CN115299405B - Method for manufacturing nude mouse transplanted tumor model - Google Patents
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- CN115299405B CN115299405B CN202210948643.3A CN202210948643A CN115299405B CN 115299405 B CN115299405 B CN 115299405B CN 202210948643 A CN202210948643 A CN 202210948643A CN 115299405 B CN115299405 B CN 115299405B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2207/12—Animals modified by administration of exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention relates to the technical field of animal model production, in particular to a method for producing animal modelsThe method for manufacturing the nude mouse transplanted tumor model comprises the following steps: selecting nude mice of 4-6 weeks old, inoculating tumor cells under aseptic condition, wherein the inoculation amount of tumor cells is (0.1-2) x10 7 200 ul/min; after the tumor is planted, an immunosuppressant is adopted to accelerate the growth of tumor bodies, wherein the addition amount of the immunosuppressant is (1-2) mg/day/patient; and continuing to culture for at least 5 days to obtain the nude mice transplantation tumor model. The invention solves the problem of lower success rate of the nude mice transplanted tumor model in the prior art. The nude mouse transplanted tumor model of the invention can widen the type range of inoculating tumor and improve the success rate of inoculating tumor body through a plurality of process control.
Description
Technical Field
The invention relates to the technical field of animal model manufacturing, in particular to a method for manufacturing a nude mouse transplanted tumor model.
Background
Due to the special constitution of the nude mice, the nude mice can be used for researching different drug-mediated nude mice transplantation tumor models, and play an important role in scientific research. Nude mice generally include nude mice and nude rats. The nude mice have poor resistance and are easy to survive in SPF environment, and the used cages, padding, feed and drinking water have toxic hepatitis and pneumonia, so that the requirements on breeding and reproduction are strict, and the like, and the nude mice are required to be subjected to strict sterilization and disinfection and are bred by adopting an isolator so as to ensure long-term survival and reproduction. The general characteristics of the nude rat are similar to those of the nude mouse, but the trunk part still has rare fur, which is not completely free of fur like the nude mouse, and the head and limbs have more fur; nude rats are susceptible to respiratory disease.
In the prior art, the nude mouse transplantation tumor model is easy to have the problems of unsuccessful tumor transplantation, intolerance of the nude mouse and the like in the manufacturing process, so that the success rate of tumor formation is low.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a method for making a nude mouse transplantation tumor model, which is used for solving the problem of low success rate of the nude mouse transplantation tumor model in the prior art. The nude mouse transplanted tumor model of the invention can widen the type range of inoculating tumor and improve the success rate of inoculating tumor body through a plurality of process control.
To achieve the above-mentioned objects and other related objects,
the invention provides a method for manufacturing a nude mouse transplanted tumor model, which comprises the following steps:
step one, selecting a nude mouse with the age of 4 to 6 weeks, inoculating tumor cells into the nude mouse under the aseptic condition, wherein the inoculation amount of the tumor cells is (0.1 to 2) x10 7 200 ul/min;
step two, adopting an immunosuppressant to accelerate tumor growth after tumor inoculation, wherein the addition amount of the immunosuppressant is (1-2) mg/day/tumor;
and thirdly, continuing to culture for at least 5 days to obtain the nude mouse transplantation tumor model.
In the manufacturing process of the nude mice transplantation tumor model, the nude mice with the age of 4-6 weeks are selected, and the nude mice with the age have certain immunity, so that the problem that the nude mice are intolerant and die prematurely after inoculation is avoided. Moreover, the immunity of nude mice at the age is not strong enough, the tumor forming process of tumor cells is not influenced, and successful tumor planting is facilitated. In addition, the nude mice at the age can adapt to the experimental period of the seed tumor, and the tumor volume of the tumor in the experimental period is ensured to be not more than 2000mm 3 。
The nude mice transplanted tumor model controls the inoculation amount of tumor cells in the manufacturing process, avoids the situation that the tumor growth speed is too high or too low, and is more beneficial to observing and researching the tumor situation of nude mice. In addition, the immunosuppressant is added after the tumor is planted, so that the rejection of the nude mice can be avoided, and the growth of tumor bodies in the nude mice can be accelerated.
Generally, since the nude mice have no thymus and have poor immunity, the whole experiment process should be ensured to be carried out under aseptic conditions as much as possible. In addition, care should be taken to sterilize the injection site prior to injection to avoid infection, resulting in failure to tumor.
Generally, under the condition of ensuring the sufficient number of cells, the cells with good growth vigor of 3-5 generations after resuscitation are selected as far as possible for collection and injection. In addition, the cells to be injected should be resuspended in serum-free medium or PBS, and the cells should be thoroughly mixed to avoid causing excessive differences in tumor volume that develop prior to drug treatment. In addition, cells should be collected and placed in an environment of 4℃or on ice to maintain the hypometabolic state of the cells.
In an embodiment of the invention, the inoculation portion of the nude mouse is at least one of a back, an armpit and a neck. The inoculation site is generally selected for injection at a site with denser blood vessels, such as the armpit (not to be injected deep in the armpit, to be optimal subcutaneously at the middle and outer sides of the armpit), the neck and back, etc., but it should be noted that the selected injection site should be ensured that the normal diet and other activities of the nude mice are not hindered after the tumor formation.
In addition, when the needle is inserted from the part slightly above the waist of the body side of the nude mouse, the distance from the inoculation point is smaller than the length of the needle head, the nude mouse can not puncture the skin or puncture the muscle layer, and the inoculation is preferably completed within 1 hour.
In an embodiment of the invention, in the inoculation process of the nude mice, a distance between an injection point and a needle insertion point after needle insertion is 0.8-1.2 cm. The distance between the injection point and the needle inlet point is controlled to avoid outflow of liquid, and the injector can be gently rocked after the liquid reaches the injection point so as to ensure that the injection is performed subcutaneously; the injection process should be guaranteed to be consistent and slow, and care should be taken to avoid bubble generation; the needle should be withdrawn slowly after injection to avoid outflow of liquid during withdrawal of the needle.
In one embodiment of the present invention, the tumor cells include a first tumor cell and a second tumor cell;
the first tumor cell comprises at least one of a liver cancer cell, a laryngeal cancer cell, a large cell lung cancer cell, an epidermoid cancer cell, an ovarian cancer cell, a breast cancer cell, a human melanoma cell, a human gastric tumor cell and a human oral epidermoid cancer cell;
the second tumor cell comprises at least one of nasopharyngeal carcinoma cell, lung cancer cell, lung adenocarcinoma cell, colon cancer cell, liver cancer cell, breast cancer cell, human neuroblastoma cell, human glioma cell, human gastric cancer cell and human prostatic cancer cell.
In one embodiment of the present invention, the first tumor cells are inoculated in an amount of 1×10 6 200 ul/min; the inoculation amount of the second tumor cells is 2x10 7 200 ul/min.
Because the first tumor cells belong to the cells which are easy to form tumor in a conventional way, the cell inoculation amount is small, and the overquick growth of the tumor cells is avoided. The second tumor cells belong to cells which are difficult to form tumor, so that the cell inoculation amount is obviously higher, and the success rate of the tumor can be improved.
In one embodiment of the invention, the second tumor cells are treated prior to inoculation as follows: second tumor cells were mixed with matrigel basal gel according to 1: (0.8-1.2) by volume ratio.
In an embodiment of the invention, a volume ratio of the second tumor cells to matrigel base gel is 1: (0.95-1.05), wherein the mixing temperature of the second tumor cells and matrigel base gum is-2-4 ℃. matrigel base gum can provide sufficient nutrition for tumor cells in nude mice, and is more beneficial for the second tumor cells to form tumors in nude mice. However, it should be noted that since matrigel base is easily coagulated at room temperature, it is ensured that the homogenized cells are placed in a low temperature environment (ice surface) during inoculation to avoid premature coagulation of matrigel base.
In one embodiment of the invention, the immunosuppressant is cyclophosphamide.
In one embodiment of the present invention, the cyclophosphamide is added in an amount of (1.8 to 2.2) mg/day. In general, cyclophosphamide is used for only two days, and injection is generally selected.
In one embodiment of the invention, the tumor volume of the tumor in the nude mice transplanted tumor model is less than or equal to 2000mm 3 。
Subcutaneously inoculated cells will typically form tumors within 1-2 weeks, typically not exceeding 1 month. In this process, the weight and state of the animal need to be closely observed for judgment.
If the cells have not yet become neoplastic beyond the expected observation time after inoculation, the cause needs to be examined from the following:
1. cell viability: cell viability is a significant cause of influencing tumor formation, and insufficient viability is not tumorigenic. Thus cells were washed with PBS and inoculated as soon as possible.
2. Animal selection: the selected nude mice should not be too old, otherwise the difficulty of tumor formation is increased.
3. Feeding environment: environmental influences on animal states, mice with poor states are easy to die and difficult to form tumors, so that nude mice are fed in an SPF-class environment.
As described above, the method for manufacturing the nude mouse transplantation tumor model has the following beneficial effects:
1. in the manufacturing process of the nude mice transplantation tumor model, the nude mice with the age of 4-6 weeks are selected, and the nude mice with the age have certain immunity, so that the problem that the nude mice are intolerant and die prematurely after inoculation is avoided. Moreover, the immunity of nude mice at the age is not strong enough, the tumor forming process of tumor cells is not influenced, and successful tumor planting is facilitated. In addition, the nude mice at the age can adapt to the experimental period of the seed tumor, and the tumor volume of the tumor in the experimental period is ensured to be not more than 2000mm 3 。
2. The nude mice transplanted tumor model controls the inoculation amount of tumor cells in the manufacturing process, avoids the situation that the tumor growth speed is too high or too low, and is more beneficial to observing and researching the tumor situation of nude mice. In addition, the immunosuppressant is added after the tumor is planted, so that the rejection of the nude mice can be avoided, and the growth of tumor bodies in the nude mice can be accelerated.
3. The nude mice transplanted tumor model can widen the type range of inoculating tumor and improve the success rate of inoculating tumor body through a plurality of process controls.
Drawings
FIG. 1 is a model of nude mouse ovarian cancer transplantable tumor of example 1.
FIG. 2 is a model of liver cancer metastasis in nude mice of example 2.
FIG. 3 is a model of nude mouse human colon cancer engraftment tumor of example 3.
FIG. 4 shows the tumor growth curves of examples 1 to 3.
Fig. 5 is a micrograph of a tumor section of the nude mouse ovarian cancer graft model of example 1.
FIG. 6 is a micrograph of a tumor section of a nude mouse liver cancer graft tumor model of example 2.
FIG. 7 is a micrograph of a tumor section of the nude mouse human colon cancer engrafting tumor model of example 3.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Example 1
A method for making a nude mouse transplanted tumor model, comprising the steps of:
s1, selecting ovarian cancer cells required for inoculation:
s11, under the condition of ensuring the sufficient number of cells, selecting cells with good growth vigor of 3-5 generations after resuscitation for collection and inoculation;
s12, re-suspending ovarian cancer cells to be inoculated by using a serum-free culture medium or PBS, and ensuring that the cells are fully and uniformly mixed so as to avoid excessively obvious difference of tumor volumes growing before drug treatment;
s13, after the cells are collected, the cells are placed in an environment of 4 ℃ or on ice so as to maintain the low metabolism state of the cells;
s2, inoculating seed tumor cells: 10 nude mice with the age of 4-6 weeks are selected, and ovarian cancer cells are inoculated to the backs of the nude mice under the aseptic condition, wherein the inoculation amount of the ovarian cancer cells is 1x10 6 200 ul/min; in the inoculation process, the needle is inserted from the part of the nude mice, which is slightly above the waist of the nude mice, so that the distance between the nude mice and the inoculation point is ensured to be smaller than the length of the needle head, and the inoculation is preferably completed within 1 hour; after needle insertion, the distance between the injection point and the needle insertion point is about 1cm, so that outflow of liquid is avoided;
s3, tumor growth: after the tumor is planted, cyclophosphamide is adopted to accelerate the growth of tumor bodies, the addition amount of cyclophosphamide is 2 mg/day/patient, and cyclophosphamide is injected for two days;
s4, tumor record: after successful tumor implantation, the tumor diameter of the tumor body reaches about 5mm or the tumor volume reaches about 50mm 3 Monitoring can be started when the tumor volume is close to 2000mm by measuring tumor every other day 3 The materials should be obtained in time, and finally the data are collected to draw a tumor growth curve, which is shown in fig. 4.
The ovarian cancer cells have a tumor formation time of 5 to 7 days and a tumor formation rate of 100%. The nude mouse ovarian cancer transplantation tumor model is shown in figure 1. The tumor body section of the nude mouse ovarian cancer transplantation tumor model is observed, and particularly as shown in fig. 5, in-vivo tumor cells, namely human ovarian cancer SKOV3 cells, can be seen in fig. 5.
It is noted that not all tumors are regular spheroids, and thus, as long as the tumor volume of the tumor tissue is guaranteed to be approximately equal, it is available for subsequent monitoring.
Example 2
A method for making a nude mouse transplanted tumor model, comprising the steps of:
s1, selecting liver cancer cells required for inoculation:
s11, under the condition of ensuring the sufficient number of cells, selecting cells with good growth vigor of 3-5 generations after resuscitation for collection and inoculation;
s12, re-suspending liver cancer cells to be inoculated by using a serum-free culture medium or PBS, and ensuring that the cells are fully and uniformly mixed so as to avoid causing excessively obvious difference of the tumor volume growing before the drug treatment;
s13, after the cells are collected, the cells are placed in an environment of 4 ℃ or on ice so as to maintain the low metabolism state of the cells;
s2, inoculating seed tumor cells: 10 nude mice with the age of 4-6 weeks are selected, and the armpit of the nude mice is inoculated with liver cancer cells under the aseptic condition, wherein the inoculation amount of the liver cancer cells is 1x10 6 200 ul/min; in the inoculation process, the needle is inserted from the part of the nude mice, which is slightly above the waist of the nude mice, so that the distance between the nude mice and the inoculation point is ensured to be smaller than the length of the needle head, and the inoculation is preferably completed within 1 hour; after needle insertion, the distance between the injection point and the needle insertion point is about 1cm, so that outflow of liquid is avoided;
s3, tumor growth: after the tumor is planted, cyclophosphamide is adopted to accelerate the growth of tumor bodies, the addition amount of cyclophosphamide is 2 mg/day/patient, and cyclophosphamide is injected for two days;
s4, tumor record: after successful tumor implantation, the tumor diameter of the tumor body reaches about 5mm or the tumor volume reaches about 50mm 3 Monitoring can be started when the tumor volume is close to 2000mm by measuring tumor every other day 3 The materials should be obtained in time, and finally the data are collected to draw a tumor growth curve, which is shown in fig. 4.
The tumor time of the liver cancer cells is 7-10 days, and the tumor rate is 100%. The model of nude mice liver cancer transplantation tumor is shown in figure 2. The tumor body section of the nude mice liver cancer transplantation tumor model is observed, and particularly as shown in fig. 6, the tumor body cell is human liver cancer cell Hep G2.
Example 3
A method for making a nude mouse transplanted tumor model, comprising the steps of:
s1, selecting human colon cancer cells required for inoculation:
s11, under the condition of ensuring the sufficient number of cells, selecting cells with good growth vigor of 3-5 generations after resuscitation for collection and inoculation;
s12, re-suspending the human colon cancer cells to be inoculated by using a serum-free culture medium or PBS, and ensuring that the cells are fully and uniformly mixed so as to avoid causing the excessively obvious difference of the tumor volumes growing before the drug treatment;
s13, mixing human colon cancer cells with matrigel base adhesive according to the volume ratio of 1: (0.95-1.05) uniformly mixing, wherein the mixing temperature of the human colon cancer cells and matrigel base gum is-2-4 ℃;
s13, after the cells are collected, the cells are placed in an environment of 4 ℃ or on ice so as to maintain the low metabolism state of the cells;
s2, inoculating seed tumor cells: 10 nude mice with the age of 4-6 weeks are selected, and human colon cancer cells are inoculated to armpits of the nude mice under the aseptic condition, wherein the inoculation amount of the human colon cancer cells is 1x10 6 200 ul/min; in the inoculation process, the needle is inserted from the part of the nude mice, which is slightly above the waist of the nude mice, so that the distance between the nude mice and the inoculation point is ensured to be smaller than the length of the needle head, and the inoculation is preferably completed within 1 hour; after needle insertion, the distance between the injection point and the needle insertion point is about 1cm, so that outflow of liquid is avoided;
s3, tumor growth: after the tumor is planted, cyclophosphamide is adopted to accelerate the growth of tumor bodies, the addition amount of cyclophosphamide is 2 mg/day/patient, and cyclophosphamide is injected for two days;
s4, tumor record: after successful tumor implantation, the tumor diameter of the tumor body reaches about 5mm or the tumor volume reaches about 50mm 3 Monitoring can be started when the tumor volume is close to 2000mm by measuring tumor every other day 3 The materials should be obtained in time, and finally the data are collected to draw a tumor growth curve, which is shown in fig. 4.
The time for the human colon cancer cells to form tumors is 7-14 days, and the rate of the tumors is 70%. The nude mouse human colon cancer engraftment model is shown in figure 3. The tumor body section of the nude mouse human colon cancer transplantation tumor model is observed, and particularly as shown in fig. 7, the in-vivo tumor cells are human colon cancer cells HT-29 in fig. 7.
In conclusion, the nude mouse transplanted tumor model of the invention can widen the range of inoculating tumor types and improve the success rate of inoculating tumor bodies through a plurality of process controls. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (5)
1. A method for making a nude mouse transplanted tumor model, comprising the steps of:
selecting nude mice of 4-6 weeks old, inoculating colon cancer cells under aseptic condition, wherein the inoculation amount of colon cancer cells is (0.1-2) x10 7 200 ul/min; wherein the inoculation part of the nude mice is at least one of the back, armpit and neck; in the nude mice inoculation process, injection points and injection points after needle insertionThe distance between the needle points is 0.8-1.2 cm;
after the tumor is planted, cyclophosphamide is adopted to accelerate the growth of tumor bodies, and the cyclophosphamide is injected for two days, wherein the addition amount of the cyclophosphamide is (1-2) mg/day/patient;
continuously culturing for at least 5 days to obtain a nude mice transplanted tumor model, wherein the tumor volume of the tumor in the nude mice transplanted tumor model is less than or equal to 2000mm 3 。
2. The method for making a nude mouse engrafted tumor model according to claim 1, wherein: the inoculation amount of the colon cancer cells is 1x10 7 200 ul/min.
3. The method for making a nude mouse engrafted tumor model according to claim 2, wherein: the colon cancer cells were treated prior to inoculation as follows: colon cancer cells were mixed with matrigel basal gel according to 1: (0.8-1.2) by volume ratio.
4. The method for making a nude mouse engrafted tumor model according to claim 3, wherein: the volume ratio of colon cancer cells to matrigel base gum is 1: (0.95-1.05), wherein the mixing temperature of the colon cancer cells and matrigel base gum is-2-4 ℃.
5. The method for making a nude mouse engrafted tumor model according to claim 1, wherein: the addition amount of cyclophosphamide is (1.8-2.2) mg/day.
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| CN104087554A (en) * | 2014-07-02 | 2014-10-08 | 张鹏 | Human thymoma cell line and application thereof |
| CN108835051A (en) * | 2018-06-01 | 2018-11-20 | 郑州大学第附属医院 | Utilize immune deficiency nude mice model constructed by lymphoma cell strain |
| CN109125740A (en) * | 2017-06-28 | 2019-01-04 | 四川大学 | A kind of novel tumor vaccine and application thereof |
| CN109422808A (en) * | 2017-08-31 | 2019-03-05 | 翟乾 | The preparation method of monoclonal antibody |
| CN110279780A (en) * | 2019-07-16 | 2019-09-27 | 北海市海金花生物技术有限公司 | A kind of Chinese Medicines with inhibition growth of tumour cell |
| CN112262818A (en) * | 2020-10-28 | 2021-01-26 | 湖南省肿瘤医院 | Epithelial ovarian cancer in-situ tumorigenic mouse model |
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| US8263823B2 (en) * | 2008-10-20 | 2012-09-11 | Adimmu Institute Inc. | Immunocompetent xenograft model |
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Patent Citations (6)
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|---|---|---|---|---|
| CN104087554A (en) * | 2014-07-02 | 2014-10-08 | 张鹏 | Human thymoma cell line and application thereof |
| CN109125740A (en) * | 2017-06-28 | 2019-01-04 | 四川大学 | A kind of novel tumor vaccine and application thereof |
| CN109422808A (en) * | 2017-08-31 | 2019-03-05 | 翟乾 | The preparation method of monoclonal antibody |
| CN108835051A (en) * | 2018-06-01 | 2018-11-20 | 郑州大学第附属医院 | Utilize immune deficiency nude mice model constructed by lymphoma cell strain |
| CN110279780A (en) * | 2019-07-16 | 2019-09-27 | 北海市海金花生物技术有限公司 | A kind of Chinese Medicines with inhibition growth of tumour cell |
| CN112262818A (en) * | 2020-10-28 | 2021-01-26 | 湖南省肿瘤医院 | Epithelial ovarian cancer in-situ tumorigenic mouse model |
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