动物造模,药效评价,诱发性淋巴瘤 z-bio 2024-09-03 477

　　（1） Chemical induced lymphoma animal model

　　The most commonly used inducer for modeling 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 through intraperitoneal injection, intravenous injection, oral or gastric administration, single or divided doses. The most commonly used method currently is intraperitoneal injection: using 8ml of DMSO and 92ml of 0.01mol/L PBS (pH=7.2) as solvents, 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). Continue with basic diet after injection and observe general changes in animal weight, diet, fur color, and activity level.

　　【 Model Features 】 ① The formation process of mouse thymic lymphoma induced by MNU is actually the malignant transformation of a single T lymphocyte TCR gene rearrangement under the action of MNU, and monoclonal proliferation, ultimately replacing normal cells in the thymus. It is a tumor derived from T cells. ② Under an optical microscope, the thymus structure was disrupted and replaced by diffuse distribution of 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.

　　The method of model evaluation and application is simple and easy to implement, with a relatively short tumorigenic period and high tumorigenic rate; Chemical carcinogens often induce tumors in multiple locations, so they are not commonly used in drug screening; From an etiological perspective, it is more similar to human tumors, so the model is often used for specific in-depth studies.

　　（2） Virus induced lymphoma animal model

　　【 Modeling Mechanism 】 Epstein Barr virus (EBV) is a double stranded DNA virus with a gene length of 172kb, which is a linear molecule in viral particles; It has B-lymphophilic characteristics and can infect B lymphocytes through EBV/c3d receptors (CD21). After entering B lymphocytes, its DNA undergoes circularization, becoming an additional component that can replicate outside the chromosomes of B lymphocytes, establishing latent infection in cells, stimulating cell proliferation and transformation, and inducing the occurrence of diseases.

　　[Method of Modeling]

　　1. For EBV separation, standard cell line B95-8, which can release EBV particles with transformation ability and transform marmoset B lymphocytes with EBV in vitro, is selected for large-scale culture. The cells are then subjected to starvation therapy and left for 7-10 days. The culture medium is collected and centrifuged at 4000r/min and 4 ℃ for 30 minutes. The supernatant is transferred to another sterile centrifuge tube and centrifuged at 12000r/min and 40C for 120-150 minutes. The supernatant is discarded and 1% fresh culture medium of the original culture medium is added. The cells are repeatedly pipetted and centrifuged at 3000r/min for 20 minutes. Filter the supernatant using a 0.22 μ m disposable filter to obtain a concentrated EBV suspension that is 100 times stronger. After packaging, store it at -80 ℃ for future 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 separation medium, and dilute the PBL to a volume of 8 × 10000000-10 × 10000000 using PRIM 1640 culture medium without calf serum. Under sterile conditions, inject 1ml/mouse of PBL suspension intraperitoneally into SCID mice. Three days after inoculation with PBL, 0.4ml of EBV suspension was intraperitoneally injected.

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

　　【 Model Characteristics 】 ① Solid lymphocyte tumor formation induced by virus, which is invasive and lethal, belongs to highly malignant non Hodgkin lymphoma. Histopathological observation shows that tumor cells are large lobed or non lobed cells, some with an immune like morphology, and some with obvious plasma cell differentiation characteristics The results of gene probe and monoclonal antibody detection showed that the induced tumor is a human EB cell-derived tumor, and contains the EB virus small nucleic acid molecule EBER-1. EB virus particles can be observed in the nucleus of the tumor cell.

　　Model evaluation and application: Normal cells are derived from peripheral venous blood of healthy adults, and specimen collection is convenient; Due to its simple genome structure, clear molecular background, ease of modification and manipulation, short experimental period (only about 2 months), and high tumor induction rate in surviving mice; The induced tumor presents as an invasive solid tumor that is easy to observe, and its histopathological morphology and growth characteristics conform to the biological characteristics of human malignant tumors; EBV is widely present in tumor cells, with a single carcinogenic factor, providing direct evidence for the causal relationship between EBV and the occurrence of normal human cell tumors.

　　（3） Animal model of lymphoma induced by ionizing radiation

　　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 irradiation and segmented irradiation are high-risk factors for the development of 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. The absorbed dose rate was 0.287Gy/min, and the absorbed dose was 1.75Gy, once a week for a total of four times to achieve modeling. The success time of the model is about 6 months.

　　【 Model Features 】 ① The original structure disappears and is replaced by tumor cells. Tumor cells are large in size, have low cell mass, are alkaline loving, have diverse nuclear abnormalities, abundant chromatin in the nucleus, obvious nucleoli, abundant free ribosomes in the cytoplasm, proliferation and expansion of smooth endoplasmic reticulum, and visible viral granules and budding phenomena. 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 induced tumor model is easy to operate, with constant target organs and carcinogens, high induction rate of tumor formation, and basically simulates the process of tumor occurrence. However, the latency period of tumor occurrence varies greatly among individuals, making it difficult to obtain animals with uniform disease course or tumor size for experimental treatment at the same time.