CN110637783B - Construction method of synovial sarcoma xenograft mouse model with healthy immunity - Google Patents
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Abstract
The invention provides a construction method of a synovial sarcoma xenograft mouse model with healthy immunity, which comprises the following steps of culturing P0 generation: resuscitating the synovial sarcoma tumor tissue block stored in liquid nitrogen environment, and transplanting into immunodeficient mouse under aseptic condition; pretreating a mouse with normal immunity by using cyclosporine; culturing Px generation: inoculating a tumor tissue block growing in a mouse from the P0 generation to the Px-1 generation into the mouse pretreated by cyclosporine to form a Px generation mouse; py generation culture: inoculating the synovial sarcoma tumor tissue block growing in the mouse of any generation from the Px generation to the Py-1 generation into the immune normal mouse until the tumor tissue grows in the body, and finishing the culture of the Py generation. The invention improves the problem that the traditional PDX model has no immune system, provides an in-vivo model for the research of tumor immunotherapy and widens the application range of the PDX model.
Description
Technical Field
The invention relates to the field of establishment of tumor transplantation mouse models, in particular to a method for establishing a synovial sarcoma xenograft mouse model with sound immunity.
Background
Synovial sarcoma (Synovial sarcoma, SS) is a highly malignant soft tissue sarcoma, accounting for about 7-10% of soft tissue sarcomas [1 ]. The SS tissues have complex origins, no clear risk factors, changeable pathological forms and difficult benign and malignant demarcation, so the diagnosis, differential diagnosis and classification become clinical problems. SS is common in men of 20-30 years old (about 1.2: 1 for men/women), occurs mainly near the large joints of the limbs, but occurs almost anywhere throughout the body, and SS hardly occurs in the joints and has no relation to normal synovial tissue. Approximately 80% of primary SS occurs in the extremities, and around 20% occurs in non-extremities such as the ribs, pericardium, lungs, and abdominal wall. The vast majority of SS migrates to the lung, with less abdominal metastasis. The previous research considers that the extensive excision of tumor bodies and surrounding excisable tissues is the first choice method for treating SS, but the recurrence rate is high, so that comprehensive treatment becomes an important means for improving the SS prognosis. This brings great mental injury to the patient and his family, and also brings heavy economic burden to the society and even the country, bringing greater social problems. Because of the existence of the heterogeneity of synovial sarcoma tissues, the basic research results based on the synovial sarcoma cell line in the past are difficult to reproduce in clinical synovial sarcoma patients, and the results largely lead to the delay of the clinical synovial sarcoma treatment level.
Therefore, the model of human-derived xenotransplantation (PDX) which is from a patient and has better homogeneity with the primary tumor of the patient has far-reaching significance, the pathogenesis of synovial sarcoma can be deeply discussed, the result of research and development based on PDX can be well repeated in the body of the patient, limited synovial sarcoma samples in the body of a clinical patient can be greatly amplified, and a xenograft mouse is used for replacing the clinical patient to carry out different combined drug screening, so that the most appropriate drug is selected, the effectiveness is greatly improved, and the side effect of the drug is reduced.
The development and application of the PDX model in recent years show that the PDX model has superiority in tumor research and clinical application compared with a tumor model established by a cell line, but the PDX model generally needs an immunodeficiency mouse as a tumor carrier for relevant research. The immune deficiency of mice has enabled the establishment and use of PDX tumor models, but has limited their use because many immune-related therapies and studies such as immune checkpoint inhibitors require the immune system of mice to be relatively normal for their performance, and the complete immune system of mice is an urgent problem to be solved by current PDX models.
In fact, the successful transplantation of the tumor of the patient into the mouse body is realized by immunizing a normal mouse at the earliest time, namely, the immune function of the normal mouse is inhibited to a certain extent by a specific mode method, so that the human tumor is successfully planted into the mouse body, but not into an immunodeficient mouse. For example, researchers have successfully established tumor models by immunizing normal rats with lethal doses of X-rays to destroy their immune system. The method can damage the organism tissue of the whole rat, thereby damaging all physiological functions of the whole rat, and the method has high death rate of the mouse and low general application value. However, as the tumor was passaged, the mitotic image and vascularization of the tumor appeared to be enhanced, suggesting a slow adaptation and change of the tumor to the environment in the mice. Then, the cortisone with large dose of adrenocortical hormone for a long time is also applied to inhibiting the immunity of normal mice, thereby creating an environment suitable for tumor transplantation. The tumor passage established by the two methods is greatly limited, and the tumor passage is difficult to continue to propagate in the mice with normal immunity, so that the application of the tumor passage is further limited.
Researchers have attempted to achieve this with antilymphocyte and antithymocyte strategies by injecting mice with either antilymphocyte serum or antilymphocyte serum and then planting human tumors in the mice. Also, researchers have attempted to grow human tumors after removal of the thymus from mice. However, the tumor xenograft model established by the methods is very low in tumor formation rate, the problem of stable passage of the tumor is not solved, and even if the tumor grows unevenly after passage, the real biological characteristics of the original tumor cannot be accurately displayed. Researchers try to inoculate the tumor of a patient under the renal capsule of an immune normal mouse, successfully establish a human-derived tumor xenograft model and perform drug sensitivity test, and the main reason of modeling is that the renal capsule part has rich blood circulation and small tumor mass, so that the tumor is easy to obtain enough nutrients, the logarithmic phase is shortened, the growth is rapid, the administration efficiency is high, the measurement is easy, and the sensitivity is high. However, the method mainly considers that the immune response caused by the initial antigen stimulation of an immune normal mouse is generally 7-10 days, and the cell-mediated immune response to the allograft is obvious at 9-12 days, so that the tumor can be proliferated within the first 6 days, so that the method still aims to test the drug sensitivity, the tumor still regresses, a microscope is needed for observing the tumor, a plurality of inconveniences exist in the experiment, and the passage and most researches still cannot be carried out.
The prior art is mainly to perform subcutaneous modeling after fresh synovial sarcoma tissues are subjected to a mincing treatment or a cell homogenization treatment. For example, CN201910462582.8 of the applicant's prior application provides a synovial sarcoma cell line hSS-005R and its progeny cell line. The method comprises the steps of taking hSS-005R cells in a synovial sarcoma cell line to carry out subcutaneous unilateral inoculation on an immunodeficiency mouse, wherein after 1-3 weeks of inoculation, a tumor begins to form and grow, and the immunodeficiency mouse 30-50 days after inoculation is a mouse model for medical research.
Disclosure of Invention
However, the homogenization treatment is considered to destroy the characteristics of the microenvironment of the tumor tissue, and the consistency of the tumor, the surrounding stroma and the patient cannot be well maintained. And the immunodeficient mouse model in the prior art still has a plurality of defects in medical application. Therefore, there is a need in the art for an immunocompromised synovial sarcoma xenograft mouse model and a method of constructing the same.
The invention firstly provides a construction method of a synovial sarcoma xenograft mouse model with sound immunity, which comprises the following steps:
step one, culturing P0 generation: collecting a synovial sarcoma patient tumor tissue block, heterogeneously transplanting a fresh patient tumor tissue block into an immunodeficiency mouse body under an aseptic condition, wherein the xenotransplantation comprises selecting an immunodeficiency mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with that of the patient, soaking the collected fresh patient tumor tissue block in an aseptic incubation liquid containing fetal calf serum, anesthetizing and fixing the mouse, implanting the patient tumor tissue block into the mouse body, suturing an incision, and breeding the mouse to enable the tumor to grow; the xenotransplantation comprises subcutaneous transplantation or orthotopic transplantation; optionally inoculating a synovial sarcoma tumor tissue mass grown in a P0 generation immunodeficient mouse into another immunodeficient mouse to form a P1 generation tumor-bearing immunodeficient mouse; optionally inoculating a synovial sarcoma tumor tissue mass grown in a P1 generation tumor-bearing immunodeficient mouse into another immunodeficient mouse to form a P2 generation tumor-bearing immunodeficient mouse; and so on;
step two, pretreating the immune normal mice with cyclosporine: injecting injection containing cyclosporine into a mouse with normal immunity or allowing the mouse with normal immunity to eat diet containing cyclosporine;
step one and step two can be finished in any order, but both need to be finished before step three;
step three, culturing Px generation: x in the Px generation is a natural number which is more than or equal to 1, and a synovial sarcoma tumor tissue block growing in the tumor-bearing immunodeficiency mouse of any generation from the P0 generation to the Px-1 generation is inoculated to the mouse prepared in the step two and pretreated by cyclosporine to form a Px generation tumor-bearing mouse; optionally inoculating a synovial sarcoma tumor tissue block growing in the Px generation tumor-bearing mouse into another mouse prepared in the second step and pretreated by cyclosporine to form a Px +1 generation tumor-bearing mouse; optionally inoculating a synovial sarcoma tumor tissue block growing in the Px +1 generation tumor-bearing mouse into another mouse prepared in the second step and pretreated by cyclosporine to form a Px +2 generation tumor-bearing mouse; and so on;
step four, Py generation culture: and y in the Py generation is a natural number which is more than or equal to x +1, a synovial sarcoma tumor tissue block growing in the tumor-bearing mouse of any generation from the Px generation to the Py-1 generation in the step three is inoculated into the immune normal mouse, and the immune normal mouse is bred until a synovial sarcoma tumor tissue grows in the immune normal mouse, so that the culture of the Py generation is completed.
In a specific embodiment, the orthotopic transplantation comprises first cutting the skin of the mouse knee joint longitudinally with a sterile ophthalmic scissors, then opening the joint capsule along the medial longitudinal incision of the patellar ligament with a microscissor or a microslade, followed by implantation of a synovial sarcoma tissue mass in the joint capsule. In one embodiment, after implantation of the synovial sarcoma tissue mass in the joint capsule, the joint capsule is sutured first, followed by suturing the skin incision; the joint capsule is a knee joint capsule.
In a specific embodiment, the synovial sarcoma patient tumor tissue mass has a unilateral length of 3mm or less, preferably 1mm or less.
In a specific embodiment, the weight of the mouse in subcutaneous transplantation or orthotopic transplantation is 8-20 g, preferably 12-15g, the sterile incubation liquid further comprises an antibiotic, preferably the sterile incubation liquid comprises an antibiotic and a DMEM medium or an RPMI-1640 medium, and more preferably the antibiotic comprises penicillin and streptomycin.
In a specific embodiment, the immunodeficient mouse is a NOD/SCID mouse and the immunodeficient mouse is a BALB/c mouse.
In a specific embodiment, the method further comprises observing the size of the synovial sarcoma in the mouse using a small animal living imager, preferably also observing whether the tumor has metastasized to the internal organs of the mouse using the small animal living imager.
In a specific embodiment, the method further comprises the step of programming the partial P0 generation tumor volume to cool and store in liquid nitrogen, and optionally the step of programming the partial P1 generation, P2 generation to Py generation tumor volume to cool and store in liquid nitrogen.
In a specific embodiment, the step of pretreating the immune-normal mice with cyclosporin in step two comprises: the skin is lightly wiped by a forceps clamping alcohol cotton ball, the prepared cyclosporine solution with the concentration of 1-2 wt% is extracted by an injector, the drug is administrated with the dosage of about 0.02-0.08 ml for each mouse, the needle point faces to the head, the needle is inserted at the lower side of the abdomen of the mouse, the skin is punctured, the needle is inserted a little in parallel, the abdominal viscera is prevented from being punctured, and then the drug is slowly injected after no liquid is pumped back; the injection is performed once a day for 3-10 days.
The invention also provides application of the mouse model obtained by the method in screening drugs for preventing or treating synovial sarcoma.
The invention also provides a construction method of the synovial sarcoma xenograft mouse model with healthy immunity, which is characterized by comprising the following steps:
step one, culturing P0 generation: firstly, recovering a synovial sarcoma patient tumor tissue block stored in a liquid nitrogen environment, then transplanting the recovered patient tumor tissue block into an immunodeficient mouse in a heterogeneous manner under an aseptic condition, wherein the heterogeneous transplantation comprises selecting an immunodeficient mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with that of the patient, soaking the recovered patient tumor tissue block in a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the patient tumor tissue block into the mouse, suturing an incision, and breeding the mouse to enable the tumor of the mouse to grow; the xenotransplantation comprises subcutaneous transplantation or orthotopic transplantation; optionally inoculating a synovial sarcoma tumor tissue mass grown in a P0 generation immunodeficient mouse into another immunodeficient mouse to form a P1 generation tumor-bearing immunodeficient mouse; optionally inoculating a synovial sarcoma tumor tissue mass grown in a P1 generation tumor-bearing immunodeficient mouse into another immunodeficient mouse to form a P2 generation tumor-bearing immunodeficient mouse; and so on;
step two, pretreating the immune normal mice with cyclosporine: injecting injection containing cyclosporine into a mouse with normal immunity or allowing the mouse with normal immunity to eat diet containing cyclosporine;
step one and step two can be finished in any order, but both need to be finished before step three;
step three, culturing Px generation: x in the Px generation is a natural number which is more than or equal to 1, and a synovial sarcoma tumor tissue block growing in the tumor-bearing immunodeficiency mouse of any generation from the P0 generation to the Px-1 generation is inoculated to the mouse prepared in the step two and pretreated by cyclosporine to form a Px generation tumor-bearing mouse; optionally inoculating a synovial sarcoma tumor tissue block growing in the Px generation tumor-bearing mouse into another mouse prepared in the second step and pretreated by cyclosporine to form a Px +1 generation tumor-bearing mouse; optionally inoculating a synovial sarcoma tumor tissue block growing in the Px +1 generation tumor-bearing mouse into another mouse prepared in the second step and pretreated by cyclosporine to form a Px +2 generation tumor-bearing mouse; and so on;
step four, Py generation culture: and y in the Py generation is a natural number which is more than or equal to x +1, a synovial sarcoma tumor tissue block growing in the tumor-bearing mouse of any generation from the Px generation to the Py-1 generation in the step three is inoculated into the immune normal mouse, and the immune normal mouse is bred until a synovial sarcoma tumor tissue grows in the immune normal mouse, so that the culture of the Py generation is completed.
In a specific embodiment, the method further comprises the steps of programmed cooling and liquid nitrogen preservation of the collected synovial sarcoma tumor tissue mass of the patient prior to the step-rapid warming resuscitation.
In a specific embodiment, the synovial sarcoma patient tumor tissue block is preserved in DMEM containing DMSO, FBS and NEAA, the temperature is reduced to the liquid nitrogen temperature by more than three sections of procedures, and the synovial sarcoma patient tumor tissue block is preserved in the liquid nitrogen temperature, and the rapid heating resuscitation refers to resuscitating frozen tissues in a constant-temperature water bath at 36-38 ℃.
Although the PDX tumor model established by transplanting the tumor tissue of a patient into an immunodeficient mouse in the prior art well maintains the biological characteristics of a primary tumor, the tumor tissue matrix components are gradually replaced by a mouse-derived matrix in the mouse in-vivo passage process, the in-vivo environment of a mouse lacking an immune system also changes the microenvironment of the tumor, and the heterogeneity of the primary tumor is gradually lost, so that the application of the model is limited.
The invention has at least the following beneficial effects: according to the method, a synovial sarcoma patient tumor specimen is planted in an immunodeficient nude mouse body, the synovial sarcoma patient tumor specimen is planted in a BALB/c mouse body with normal immunity after passage, cyclosporin is used for immunosuppression, and cyclosporin drug intervention can be removed after passage, so that a PDX model can be successfully established in the mouse body with normal immunity, the problem that a traditional PDX model has no immune system is solved, an in-vivo model is provided for the research of tumor immunotherapy, and the application range of the PDX model is widened.
Drawings
FIG. 1 is a photograph of tumors formed in immunocompromised BALB/c mice.
FIG. 2 is a comparison of synovial sarcoma of immunocompromised BALB/c mice and pathological images of patient tumors.
FIG. 3 shows the results of CD4+ T cell staining of patient tumors and paraneoplastic tissues and tumor and paraneoplastic tissues of various mouse generations.
FIG. 4 shows the results of CD8+ T cell staining of patient tumors and paraneoplastic tissues and tumor and paraneoplastic tissues of various mouse generations.
FIG. 5 shows the results of CD68+ macrophage staining of patient tumors and paraneoplastic tissues and tumor and paraneoplastic tissues of various mouse generations.
FIG. 6 shows the result of PD-1 positive cell staining of tumor and paraneoplastic tissues of patients and tumor and paraneoplastic tissues of various generations of mice.
FIG. 7 shows the result of PD-L1 positive cell staining of patient tumor and paraneoplastic tissue and tumor and paraneoplastic tissue of various mouse generations.
Detailed Description
Approved by the ethical committee of hospitals, patients sign an informed consent before sample collection, collected tissues are subjected to histopathological examination, and tumor tissue blocks of synovial sarcoma patients, which are pathologically diagnosed, are stored in sterile physiological saline and placed on ice. Dividing the tissue block into 3 parts, and directly treating (see below for details) the tissue block under aseptic conditions in part 1 to perform xenografting into immunodeficient mice; part 2 was placed in DMEM (a medium containing various amino acids and glucose) supplemented with 10% DMSO (dimethyl sulfoxide), 90% FBS (fetal bovine serum) and 1% NEAA (non-essential amino acids), gradient-frozen to-80 ℃, and placed in liquid nitrogen for storage; and the part 3 is put into liquid nitrogen for quick freezing and then is put into the liquid nitrogen for preservation. Wherein, part 1 is used for PDX modeling, part two is used for resuscitation and inoculation, and part three is used for gene detection of censorship.
Step one, culturing P0 generation-subcutaneous inoculation: selecting 10 NOD/SCID mice with 4 weeks of age, keeping the sex of the mice consistent with that of the patients and the weight of the mice is about 12-15g, fresh tumor tissue (or synovial sarcoma patient tumor tissue frozen and resuscitated with liquid nitrogen) was soaked in sterile FBS + penicillin and streptomycin (final penicillin concentration 100U/ml, final streptomycin concentration 100U/ml) and cut into 1 × 1mm pieces with sterile scissors. Mice were anesthetized and fixed, skin incisions were made near the axilla on the right side, tumor tissue blocks were implanted subcutaneously (P0), and surgical incisions were sutured. Mice were observed daily for tumor growth. Tumors grow to diameters exceeding 5 mm. Tumor volume was calculated by measuring tumor volume 2 times per week with a vernier caliper and recording tumor length (a) and width (b) (V ═ a × b)2/2). Meanwhile, a small animal living body imager is adopted to observe the viscera transfer condition, and the volume V of the tumor body is increased to 1500mm3When the time is about 42 days later, the animal is dissected, the tumor body is stripped and weighed, part of tumor mass is inoculated and passed, and the rest is placed into liquid nitrogen for preservation.
Step one is either in-situ implantation: 10 NOD/SCID mice with 4 weeks of age are selected, the sex of the mice is consistent with that of patients, and the weight of the mice is about 12-15 g. Fresh sterile tumor tissue (or tumor tissue after liquid nitrogen preservation and resuscitation) was cut into 1 x 1mm pieces with sterile scissors and stored in pure FBS. After mice were anesthetized, the mouse hairs around the knee joints were shaved and disinfected with 75% ethanol. Fixing the experimental mouse on an operating table, cutting the skin of the knee joint of the mouse by a sterilized ophthalmic scissors longitudinally by about 1cm, and opening the joint capsule along the longitudinal incision on the inner side of the patellar ligament by using a pair of microscissors or a microslip. The minced tumor tissue mass is implanted into the joint capsule. The joint capsule was sutured with 8-0 surgical sutures and the skin incision was sutured with 6-0 surgical sutures. The post-operative mice were placed on a 37 ℃ hotbed until resuscitated, and penicillin was routinely administered intraperitoneally to prevent post-operative infection. The mice after operation are put into a cage for feeding without limiting the movement of the mice, and can be fed with food and water at will. The synovial sarcoma tumorigenesis condition is observed every week, the synovial sarcoma size and the visceral metastasis condition (whether the tumor is transferred to the viscera of the mouse) are observed by using a small animal living body imager every 3 weeks, and the tumor volume is increased to 1500mm3Dissecting knee joint at 45-60d, stripping tumor, weighing, inoculating part of tumor mass, and storing in liquid nitrogen. The process of preserving tissue mass or whole tumor body in liquid nitrogen includes programmed cooling, specifically at 4 deg.C to-20 deg.CSeveral temperature gradients of DEG C, -80 ℃ (special freezer refrigerator) and liquid nitrogen.
Step two, pretreatment of mice used in Px generation, namely pretreatment of immune normal BALB/c mice by cyclosporine: the skin on the opposite side of the injection site is lightly wiped by a forceps-clamped alcohol cotton ball, a prepared 1.57% cyclosporine solution is extracted by a 1ml syringe, the drug is administered at the dose of 7.0mg/kg (about 0.05ml is needed for each mouse on average), the needle point faces the head, the needle is inserted into the lower side of the abdomen of the mouse, the skin is punctured, the needle is inserted a little in parallel, the abdominal organs are prevented from being punctured, and the drug is slowly injected after no liquid is pumped back. Once daily, injections were administered for 7 days.
The first step and the second step can be completed in any sequence, but both steps need to be completed before the third step.
Step three, culturing Px generation, namely, passage of tumor tissues of synovial sarcoma xenograft mice to BALB/c mice pretreated by cyclosporine: inoculating the well-grown P0 generation xenograft to a tumor-bearing mouse in the step one until the tumor volume is increased to 1500mm3And (3) killing the mice at the time of 42d, completely stripping the tumors, inoculating the mice in the right armpit of the immune normal BALB/c mice inhibited by the cyclosporine (P1) as before, namely inoculating the mice in the mice pretreated by the step two, observing the growth condition of the tumors of the mice every day, measuring the length and the width of the tumors by using a vernier caliper as before, calculating the volume of the tumors, observing the visceral metastasis condition by using a living body imaging instrument of the mice, dissecting the animals after 42d, stripping the tumors, weighing part of the tumors, carrying out passage by analogy (P2, P3 and the like), and preserving the rest in liquid nitrogen.
Step four, Py generation culture- -passage of synovial sarcoma xenograft mouse tumor tissue to an immune normal BALB/c mouse: taking a well-grown xenograft BALB/c tumorigenic mouse pretreated by cyclosporine, namely a Px generation mouse inoculated in the step three, and when the tumor volume is increased to 1500mm3At the time of 42 days or 42 days, killing the mice, completely stripping the tumor, inoculating the mice to the right armpit of an immune normal BALB/c mouse as described above, observing the growth condition of the tumor of the mice every day, measuring the length and the width of the tumor body by using a vernier caliper as described above, calculating the volume of the tumor body, observing the visceral metastasis condition by using a small animal living body imager, dissecting the animals after 42 days, stripping the tumor body, weighing, and partially stripping the tumor bodyAnd (5) carrying out passage in the same way, and placing the rest into liquid nitrogen for preservation.
In the invention, the tumor tissue of the synovial sarcoma xenograft mouse is passaged to a BALB/c mouse pretreated by cyclosporine, and then can be passaged to an immune normal BALB/c mouse which is not pretreated by cyclosporine to continue forming tumor, so the invention successfully establishes an immune-sound synovial sarcoma xenograft mouse model.
Resuscitating and inoculating primary and passage tumor tissues of synovial sarcoma: the frozen P0, P1 and P2 in liquid nitrogen are taken to replace synovial sarcoma tumor tissues, quickly re-warmed to normal temperature, and inoculated to the oxter and joint cavity of the mouse respectively, and the method is the same as the method.
Because synovial sarcoma tissue of patients is a precious material for research, tumor tissue of tumor-bearing mice needs to be passaged to generate more tumor-bearing mice for experiments. The reason why the tumor tissue is frozen and thawed is that too large tumor masses cannot be used in each modeling experiment, and the tumor masses left for seed storage, namely frozen and thawed, can be used in the next experiment.
In this subject group, 3 synovial sarcoma patients were collected, and a PDX model was established by subcutaneous or in situ tumor formation, 3 cases of tumor formation. That is, after the four steps of cultivation, the invention successfully prepares a synovial sarcoma xenograft mouse model with healthy immunity. FIG. 1 is a photograph of tumors from BALB/c mice immunized in the Py generation, which no longer require pretreatment with cyclosporin, according to the above method. FIG. 2 is a graph comparing synovial sarcoma of immunocompromised BALB/c mice corresponding to FIG. 1 with pathological images of tumors of patients. As can be seen from FIG. 2, the tumors in BALB/c mice were consistent with the patient tumors in terms of cell morphology, protein expression, etc., as compared to the pathology of the patient tumors.
As shown in FIGS. 3-7, the staining results of the tumor tissue and the immune cells infiltrated into the para-carcinoma tissue, namely CD4+ T cells, CD8+ T cells, CD68+ macrophages, and PD-1 positive cells and PD-L1 positive cells thereof, are respectively shown in the 200X views. The staining results of tumor and paratumor tissues of patients and mice of each generation are shown in the figure. The graphs show that there was more corresponding infiltration of immune cells in both the third generation P3 '(cyclosporine, CsA) and fourth generation P4' (cyclosporine, CsA) groups, but still less infiltration compared to patient results. Whereas the fourth generation P4' (control, Ctrl) had a similar degree of immune cell infiltration as the patients. Wherein, the cyclosporin group refers to the Px generation corresponding to the cyclosporin-treated mice in the present invention, and the control group refers to the Py generation corresponding to the non-cyclosporin-treated immunocompromised mice in the present invention.
In the invention, tumor tissues of a patient are planted in an immunodeficiency mouse, synovial sarcoma tissues growing from the immunodeficiency mouse are planted in an immune normal mouse pretreated by cyclosporine, and synovial sarcoma tissues in the mouse pretreated by cyclosporine are planted in an immune normal mouse not pretreated by cyclosporine, so that the immune healthy synovial sarcoma xenograft mouse model is obtained. The above steps are all absent, and any absence of one of the steps can result in unsuccessful modeling.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions and substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (10)
1. A method for constructing an immunocompromised synovial sarcoma xenograft mouse model, comprising the steps of:
step one, culturing P0 generation: firstly, recovering a synovial sarcoma patient tumor tissue block stored in a liquid nitrogen environment, then transplanting the recovered patient tumor tissue block into an immunodeficient mouse in a heterogeneous manner under an aseptic condition, wherein the heterogeneous transplantation comprises selecting an immunodeficient mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with that of the patient, soaking the recovered patient tumor tissue block in a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the patient tumor tissue block into the mouse, suturing an incision, and breeding the mouse to enable the tumor of the mouse to grow; the xenotransplantation comprises subcutaneous transplantation or orthotopic transplantation; inoculating a synovial sarcoma tumor tissue block growing in a P0 generation immunodeficient mouse into another immunodeficient mouse to form a P1 generation tumor-bearing immunodeficient mouse; inoculating a synovial sarcoma tumor tissue block growing in a P1 generation tumor-bearing immunodeficiency mouse into another immunodeficiency mouse to form a P2 generation tumor-bearing immunodeficiency mouse; and so on;
step two, pretreating the immune normal mice with cyclosporine: injecting injection containing cyclosporine into a mouse with normal immunity or allowing the mouse with normal immunity to eat diet containing cyclosporine;
step one and step two are completed in any sequence, but both need to be completed before step three;
step three, culturing Px generation: x in the Px generation is a natural number which is more than or equal to 1, and a synovial sarcoma tumor tissue block growing in the tumor-bearing immunodeficiency mouse of any generation from the P0 generation to the Px-1 generation is inoculated to the mouse prepared in the step two and pretreated by cyclosporine to form a Px generation tumor-bearing mouse; inoculating a synovial sarcoma tumor tissue block growing in the Px generation tumor-bearing mouse into a mouse which is prepared in the other step II and is pretreated by cyclosporine to form a Px +1 generation tumor-bearing mouse; inoculating a synovial sarcoma tumor tissue block growing in the Px +1 generation tumor-bearing mouse body into another mouse prepared in the second step and pretreated by cyclosporine to form a Px +2 generation tumor-bearing mouse; and so on;
step four, Py generation culture: and y in the Py generation is a natural number which is more than or equal to x +1, a synovial sarcoma tumor tissue block growing in the tumor-bearing mouse of any generation from the Px generation to the Py-1 generation in the step three is inoculated into the immune normal mouse, and the immune normal mouse is bred until a synovial sarcoma tumor tissue grows in the immune normal mouse, so that the culture of the Py generation is completed.
2. The method of claim 1, further comprising the steps of programmed cooling and liquid nitrogen preservation of the collected synovial sarcoma tumor tissue mass prior to the step-flash resuscitation.
3. The method according to claim 2, wherein the synovial sarcoma patient tumor tissue block is preserved in DMEM containing DMSO, FBS and NEAA, and the temperature is reduced to liquid nitrogen temperature by more than three sections of procedures and preserved in the liquid nitrogen temperature, and the rapid heating recovery is to put the frozen tissue into a constant temperature water bath at 36-38 ℃ for recovery.
4. The method of claim 1, wherein the immunodeficient mouse is a NOD/SCID mouse and the immunocompromised mouse is a BALB/c mouse.
5. The method according to claim 1, wherein the weight of the mouse is 8-20 g in the subcutaneous transplantation or the orthotopic transplantation, the sterile incubation solution comprises antibiotics and DMEM medium or RPMI-1640 medium, and the antibiotics comprise penicillin and streptomycin.
6. The method of claim 1, wherein the orthotopic transplantation comprises implantation of a synovial sarcoma tissue mass in a mouse knee capsule.
7. The method of claim 1, further comprising observing the size of the synovial sarcoma in the mouse using a small animal in vivo imager.
8. The method of claim 1, further comprising programming the partial P0 generation tumor volume to be reduced in temperature and stored in liquid nitrogen, and comprising programming the partial P1 generation, P2 generation to Py generation tumor volume to be reduced in temperature and stored in liquid nitrogen.
9. The method of claim 1, wherein the step of pretreating the immunocompromised mouse with cyclosporin in step two comprises: the skin is lightly wiped by a forceps-clamped alcohol cotton ball, and a prepared cyclosporine solution with the concentration of 1-2 wt% is extracted by an injector and administered at the dose of 0.02-0.08 ml for each mouse; the injection is taken once a day and is continuously injected for 3-10 days.
10. The method of claim 1, wherein the synovial sarcoma patient tumor tissue mass is unilateral and less than or equal to 3mm in length.
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