CN113197156A - Prostate cancer PDX model construction method - Google Patents
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- A—HUMAN NECESSITIES
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to the technical field of experimental animal models, and particularly discloses a prostate cancer PDX model construction method. The construction method comprises the following steps: the ratio of the cut animal tissue blocks to the cells is 2 x 106‑5×106And uniformly mixing the seminal vesicle cell suspension per mL to obtain a mixed solution, then mixing the mixed solution with matrix glue and transplanting the mixed solution into an animal body, and then transplanting the tumor cell solution into the animal body for passage. The prostate cancer PDX model obtained by the construction method has higher similarity with clinical prostate cancer, the tumor formation rate is high after the tumor cell solution is transplanted, the original microenvironment of a tumor tissue is reserved, the operation method is simple, the cost is lower, and the method can be widely applied to individualized accurate diagnosis and treatment of the prostate cancer, screening of new anti-cancer drugs for clinical application and research of the prostate cancer.
Description
Technical Field
The invention relates to the technical field of experimental animal models, in particular to a prostate cancer PDX model construction method.
Background
Tumors are the most serious group of diseases endangering human health and are one of the leading causes of death in humans. According to data statistics of the world health organization, the number of new tumor cases is 1270 ten thousand in 2008, and the number of tumor deaths is 760 ten thousand in the world. At present, about 900 million new tumor patients are added in the world every year, the worldwide tumor incidence is estimated to increase by about 60 percent compared with the current situation by 2025, and the number of the new tumor patients in the world reaches 1500 million. The morbidity and mortality of tumors in China are the first in the world, the number of people dying from tumors in each year exceeds 160 ten thousand, the tumor morbidity is about 200/10 ten thousand, new cases of the disease are as high as 220 ten thousand every year, and one of every 4 people dying in China is the first to die from tumors and live at the first cause of death. Prostate cancer is a malignant tumor with a high incidence in men. The incidence of prostate cancer is also listed in the prostate of the incidence of male malignant tumors in China, and in recent years, the incidence also tends to increase year by year.
The PDX model (PDX) is a new generation of human tumor xenograft model established by inoculating surgically excised tumor tissue of patients into immunodeficient mice. The model retains the histological and genetic characteristics of primary tumors and maintains the heterogeneity of the tumors of patients, and the pharmacodynamic result has high correlation with clinic. The PDX model can be widely applied to the development of new drugs, particularly to the clinical trial patient screening of target drugs and the research of predictive biomarkers. Compared with the traditional cell line transplantation model, the PDX model is not cultured in vitro, so that the genetic characteristics and heterogeneity of the primary tumor are well maintained, the clinical predictability of the experimental result is better, and the practical situation of clinical patients can be reflected better. The PDX model has the advantage that the characteristics of tumor pathology, tumor genetics, etc. can be reproduced completely. In addition, the PDX model can be used for accurately screening targeted and chemotherapeutic drugs to establish an optimal drug regimen. The method is used for real-time monitoring of the treatment effect of the drug, research of the drug resistance mechanism of the tumor and the like.
At present, the types of solid tumor animal models are limited, and the requirements of preclinical research cannot be met, so that the prediction of clinical curative effect and preclinical research are hindered, and therefore, a high-efficiency and stable tumor PDX model is required to be established. In order to overcome the difficulty that the animal model is limited in the clinical research and treatment of the prostate cancer, the invention constructs the human tumor xenograft model of the prostate cancer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for constructing a prostate cancer PDX model, the prostate cancer PDX model obtained by the construction method has higher similarity with clinical prostate cancer, the tumor formation rate after the transplantation of the tumor cell solution is high, the original microenvironment of a tumor tissue is reserved, the operation method is simple, the cost is lower, and the method can be widely applied to the individualized accurate diagnosis and treatment of the prostate cancer, the screening of new anti-cancer clinical drugs and the research of the prostate cancer.
In order to achieve the purpose, the invention adopts the technical scheme that:
a prostate cancer PDX model construction method comprises the following steps:
the ratio of the cut animal tissue blocks to the cells is 2 x 106-5×106And uniformly mixing the seminal vesicle cell suspension per mL to obtain a mixed solution, then mixing the mixed solution with matrix glue and transplanting the mixed solution into an animal body, and then transplanting the tumor cell solution into the animal body for passage.
As a preferred embodiment of the method for constructing the prostate cancer PDX model, the seminal vesicle cell ratio in the seminal vesicle cell suspension is 2 x 106one/mL.
As a preferable embodiment of the construction method of the prostate cancer PDX model, the size of the sheared animal tissue block is 10-30mm3。
As a preferred embodiment of the construction method of the prostate cancer PDX model, the volume of the sheared animal tissue block and the seminal vesicle cell suspension is 1: 1.
As a preferred embodiment of the method for constructing a prostate cancer PDX model according to the present invention, the specific steps of transplanting tumor cells into an animal for passaging are as follows: obtaining tumor tissue, flushing and cutting to obtain tumor tissue blocks, adding a tissue freezing medium into the tumor tissue blocks to obtain a tumor cell solution, reversing and uniformly mixing, refrigerating and freezing for standing, recovering and culturing the frozen tumor cell solution according to a vitrification recovery process, and finally transplanting the recovered tumor cell solution into an animal body for passage.
As a preferred embodiment of the prostate cancer PDX model construction method, a tumor cell solution is subjected to shake culture at 35-38 ℃ after being recovered according to a vitrification recovery process, the rotation speed of the shake culture is 20-40r/min, and the shake time is 20-40 min.
As a preferred embodiment of the construction method of the prostate cancer PDX model, the time for reverse mixing is 1-10 min.
As a preferred embodiment of the prostate cancer PDX model construction method, the tumor cell solution is reversed and mixed evenly, and then is refrigerated for 10-30min at the temperature of 2-8 ℃.
As a preferable embodiment of the construction method of the prostate cancer PDX model, the time of freezing and placing is 10-30 min.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a construction method of a prostate cancer PDX model, which constructs a high-efficiency stable prostate cancer PDX model, overcomes the difficulty that the animal model is limited in clinical research and treatment of prostate cancer, and the model grows depending on the reproductive system of a mouse and has higher similarity with the clinical prostate cancer; by adopting the construction method, the tumor formation rate is high after the tumor cells are transplanted, the original microenvironment of the tumor tissue is reserved, the construction method is simple, the cost is lower, and the construction method can be widely applied to the individualized accurate diagnosis and treatment of the prostate cancer, the screening of new anti-cancer drugs for clinical application and the research of the prostate cancer.
Drawings
FIG. 1 is a graph of tumor volume proliferation trend for a PDX model of prostate cancer;
FIG. 2 is a graph of HE staining of primary P0 tissue in a patient;
FIG. 3 is a graph showing HE staining of mouse P1 tumor tissue.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
Embodiment 1, method for constructing prostate cancer PDX model
A prostate cancer PDX model construction method comprises the following steps:
1) cell extraction: taking one healthy B-NOD mouse, making T-shaped incision on lower abdomen, picking seminal vesicle gland, stripping surface fascia, placing on 200 mesh sieve for soft grinding, collecting seminal vesicle gland cells, mixing with a small amount of DMEM to obtain seminal vesicle gland cell suspension, and adjusting the proportion of seminal vesicle gland cells in seminal vesicle gland cell suspension to 2 × 106Per mL concentration;
2) resuscitating animal tissue pieces, and cutting into pieces of 10-30mm3Uniformly mixing the sheared animal tissue block and seminal vesicle cell suspension according to the volume ratio of 1:1 to obtain a tissue block containing seminal vesicle cells;
3) transferring the tissue block containing the seminal vesicle gland cells to a culture dish filled with EPS for culture to obtain a mixed solution, sealing, placing on an ice surface, and transferring to an animal room. In a biological safety cabinet, mixing the mixed solution and matrigel according to the volume ratio of 1:1 and transplanting the mixture to the subcutaneous part of a mouse;
4) killing tumor-bearing mice with well-grown tumor mass under anesthesia, removing tumor tissue under aseptic condition, flushing the tumor tissue with HEPS solution within 30min, and cutting the tumor tissue to 2-3mm3And (3) obtaining a tumor tissue block, selecting a pink fresh viable tumor tissue block, placing the pink fresh viable tumor tissue block in a DMEM (DMEM) culture solution for storage, and directly performing tissue cryopreservation operation when the pink fresh viable tumor tissue block cannot be transplanted to a host animal within 48 hours. Adding 2mL of tissue cryopreservation liquid into every 2-3 small tumor tissue blocks, placing the tissue cryopreservation liquid into a 2mL sterilizing cryopreservation tube to obtain tumor cell solution, reversing and uniformly mixing the tumor cell solution for 3 minutes, placing the tumor cell solution into a 2-8 ℃ refrigerator for standing for 20 minutes, then gently reversing and uniformly mixing the tumor cell solution for 4-5 times, transferring the tumor cell solution into a-20 ℃ refrigerator for freezing for 20 minutes, then transferring the tumor cell solution into a-80 ℃ refrigerator for overnight, finally transferring the tumor cell solution into a liquid nitrogen environment for long-term cryopreservation, then taking out the cryopreservation tube from a liquid nitrogen tank, recovering the tumor cell solution according to a vitrification recovery process, performing shaking culture at the temperature of 30r/min for 30 minutes at 37 ℃, and finally transferring the recovered tumor cell solution into an animal body for passage to obtain the prostate cancer PDX model.
Pretreatment of experimental animals: selecting qualified SPF grade B-NOD mice, administering testosterone of 40mg/kg, intragastrically inoculating the mice the next day, and administering 1 time per week after inoculation.
Embodiment 2, prostate cancer PDX model construction method
A prostate cancer PDX model construction method comprises the following steps:
1) cell extraction: taking one healthy B-NOD mouse, making T-shaped incision on lower abdomen, picking seminal vesicle gland, stripping surface fascia, placing on 200 mesh sieve for soft grinding, collecting seminal vesicle gland cells, mixing with a small amount of DMEM to obtain seminal vesicle gland cell suspension, and adjusting the proportion of seminal vesicle gland cells in seminal vesicle gland cell suspension to 3 × 106Per mL concentration;
2) resuscitating animal tissue pieces, and cutting into pieces of 10-30mm3Uniformly mixing the sheared animal tissue block and seminal vesicle cell suspension according to the volume ratio of 1:1 to obtain a tissue block containing seminal vesicle cells;
3) transferring the tissue block containing the seminal vesicle gland cells to a culture dish filled with EPS for culture to obtain a mixed solution, sealing, placing on an ice surface, and transferring to an animal room. In a biological safety cabinet, mixing the mixed solution and matrigel according to the volume ratio of 1:1 and transplanting the mixture to the subcutaneous part of a mouse;
4) killing tumor-bearing mice with well-grown tumor mass under anesthesia, removing tumor tissue under aseptic condition, flushing the tumor tissue with HEPS solution within 30min, and cutting the tumor tissue to 2-3mm3And (3) obtaining a tumor tissue block, selecting a pink fresh viable tumor tissue block, placing the pink fresh viable tumor tissue block in a DMEM (DMEM) culture solution for storage, and directly performing tissue cryopreservation operation when the pink fresh viable tumor tissue block cannot be transplanted to a host animal within 48 hours. Adding 2mL of tissue cryopreservation liquid into every 2-3 small tumor tissue blocks, placing the tissue cryopreservation liquid into a 2mL sterilizing cryopreservation tube to obtain tumor cell solution, reversing and uniformly mixing the tumor cell solution for 3 minutes, placing the tumor cell solution into a 2-8 ℃ refrigerator for standing for 20 minutes, then gently reversing and uniformly mixing the tumor cell solution for 4-5 times, transferring the tumor cell solution into a-20 ℃ refrigerator for freezing for 20 minutes, then transferring the tumor cell solution into a-80 ℃ refrigerator for overnight, finally transferring the tumor cell solution into a liquid nitrogen environment for long-term cryopreservation, then taking out the cryopreservation tube from a liquid nitrogen tank, recovering the tumor cell solution according to a vitrification recovery process, performing shaking culture at the temperature of 30r/min for 30 minutes at 37 ℃, and finally transferring the recovered tumor cell solution into an animal body for passage to obtain the prostate cancer PDX model.
Pretreatment of experimental animals: selecting qualified SPF grade B-NOD mice, administering testosterone of 40mg/kg, intragastrically inoculating the mice the next day, and administering 1 time per week after inoculation.
Embodiment 3, method for constructing prostate cancer PDX model
A prostate cancer PDX model construction method comprises the following steps:
1) cell extraction: taking one healthy B-NOD mouse, making T-shaped incision on lower abdomen, picking seminal vesicle gland, stripping surface fascia, placing on 200 mesh sieve for soft grinding, collecting seminal vesicle gland cells, mixing with a small amount of DMEM to obtain seminal vesicle gland cell suspension, and adjusting the proportion of seminal vesicle gland cells in seminal vesicle gland cell suspension to 4 × 106Per mL concentration;
2) resuscitating animal tissue pieces, and cutting into pieces of 10-30mm3Uniformly mixing the sheared animal tissue block and seminal vesicle cell suspension according to the volume ratio of 1:1 to obtain a tissue block containing seminal vesicle cells;
3) transferring the tissue block containing the seminal vesicle gland cells to a culture dish filled with EPS for culture to obtain a mixed solution, sealing, placing on an ice surface, and transferring to an animal room. In a biological safety cabinet, mixing the mixed solution and matrigel according to the volume ratio of 1:1 and transplanting the mixture to the subcutaneous part of a mouse;
4) killing tumor-bearing mice with well-grown tumor mass under anesthesia, removing tumor tissue under aseptic condition, flushing the tumor tissue with HEPS solution within 30min, and cutting the tumor tissue to 2-3mm3And (3) obtaining a tumor tissue block, selecting a pink fresh viable tumor tissue block, placing the pink fresh viable tumor tissue block in a DMEM (DMEM) culture solution for storage, and directly performing tissue cryopreservation operation when the pink fresh viable tumor tissue block cannot be transplanted to a host animal within 48 hours. Adding 2mL of tissue cryopreservation solution into 2mL of small tumor tissue blocks per 2-3 tumor tissue blocks, placing in a 2mL sterilizing cryopreservation tube to obtain tumor cell solution, reversing and mixing for 3 min, placing in a 2-8 deg.C refrigerator for standing for 20min, then gently reversing and mixing for 4-5 times, transferring into a-20 deg.C refrigerator for freezing for 20min, transferring into a-80 deg.C refrigerator for overnight, transferring into liquid nitrogen environment for long-term cryopreservation, taking out the cryopreservation tube from the liquid nitrogen tank, recovering according to vitrification recovery process, performing shake culture at 37 deg.C for 30min at 30r/min, and finally subjecting to freeze-thawAnd transplanting the recovered tumor cell solution into an animal body for passage to obtain a prostate cancer PDX model.
Pretreatment of experimental animals: selecting qualified SPF grade B-NOD mice, administering testosterone of 40mg/kg, intragastrically inoculating the mice the next day, and administering 1 time per week after inoculation.
Embodiment 4 method for constructing prostate cancer PDX model
A prostate cancer PDX model construction method comprises the following steps:
1) cell extraction: taking one healthy B-NOD mouse, making T-shaped incision on lower abdomen, picking seminal vesicle gland, stripping surface fascia, placing on 200 mesh sieve for soft grinding, collecting seminal vesicle gland cells, mixing with a small amount of DMEM to obtain seminal vesicle gland cell suspension, and adjusting the proportion of seminal vesicle gland cells in seminal vesicle gland cell suspension to 5 × 106Per mL concentration;
2) resuscitating animal tissue pieces, and cutting into pieces of 10-30mm3Uniformly mixing the sheared animal tissue block and seminal vesicle cell suspension according to the volume ratio of 1:1 to obtain a tissue block containing seminal vesicle cells;
3) transferring the tissue block containing the seminal vesicle gland cells to a culture dish filled with EPS for culture to obtain a mixed solution, sealing, placing on an ice surface, and transferring to an animal room. In a biological safety cabinet, mixing the mixed solution and matrigel according to the volume ratio of 1:1 and transplanting the mixture to the subcutaneous part of a mouse;
4) killing tumor-bearing mice with well-grown tumor mass under anesthesia, removing tumor tissue under aseptic condition, flushing the tumor tissue with HEPS solution within 30min, and cutting the tumor tissue to 2-3mm3And (3) obtaining a tumor tissue block, selecting a pink fresh viable tumor tissue block, placing the pink fresh viable tumor tissue block in a DMEM (DMEM) culture solution for storage, and directly performing tissue cryopreservation operation when the pink fresh viable tumor tissue block cannot be transplanted to a host animal within 48 hours. Adding 2mL of tissue cryopreservation solution into 2mL of small tumor tissue blocks, placing in 2mL of sterilized cryopreservation tube to obtain tumor cell solution, mixing for 3 min, standing in 2-8 deg.C refrigerator for 20min, mixing for 4-5 times, freezing in-20 deg.C refrigerator for 20min, transferring to-80 deg.C refrigerator for overnight, transferring into liquid nitrogen environment for long-term cryopreservation, and taking out the cryopreservation tube from liquid nitrogen tankAfter recovery according to a vitrification recovery process, carrying out shaking culture at 37 ℃ for 30min at a speed of 30r/min, and finally transplanting the recovered tumor cell solution into an animal body for passage to obtain a prostate cancer PDX model.
Pretreatment of experimental animals: selecting qualified SPF grade B-NOD mice, administering testosterone of 40mg/kg, intragastrically inoculating the mice the next day, and administering 1 time per week after inoculation.
Examples of the experiments
Observation and body weight: changes in the tumor size of implanted tumor masses were observed or measured weekly in the prostate cancer PDX model prepared in example 1, and the body weight of mice was measured. Tumor diameter measurement: subcutaneous implantation the long and short diameters of the tumor mass were measured using a vernier caliper, as follows: the change in tumor volume was recorded as (major diameter × minor diameter 2)/2. The results showed that the tumor tissue volume was 259mm 6 weeks after inoculation3The above.
And (3) pathological detection: referring to FIG. 1, the tumor mass to be treated is as long as about 800mm3And performing autopsy after the mice are sacrificed, observing lesion metastasis, stripping subcutaneous tumor tissues, preserving more than half of the tissue quantity for vitrification cryopreservation, and cutting and storing the rest tissues according to the detection requirements.
Referring to FIGS. 2-3, HE staining confirmed tumor structure and nuclear fission images were observed to confirm tumor tissue viability. After the mice are cultured in vivo 6 weeks after inoculation, the glandular cells in the tissues are massively proliferated, acinar tissue structures can still be seen, non-tumor transparent cells disappear, and matrixes are gradually replaced by murine tissues.
The invention constructs a high-efficiency stable prostate cancer PDX model, overcomes the difficulty that the animal model is limited in the clinical research and treatment of the prostate cancer, and the invention grows depending on the reproductive system of a mouse and has higher similarity with the clinical prostate cancer; by adopting the construction method, the tumor formation rate is high after the tumor cells are transplanted, the original microenvironment of the tumor tissue is reserved, the construction method is simple, the cost is lower, and the construction method can be widely applied to the individualized accurate diagnosis and treatment of the prostate cancer, the screening of new anti-cancer drugs for clinical application and the research of the prostate cancer.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A prostate cancer PDX model construction method is characterized by comprising the following steps:
the ratio of the cut animal tissue blocks to the cells is 2 x 106-5×106And uniformly mixing the seminal vesicle cell suspension per mL to obtain a mixed solution, then mixing the mixed solution with matrix glue and transplanting the mixed solution into an animal body, and then transplanting the tumor cell solution into the animal body for passage.
2. The method for constructing a prostate cancer PDX model according to claim 1, wherein the proportion of seminal vesicle cells in said seminal vesicle cell suspension is 2 x 106one/mL.
3. The method of constructing a prostate cancer PDX model of claim 1, wherein said minced animal tissue mass has a size of 10-30mm3。
4. The method for constructing a prostate cancer PDX model according to claim 1, wherein the volume of the minced animal tissue mass and the seminal vesicle cell suspension is 1: 1.
5. The method for constructing a prostate cancer PDX model according to claim 1, wherein said step of transplanting tumor cells into an animal for passaging comprises the steps of: obtaining tumor tissue, flushing and cutting to obtain tumor tissue blocks, adding a tissue freezing medium into the tumor tissue blocks to obtain a tumor cell solution, reversing and uniformly mixing, refrigerating and freezing for standing, recovering and culturing the frozen tumor cell solution according to a vitrification recovery process, and finally transplanting the recovered tumor cell solution into an animal body for passage.
6. The method for constructing a prostate cancer PDX model according to claim 5, wherein the tumor cell solution is shake-cultured at 35-38 ℃ after being recovered according to a vitrification recovery process, the rotation speed of shake culture is 20-40r/min, and the shake time is 20-40 min.
7. The method for constructing a prostate cancer PDX model according to claim 5, wherein said inversion mixing time is 1-10 min.
8. The method for constructing a prostate cancer PDX model according to claim 5, wherein said tumor cell solution is refrigerated for 10-30min at a temperature of 2-8 ℃ after being inverted and mixed uniformly.
9. The method for constructing a PDX model for prostate cancer according to claim 5, wherein said freezing is performed for a period of time ranging from 10min to 30 min.
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