动物造模,药效评价,诱发性淋巴瘤 z-bio 2024-07-08 398

　　（1） Chemically induced lymphoma animal model

　　The most commonly used inducer is N-methylnitrosourea (MNU). Its carcinogenic mechanism is related to the formation of O6 methylguanine, which leads to GyA point mutations during DNA replication.

　　【 Modeling Method 】 Multiple strains of mice can be induced to develop thymic lymphoma by intraperitoneal injection, intravenous injection, oral administration, or gastric lavage, with single or partial administration. The most commonly used method currently is intraperitoneal injection: using 8ml of DMSO 92ml of 0.01mol/L PBS (pH=7.2) as the solvent, MNU is prepared into an induction solution with a concentration of 10mg/ml, ready to use. Select 6-8 week SPF C57BL/6 mice, both male and female. Intraperitoneal injection of MNU induction solution (50mg/kg body weight) was administered twice (at weeks 0 and 8, respectively). After the injection begins, continue with the basic diet and observe general changes in the animal's weight, diet, fur color, and activity level.

　　【 Model Features 】 ① The formation process of mouse thymic lymphoma induced by MNU is actually a tumor of T cell origin, which undergoes malignant transformation and monoclonal proliferation under the action of MNU after a rearrangement of the TCR gene of a single T lymphocyte, ultimately replacing normal cells in the thymus. ② Under an optical microscope, the thymus structure is disrupted and replaced by diffusely distributed medium-sized lymphoid tumor cells Single or partial administration by intraperitoneal injection, intravenous injection, oral administration or intragastric administration can induce lymphoma in different strains of animals, but the incidence rate is different.

　　【 Model evaluation and application 】 The method is simple and easy to implement, with a relatively short tumor formation cycle and a high tumor formation rate; Chemical carcinogens often induce multiple site tumors, so they are not commonly used in drug screening; From a etiological perspective, it is more similar to human tumors, so this model is often used for specific in-depth research.

　　（2） Animal model of virus induced lymphoma

　　【 Modeling Mechanism 】 Epstein Barr virus (EBV) is a double stranded DNA virus with a gene length of 172kb, appearing as a linear molecule in virus particles; It has the characteristic of B lymphocytes and can infect B lymphocytes through the EBV/c3d receptor (CD21). After entering B lymphocytes, its DNA undergoes cyclization, becoming an additional component of B lymphocytes that can be replicated outside the chromosome. It establishes latent infection in cells, stimulates cell proliferation and transformation, and induces the occurrence of diseases.

　　【 Modeling Method 】

　　1. EBV isolation: Standard cell line B95-8, which can release EBV particles with transformative ability and transform marmoset B lymphocytes with EBV in vitro, was cultured on a large scale. The cells were subjected to starvation therapy for 7-10 days, and the culture medium was collected. The supernatant was centrifuged at 4000 r/min and 4 ℃ for 30 minutes. The supernatant was transferred to another sterile centrifuge tube, and centrifuged at 12 000 r/min and 40 ℃ for 120-150 minutes. The supernatant was discarded and 1% fresh culture medium of the original culture medium was added. The cells were repeatedly pipetted and centrifuged at 3000 r/min for 20 minutes. Filter the supernatant with a disposable 0.22 μ m filter to obtain a concentrated EBV suspension that is 100 times concentrated. After packaging, store it at -80 ℃ for later use.

　　2. Lymphocyte isolation and inoculation with fresh blood from healthy adults, using EBVIgA rapid detection kit to detect VCA IgA antibody titers. Separate lymphocytes (PBL) using lymphocyte isolation medium, and dilute PBL to 8 × 10000000~10 × 10000000 using PRIM 1640 culture medium without calf serum. Under sterile conditions, inject 1ml/mouse of PBL suspension into the abdominal cavity of SCID mice. After 3 days of vaccination with PBL, 0.4ml of EBV suspension was intraperitoneally injected.

　　3. During the replication process of the model, the host may die due to graft-versus-host reactions, leading to a lack of stability in the replicated model. Cyclosporin A can be used to inhibit the above reactions. When used, 0.9% NaCl injection is diluted at 1mg/ml, and PBL is injected. Each SCID mouse is injected intraperitoneally at a dose of 10mg/(kg · d) for 2 consecutive days, with 15mg/(kg · d) starting from the 3rd day and injected every other day, a total of 11 times.

　　【 Model features 】 ① Viral induced solid lymphocytic tumor formation, which is invasive and lethal, belongs to non Hodgkin's lymphoma with high malignancy. Observation of histopathology reveals that tumor cells are large fissures and non fissured cells, some of which exhibit immunoblastic like morphology, while others exhibit distinct plasma cell differentiation characteristics The results of gene probe and monoclonal antibody detection indicate that the induced tumor is a human EB cell-derived tumor, and contains EBER-1, a small nucleic acid molecule of EB virus. EB virus particles can be observed in the nucleus of the tumor.

　　【 Model evaluation and application 】 Normal cells are derived from peripheral venous blood of healthy adults, and specimen collection is convenient; Due to the simple structure of the virus genome, clear molecular background, easy modification and operation, short experimental period (only about 2 months), and high tumor induction rate in surviving mice; Inducing tumors exhibit invasive solid tumors that are easy to observe, and their histopathological morphology and growth characteristics conform to the biological characteristics of human malignant tumors; EBV is widely present in tumor cells and has a single carcinogenic factor, which can provide direct evidence of the causal relationship between EBV and the occurrence of normal human cell tumors.

　　（3） Animal model of lymphoma induced by ionizing radiation

　　[Modeling mechanism] Ionizing radiation can directly cause DNA damage. Promote the occurrence and development of various tumors in humans and animals. Numerous studies have shown that the thymus, as an important component of the immune system, is a target organ for radiation induced carcinogenesis. Radiation induced thymic lymphoma in mice has also become one of the classic animal models for studying radiation induced carcinogenesis. nothing

　　Both acute and fractional irradiation are high-risk factors for developing thymic lymphoma.

　　【 Modeling Method 】 Inbred BALB/c mice, female, weighing 18-22g, were subjected to whole-body irradiation using a deep X-ray therapy machine with an absorption dose rate of 0.287Gy/min and an absorption dose of 1.75Gy, once a week. A total of four doses were used to establish the model. The success time of the model is about 6 months.

　　【 Model Features 】 ① The original structure has disappeared and been replaced by tumor cells. The tumor cells are large in size, with low cell mass, basophilic, and diverse cell nuclei. There is often more chromatin in the nucleus, with obvious nucleoli. The cytoplasm is rich in free ribosomes, and the smooth endoplasmic reticulum proliferates and expands. Viral particles and budding phenomena can be seen. The low degree of cell differentiation, fast growth, and vigorous proliferation are consistent with the characteristics of tumor cells. ② Tumor cells exhibit infiltration and metastasis behavior, and are transplantable in homologous mice.

　　【 Model Evaluation and Application 】 The inducible tumor model is easy to operate, with constant target organs and carcinogens, and a high induction rate of tumor formation. It basically simulates the process of tumor occurrence. However, the latent period of tumor occurrence varies greatly among individuals, making it difficult to obtain animals with a uniform disease course or tumor size simultaneously for experimental treatment.