescent quantitative PCR shows that the mRNA expression of CTLA-4Ig and PD-L1 genes is increased by 10 relative to that of EMSCs without gene modification5Doubling the weight; westernblotting also detected protein expression of the PD-L1 gene. The results are shown in FIG. 2.
Example 11
Therapeutic effect of CP EMSCs cell preparation 1 on bleomycin-induced pulmonary fibrosis
1 establishment, grouping and intervention of pulmonary fibrosis model
32 male C57BL/6 mice with the age of 6-8 weeks are taken, 24 of the mice are subjected to intraperitoneal injection of 1.5 mu g/mL bleomycin, 1 time per week and 3 weeks of continuous administration to establish a pulmonary fibrosis mouse model, and whether the model construction is successful is judged by detecting the expression of the lung tissue alpha-SMA protein. In addition, 8 control groups were administered with saline. 24 mice given 1.5. mu.g/mL bleomycin were randomly divided into a model group, an EMSCs group and a CP EMSCs group 3 days after the first administration, 8 mice in each group were given the EMSCs group and CP EMSCs to the cell preparation 1 obtained in example 2 by tail vein injection, and the control group and the model group were given 0.9% sodium chloride sterilized by tail vein injection for 1 intervention per week for 3 consecutive interventions. And on day 21 cervical dislocation was sacrificed and the material was taken for the following experiment. And the cell preparations 2 to 8 prepared in examples 3 to 9 were subjected to intravenous microinjection by the same method, and the treatment results were examined by the same method.
2 protein immunoblotting
2.1 protein extraction and quantitation
Taking lung tissues, carrying out tissue grinding to extract total protein, adding a proper amount of lysate (containing 1% PMSF), and incubating for 30 minutes on ice; carrying out refrigerated centrifugation at 12000rpm for 30 minutes, and transferring the supernatant into a new centrifuge tube; taking 2 mu L for protein quantification, adding a proper amount of Loading Buffer into the rest proteins, uniformly mixing, and then heating and denaturing at 100 ℃ for 5 minutes and storing at-20 ℃. The protein quantification process was as follows: diluting a sample to be detected by 50 times of lysis solution, adding 100 mu L of lysis solution into a 96-hole cell culture plate, respectively taking solution A and solution B (the ratio of the solution A to the solution B is 1:50) in a centrifuge tube according to the specification of the BCA method protein concentration determination kit, gently blowing and beating the solution A and the solution B uniformly by using a pipette to prepare a mixed solution, and adding 100 mu L of the mixed solution into each hole of the sample adding hole; covering a film, incubating in water bath at 37 ℃ for 30 minutes, and detecting the absorbance value under the wavelength of 570nm of an enzyme-labeling instrument; and drawing a standard curve according to the absorbance value and calculating the protein concentration of each sample to be detected.
2.2 protein electrophoresis and immunoblotting
Preparing 10% separation gel, preparing concentrated gel after the separation gel is solidified, adding the concentrated gel to the upper layer of the separation gel, inserting a comb, and slightly pulling out the comb after the gel is solidified; adding a protein Marker and a sample into each hole of 10% SDS-PAGE respectively, and after electrophoresis at 80V for 30 minutes, carrying out electrophoresis at 120V for 1.5 hours; rotating the membrane (nitrocellulose membrane) for 90 minutes under the condition of constant pressure of 100V at room temperature; taking down the membrane, marking the front side and the back side, and blocking the membrane for 2 hours at room temperature by using 5% BSA; washing with 1 × TBS-T for 5min for 3 times; diluted to working concentration with 5% BSA according to antibody instructions, hybridized to nitrocellulose membrane overnight at 4 ℃; washing with 1 × TBS-T for 5min for 3 times; 1, diluting the secondary antibody at 1000, and hybridizing at room temperature for 1 hour; washing with 1 × TBS-T for 5min for 3 times; exposing; scan and calculate the grey values for each histone band with the software Image J (v 1.45).
3. Real-time fluorescent quantitative PCR
3.1 Total RNA extraction and reverse transcription
Taking a small amount of tissue, grinding, and adding a proper amount of trizol for cracking; standing at room temperature for 3 minutes, and transferring the lysate to a clean enzyme-free microcentrifuge tube after liquefaction. 0.2mL of precooled chloroform was added, mixed by rapid inversion of the mixture from top to bottom, allowed to stand on ice for 5 minutes to allow the protein complex to be completely cleaved, and centrifuged (4 ℃, 13000g, 15 minutes). Absorbing the upper aqueous phase, transferring to another enzyme-free centrifuge tube, adding 400 μ L isopropanol, rapidly and forcibly reversing up and down, mixing, standing at-20 deg.C for 30 min, and precipitating RNA. Centrifuging (4 ℃, 13000g, 10 minutes), leading a little white precipitate to be seen at the bottom of the tube, removing the supernatant, adding 1mL of 70% ethanol, reversing the mixture from top to bottom, washing to remove impurities, and placing on ice for 5 minutes. Centrifuging (4 deg.C, 7600g, 5 min), sucking dry ethanol, drying at room temperature for 5min until RNA precipitate is transparent, and adding 30 μ L nuclease-free water to dissolve RNA. Taking 1 microliter of RNA to determine the concentration and purity of the RNA, wherein the 260/280 ratio is between 1.8 and 2.0, which indicates that the RNA quality is good, and then synthesizing cDNA according to the procedure in the specification of a reverse transcription kit, wherein the cDNA is used as a template for real-time fluorescence quantitative PCR.
3.2 real-time fluorescent quantitative PCR
The reaction system contained in a real-time reaction system of 15. mu.L: 7.5. mu.L of SYBR Green (2X), 0.6. mu.L (10. mu. mol/L) of each of the upstream and downstream primers for IL-6, MCP-1, TGF-. beta.or IL-1. beta. genes in lung tissue, 1.5. mu.L of 1:15 diluted cDNA, 4.5. mu.L of L H2O, 0.3. mu.L ROX. Amplifying for 40 cycles in a fluorescent quantitative PCR instrument, wherein the temperatures and the times required by denaturation, annealing and extension are respectively 95 ℃ and 30 s; 30s at 60 ℃; 72 ℃ for 90 s. The assay was repeated 3 times for each sample. The relative amount of mRNA is expressed as Ct value, and the average relative expression amount is represented by 2－△△CtAnd (5) analyzing a calculation method.
4. Hematoxylin-eosin (HE) staining of lung tissue
After the materials are taken, lung tissues are fixed by 10% formic acid, dehydrated by a conventional method, embedded by paraffin, wax blocks are prepared, paraffin samples are sliced by a paraffin slicer to be 3.5 mu m thick, and the slices are continuously sliced, wherein the staining procedure comprises baking the slices for 20 minutes, drying xylene for 2 times multiplied by 10 minutes, absolute ethyl alcohol for 2 times multiplied by 2 minutes, 95% ethyl alcohol for 1 minute, 80% ethyl alcohol for 1 minute, 70% ethyl alcohol for 1 minute, washing with water for 1 minute, hematoxylin for 8 minutes, hematoxylin for 10 minutes, washing with water for 2 times multiplied by 1 minute, 0.5% hydrochloric acid for 10 seconds, washing with water for 10 minutes, eosin for 2 minutes, washing with water for 1 minute, 80%, 85% and 90% ethyl alcohol for 5 seconds respectively, 95% ethyl alcohol for 1 minute, absolute ethyl alcohol for 2 times multiplied by 2 minutes, absolute ethyl alcohol for 3 minutes, xylene for 2 times multiplied by 2 minutes, and after the staining is finished, neutral gum sealing is directly used for microscopic examination.
5. Statistical method
The Image J software is used for processing and analyzing the Image data, the statistical software SPSS17.0 is used for statistical processing and analyzing other data, and the difference that P is less than 0.05 has statistical significance.
6. Results and analysis
6.1 Effect of CP EMSCs cell preparation 1 on fibrosis status of Lung tissue
The HE staining result shows that the lung tissue structure of the control mouse is clear, and the alveolar septal broadening is not seen. The lung tissue structure of the model group mice is seriously damaged, part of alveoli are fused, the alveoli interval is obviously thickened, and a large amount of wide-band and flaky collagen fibers appear in the pulmonary interstitium, so that the pulmonary fibrosis is diffuse. The lung tissue of the mice in the CP EMSCs group still has fibrosis, but the fibrosis degree is obviously reduced compared with that of the mice in the model group and the EMSCs group, diffuse fibrosis is not seen, and the lesion sites are in focal distribution. The results are shown in FIG. 3.
6.2 Effect of CP EMSCs cell preparation 1 on expression of alpha-SMA in Lung tissue
As shown in the attached figure 4, compared with a normal control group, the expression level of the mouse lung tissue fibrosis marker alpha-SMA protein in the model group is obviously increased, which indicates that the mouse pulmonary fibrosis model is successfully established. Compared with the model group and the EMSCs group, the expression level of the alpha-SMA protein in the whole lung tissue of the mouse in the CP EMSCs group is obviously reduced, which shows that the CP EMSCs composition can effectively relieve pulmonary fibrosis and reduce the dosage of the EMSCs.
3.3 EMSCs cell preparation 1 genes associated with fibrosis of Lung tissue
As can be seen from FIG. 5, the mRNA expression levels of various inflammatory chemokines in lung tissues of mice in the model group and the EMSCs group were significantly increased compared to the control group, indicating that the low dose of EMSCs had a limited effect on relieving lung inflammation. Compared with the model group and the EMSCs group, the expression of the mouse lung tissues IL-6, MCP-1, TGF-beta and IL-1 beta in the CP EMSCs group is obviously reduced (p is less than 0.05, p is less than 0.01), which indicates that the CP EMSCs composition plays an important role in the process of treating pulmonary fibrosis inflammation, can obviously reduce the using dosage of the EMSCs and enhance the treatment effect.
In the field of cell therapy, the cell concentration is high and the effect is good within a certain range generally. According to the application, the cell preparation 1 prepared in the embodiment 2 is selected, because the cell activity of the early cell is better than that of the cell in the later passage stage, and the cell concentration in the cell preparation 1 is low, compared with the EMSCs with the same concentration in a control group, the cell preparation can well show that the overexpression inhibition factor has the effect of enhancing the treatment effect of the EMSCs.
The results of the above examples show that the bleomycin-induced pulmonary fibrosis mouse model can significantly improve pulmonary fibrosis symptoms and down-regulate the expression level of the α -SMA protein in lung tissue by the intervention treatment of the low-dose CP EMSCs cell preparation. In addition, the CP EMSCs composition plays an important role in treating pulmonary fibrosis inflammation process, and is specifically shown in the aspect of down-regulating gene expression of lung tissues such as IL-6, MCP-1, IL-1 beta, TGF-beta and the like. The EMSCs composition at the equivalent dose did not show the effect of improving pulmonary fibrosis at all. Therefore, the CP EMSCs can reduce the using amount of the EMSCs in the treatment of pulmonary fibrosis and enhance the treatment effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. Application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparing medicines for preventing and/or treating pulmonary fibrosis.
2. The use according to claim 1, wherein the method for constructing the endometrial stem cells over-expressing CTLA-4Ig/PD-L1 comprises the following steps:
1) extracting RNA of the spleen of the animal, and obtaining cDNA through reverse transcription;
2) respectively amplifying CTLA-4Ig gene and PD-L1 gene by using the cDNA as a template to obtain CTLA-4Ig gene segment and PD-L1 gene segment;
3) cloning the CTLA-4Ig gene fragment and the PD-L1 gene fragment to a pLenti CMV GFP Puro (658-5) vector to obtain a recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro;
4) mixing the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, PMD2 and PSPAX2, treating eukaryotic cells under the action of a transfection reagent, culturing and collecting supernatant;
5) infecting the supernatant into endometrial stem cells, and performing cell culture to obtain the endometrial stem cells over-expressing CTLA-4 Ig/PD-L1.
3. The use of claim 2, wherein the primer for amplifying the CTLA-4Ig gene in the step 2) has a nucleotide sequence shown in SEQ ID NO:1 and the nucleotide sequence of the forward primer shown in SEQ ID NO:2, and a reverse primer;
the primer for amplifying the PD-L1 gene has a nucleotide sequence shown as SEQ ID NO:3 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:4, or a reverse primer as shown in figure 4.
4. The use according to claim 2, wherein the mass ratio of the recombinant vector pLenti CMV CTLA-4Ig IRES PD-L1 Puro, PMD2 and PSPAX2 in step 4) is 3: 1.5-2.5: 2.5 to 3.5.
5. The use according to claim 2, wherein the endometrial stem cells in step 5) are selected from any one of primary to tenth generation of endometrial stem cells.
6. The use according to any one of claims 2 to 5, wherein the infection in step 5) is carried out under the action of a transfection enhancing agent; the transfection enhancing agent comprises polybrene reagent.
7. The use of claim 1, wherein the pulmonary fibrosis comprises pulmonary fibrosis induced by a drug;
the drug comprises bleomycin and/or lipopolysaccharide.
8. The use of claim 1 or 7, wherein the medicament comprises an injectable formulation; the injection is a sodium chloride solution containing endometrial stem cells over-expressing CTLA-4 Ig/PD-L1;
the mass volume percentage of the sodium chloride solution is 0.8-1.0%.
9. A cell preparation for preventing and/or treating pulmonary fibrosis, comprising a sodium chloride solution containing endometrial stem cells overexpressing CTLA-4 Ig/PD-L1;
the mass volume percentage of the sodium chloride solution is 0.8-1.0%.
10. The cell preparation of claim 9, wherein the density of the endometrial stem cells overexpressing CTLA-4Ig/PD-L1 is 2.0 x 105～4.0×105one/mL.
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