动物造模,药效评价,肿瘤模型 z-bio 2024-07-30 417

　　There are many methods to replicate animal tumors, such as irradiating experimental animals with radiation or injecting radioactive isotopes intravenously or locally; Using various chemical carcinogens (alkylating agents, polycyclic aromatic hydrocarbons, aromatic amines, amino azo dyes, nitrosamines); Use plant toxins (such as cyclamate, safrole, etc.); Using metals such as chromium, nickel, arsenic, cadmium, etc; Using RNA and DNA tumor viruses; The use of various carcinogenic mycotoxins (among which aflatoxin has the strongest carcinogenic effect) can induce various tumors.

　　The number of induced tumor models ranks first among induced animal models. Generally, carcinogens are used to induce tumors in animals through oral administration, injection, burial, and application.

　　There are also many reports on viruses that can induce animal tumors, such as mouse leukemia virus (MLV), chicken leukemia virus (ALV), and cat leukemia virus (FLV), which can cause leukemia in mice, chickens, and cats, respectively. Rous chicken tumor virus can cause tumors in various animals such as field mice, chickens, ducks, quails, monkeys, and snakes. Feline sarcoma botulinum toxin (FSV) can cause sarcoma in rats, cats, dogs, and monkeys. Human adenovirus can induce sarcoma and lymphoma in mice and field mice.

　　（1） Induced tumor animal model

　　1. Hepatocellular carcinoma induced by diethylnitrosamine (DEN) in rats: A closed group of rats weighing around 250g, male or female, was selected. Feed in cages according to gender. In addition to providing regular food, feed with carcinogenic substances by gavage with 0.25% DEN aqueous solution at a dose of 10mg/kg once a week. For the remaining 5 days, place 0.025% DEN aqueous solution in a water bottle and let it drink freely. It takes about 4 months to induce liver cancer. Or simply mix 0.005% into drinking water and take orally for 8 months to induce liver cancer. 4-2 methylaminoazobenzene (DBA) induces liver cancer in rats: Rats are fed with a diet containing 0.06% DBA, and the vitamin B2 in the diet should not exceed 1.5-2mg/kg. A large amount of liver cancer is successfully induced from April to June. 2-Acetylamino acid (2AAF) induces liver cancer in mice, dogs, cats, chickens, and rabbits: Adult rats are fed a standard diet containing 0.03% 2AAF. On average, 2-3mg of 2AAF per day (or 2AAF mixed with oil and fed orally) can lead to 80-90% of animals developing liver tumors after 3-4 months. Diethyl nitrosamine induced liver cancer in rats: administered at a dose of 0.3-14mg/kg body weight per day, mixed with feed or water. After 6-9 months, 255/300 rats developed liver cancer. Induction of mouse liver cancer by imino azotoluene (OAAT): Apply 1% OAAF benzene solution (about 0.1ml containing 1mg) to the skin between the two shoulder blades of the animal, once every other day, 2-3 drops each time, usually 100 times. The first liver tumor appears 7-8 weeks after the experiment, and about 55% of mouse liver tumors can be induced after 7 months or more. Alternatively, 2.5mg of OAAT can be dissolved in sunflower seed oil and subcutaneously injected into C3H mice four times, with a 10 day interval between each day, to induce liver cancer. Aflatoxin induces liver cancer in rats: The daily feed contains 0.001-0.015ppm, and after being mixed with the feed for 6 months, the liver cancer induction rate reaches 80%.

　　2. Gastric cancer induced by methylcholanthracene in mice: About 20g of mice were taken, and sterile surgery was performed to puncture the glandular gastric mucosa surface

　　Hanging methylcholanthracene (MC) wire junction. The knot containing MC is made of ordinary thin wire, tied at one end, and placed in a small glass test tube containing MC. It is slightly heated on an alcohol lamp to allow MC to liquefy and infiltrate into the knot. Immerse 10-20 wires in 20-methylcholanthracene at an MC concentration of 0.05-0.1g. Successful gastric cancer can be induced 4-8 months after surgical thread embedding. Using asymmetric nitrosamines at a dose of 0.25ml/kg body weight, all animals developed anterior gastric papillary carcinoma after 3 months, and 85-100% developed anterior gastric cancer after 7-8 months. Kunming is the most sensitive. The A series is second, while the 615 series mice have the poorest sensitivity. In addition, methyl nitrosoacetate urea can be added to the drinking water of BD rats at a dose of 2mg/kg body weight, 5 times a week. After 520 days, all rats developed adenocarcinoma.

　　3. Induction of esophageal cancer in rats by methylbenzylnitrosamine (MBNA): Wistar rats weighing over 100g were given drinking water containing MBNA, and MBNA was added to the feed to achieve a daily intake of 0.75-1.5mg/kg body weight. 80-100 days can induce esophageal cancer. Dihydrosafrole can also be used, which is a seasoning for making beer. Adding 2500 to 10000 parts per million (2500-10000 ppm) of safrole to rat feed can cause 20-75% of esophageal cancer. Animals were orally fed with 0.2% or 0.005% methylbenzylnitrosamine aqueous solution once a day at a dose of 1mg/kg body weight. On the 27th day, one case of esophageal papilloma was detected, and on the 154th day, the first case of esophageal cancer was discovered. The incidence of esophageal cancer at 11 months was 53%.

　　4. Induction of lung cancer in mice by diethylnitrosamine (DEN): Mice were subcutaneously injected with 1% DEN aqueous solution once a week, at a dose of 56mg/kg each time. When the total dose of DEN reached 868mg and the observation time was about 100 days, the cancer incidence rate could reach 40%. When the total dose of DEN reaches 1176mg and the observation time is about six months; The cancer incidence rate can reach 94%. Uratan induced lung adenocarcinoma: Mice (A series, 1-11/2 months old) are more sensitive to it. Each mouse is injected with 0.1-0.3ml of 10% Uratan physiological saline solution intraperitoneally, with an interval of 3-5 days between injections, for a total of 2-3 months. The dosage for each mouse is about 100mg. After 3 months of injection, the incidence of lung adenocarcinoma is 100%, and most of them are multiple. This type of induced tumor is benign. In addition, lung cancer can be induced by intratracheal injection of benzo [a] pyrene, ammonium sulfate aerosols, methylcholanthracene, and other substances. Injecting 3,4 benzo [a] pyrene (a mixture of 3-15mg benzo [a] pyrene and an equal amount of Fe2O3) into the trachea of monkeys once a week for a total of 10 times, 2 out of 6 monkeys induced squamous cell carcinoma of the lungs. Someone also administered sulfuric acid amine gas solvent to 100 rats for inhalation, and after 13 months, all rats developed lung adenocarcinoma. Mix methylcholanthracene with 0.2% gelatin as a suspension and inject 0.1ml (containing 5mg methylcholanthracene) into the trachea of golden rats once a week for a total of 6 times. After 53 weeks, 62.5% of the animals developed lung cancer.

　　6. Female mice with cervical cancer were taken, and cotton yarn with 0.1mg MC attached was tied to the animal without anesthesia. With the help of a vaginal dilator and a ground curved needle, the thread was inserted into the cervix. Thread through the back of the right uterine horn to fix the knot at the cervical opening. The other end of the thread is fixed to the back muscle, sutured to the skin, and after hanging the thread, penicillin is injected continuously for 2-3 days starting from the same day. To prevent postoperative infection. After a certain period of time (about six months), the animals were euthanized, and the cervical tissue was fixed in 10% formalin, embedded in paraffin, and sliced continuously.

　　7. Four week old male rats with colon cancer were subcutaneously injected with dimethylbenzylhydrazine (DMH) once a week for 21 consecutive weeks, at a dose of 1mg/kg each time. Animals were euthanized 1-4 weeks after the last administration. The descending colon was fixed with Bouin solution, dehydrated, embedded in paraffin, and sliced. The DMH used is first prepared into a mother liquor containing 400mg per 100ml, and 37mg of EDTA is added. The pH is adjusted to 6.5 using sodium hydroxide (0.1N) solution for later use.

　　（2） Transplanted tumor animal model

　　Most of the anti-tumor drugs currently used in clinical practice are discovered through screening in animal transplantation tumor trials. The advantages of using animal transplantation tumor screening drugs are: allowing a group of animals to be simultaneously inoculated with the same amount of tumor cells, with relatively consistent growth rates, small individual differences, a survival rate of nearly 100%, similar effects on the host, easy to objectively judge efficacy, continuous transplantation in the same or the same strain of animals, long-term retention for experimental use, generally short experimental periods, and easy control of experimental conditions. Therefore, currently most anti-tumor drug screening uses animal transplant tumors as screening models. At present, there are about 400 animal transplant tumors preserved in the world, but only 20-30 are commonly used in screening experiments. According to statistics from 1984, China has established 64 common tumor strains of various animals and humans within the same species of animals. For example, mouse lung adenoma (HP615), mouse cervical tumor 27 (U27), mouse brain tumor 22 (B22), mouse lymphocytic leukemia (L615), nude mouse human liver tumor transplant tumor, and human brain malignant glioma (NCS-1).

　　Animal tumors can be cultured into the desired tumor cell lines through transplantation and passage. Tumor cell line is a tumor cell model with stable histological type and growth characteristics, and can be continuously passaged in homologous or homologous animals. Tumor transplantation in healthy animals is equivalent to live tissue culture, which can preserve tumor species for long-term use in experiments.

　　Two methods of transplantation are commonly used in experiments: ascites tumor and solid tumor. For tumors that can produce ascites, a certain number of cells can be injected into the recipient animal's abdominal cavity to form ascites tumors or produce ascites. Solid tumor transplantation is also performed under sterile conditions, where the solid tumor is cut into small pieces of 2-3mm and implanted subcutaneously in the recipient animal.

　　Autologous homologous animal tumor transplantation does not produce drainage phenomenon. When transplanting animals of the same species, methods such as injecting adrenal cortex hormones, anti-tumor drugs, and appropriate amounts of radiation can be combined to reduce host immune rejection. Xenogeneic tumor transplantation began with Leidy (1834) and was quite difficult. In the past 50 years, the following methods have been commonly used for allogeneic transplantation: ① Inoculation under the skin or mucosa, which has the advantage of easy observation, but has a large rejection effect and poor efficacy. ② Animal tumors were transplanted into the chicken embryo membrane. The characteristic is that it is relatively easy to survive, but there have been no successful reports of human tumors Human tumors were inoculated into the anterior chamber of rats, guinea pigs, and rabbits. The disadvantage is that cells cannot be passaged Transplanted into animal brains. Tumors grow rapidly, but they are difficult to observe. In 1983, Bodgen et al. screened for new drugs by transplanting human tumors under the renal capsule of thymus free rats. The entire experiment only took 11 days and had a high success rate. This work brought good news to drug screening for clinical patients.

　　Since the 1960s, hundreds of transplantable human tumors have been established abroad, which can prevent the formation and functional degradation associated with passage. In 1969, Rygaaid successfully transplanted human tumors into thymus free nude mice for the first time, opening up a new era for xenograft tumor transplantation. Due to the lack of T lymphocyte function, nude mice are an ideal tumor transplantation model material.