Tumor animal model establishment experiment

(1) Evaluation of the efficacy of anti-tumor immunotherapy;

(2) as a screening model for anti-tumor drugs;

(3) Provide a better research platform for tumor metastasis research;

(4) Provide a good experimental tool for the development of anti-tumor metastatic drugs.

Second, induced tumor animal model

Principles of experimental methods: Induced tumor animal models refer to tumors induced by researchers using chemical carcinogens, radiation, and oncogenic viruses.

Experimental materials: tumor cells, mouse sex

Reagents, kits: serum-free medium, 3% neutral methanol, paraffin

Instruments, consumables : cryogenic centrifuge, blood cell counter, vernier caliper

Experimental steps:

1, liver cancer

(1) Diethylnitrosamine (DEN) induces liver cancer in rats

1 Take a closed group of rats with a body weight of about 250 g, male or female;
2 Cage by sex. In addition to ordinary food, the carcinogen, that is, with 0.25% DEN aqueous solution, the dose is 10 mg / kg, once a week, the remaining 5 days with 0.025% DEN aqueous solution into the water bottle, let it freely drink;
3 A total of about 4 months can induce liver cancer;
4 It is also possible to induce liver cancer by inhaling the mouth with 0.005% of the drinking water for 8 months.

(2) 4-2 methylaminoazabenzene (DBA) induced liver cancer in rats

1 Feed the rats with feed containing 0.06% DBA, and the vitamin B2 in the feed should not exceed 1.5~2 mg/kg;
From April to June, a large number of liver cancers were successfully induced.

(3) 2-acetylamino acid (2AAF) induces liver cancer in mice, dogs, cats, chickens and rabbits

1 Adult rats are given 0.03% 2AAF standard feed;
2 An average of 2 to 3 mg 2AAF per day (2AAF can also be mixed in oil), and 80 to 90% of animals develop liver tumors after 3 to 4 months.

(4) Diethylnitrosamine-induced liver cancer in rats

1 The dosage is 0.3~14 mg/kg body weight per day, mixed with feed or drinking water;
Liver cancer occurred in 255/300 rats after 2 to 6 months.

(5) Iminoazotoluene (OAAT) induced mouse liver cancer

1 Apply 1% OAAF benzene solution (about 0.1 ml containing 1 mg) to the skin between the shoulders of the animal, once every other day, 2 to 3 drops each time, usually 100 times.
2 The first liver tumor appeared 7 to 8 weeks after the experiment, and the liver tumor of the mouse was induced by about 55% for more than 7 months.
3 or 2.5 mg OAAT dissolved in sunflower oil, subcutaneous injection of C3H mice 4 times, 10 days apart, can also induce liver cancer.

(6) Aflatoxin-induced liver cancer in rats

The daily feed contains 0.001 to 0.015 ppm, and after 6 months of feeding in the feed, the rate of liver cancer induction is 80%.

2, stomach cancer

(1) Methylcholine induced mouse gastric cancer

1 Take about 20 g of mice, under the sterile surgery, the methyl cholestyramine (MC) line knot is worn on the glandular mucosa.
2 The wire knot containing MC is made of ordinary thin wire. After knotting at one end, the wire knot is placed in a small glass test tube containing MC, and the temperature is slightly heated on the alcohol lamp to make the MC liquefy and penetrate into the knot. The concentration of MC is 0.05 to 0.1 g. 20-methyl choline is immersed in 10 to 20 lines.
3 Successful gastric cancer can be induced 4 to 8 months after embedding.

(2) Asymmetric nitrosamine induced gastric cancer in mice

The dose was 0.25 ml/kg body weight. All animals developed pre-gastric papillary carcinoma after 3 months, and 85-100% of the pre-clinical gastric cancer occurred after 7-8 months. Kunming is the most sensitive. In line A, the 615 line mice were the least sensitive.

(3) Methylnitrosoacetate urea induced gastric cancer in mice

Urea nitrosylacetate urea was added to the drinking water of BD rats at a dose of 2 mg/kg body weight for 5 times a week. After 520 days, all the rats developed adenocarcinoma.

3, esophageal cancer

(1) Methylbenzylnitrosamine (MBNA) induces esophageal cancer in rats

1 Take Wistar rats weighing more than 100 g;
2 to consume drinking water containing methylbenzyl nitrosamine, and incorporate MBNA into the feed to achieve a daily intake of 0.75 ~ 1.5 mg / kg body weight;
3 80 to 100 days can induce esophageal cancer.

(2) Dihydrosafrole induces esophageal cancer in rats

Dihydroxanthin is a condiment for the preparation of beer. Adding 2,500 to 10,000 (2,500 to 10,000 ppm) of baicalein to the rat diet can cause 20 to 75% of esophageal cancer.

(3) Methylbenzylnitrosamine induces esophageal cancer in rats

1 Oral administration of 0.2% or 0.005% aqueous solution of methylbenzylnitrosamine to the animals once a day, the rat perfusion dose is 1 mg / kg body weight;
One case of esophageal papilloma was found on the 27th day, and the first case of esophageal cancer was found in 154 days. The incidence of esophageal cancer was 53% in 11 months.

4, lung cancer

(1) Diethylnitrosamine (DEN) induced mouse lung cancer

1 mice were injected subcutaneously with 1% DEN aqueous solution once a week, each dose of 56 mg / kg, the total dose of DEN reached 868 mg;
2 When the observation time is about 100 days, the cancer rate can reach 40%.
3 The total dose of DEN reached 1176 mg, and the observation time was about half a year; the cancer rate was 94%.

(2) Uratan-induced lung adenocarcinoma

1 Mice (A line, 1～11/2 months old) are more sensitive than rats, each time 10% urethane physiological saline solution is injected into each abdominal cavity 0.1-0.3 ml, and re-injected at intervals of 3 to 5 days. For 3 months, the amount of each mouse is about 100 mg;
2 The incidence of lung adenocarcinoma was 100% 3 months after the injection, and most of them were multiple, and the induced tumor was benign.

(3) Benzopyrene-induced lung adenocarcinoma

1 monkey intratracheal injection of 3,4-benzopyrene (benzoxan 3 ~ 15 mg and an equal amount of Fe ₂ O ₃ mixture), once a week, a total of 10 times;
Two of the 2 6 monkeys induced squamous cell carcinoma of the lung.

(4) Ammonium sulfate aerosol induced lung adenocarcinoma

1 Inhaled 100 rats with an amine sulfate solvent;
2 lung adenocarcinoma occurred in all rats after 13 months.

(5) Methylcholine-induced lung adenocarcinoma

1 Mix the methyl choline with 0.2% gelatin as a suspending agent and inject it into the trachea of ​​the golden hamster once a week, 0.1 ml (containing methyl choline 5 mg) once a week for 6 times;
2 53 weeks later, 62.5% of the animals developed lung cancer.

5, nasopharyngeal carcinoma

(1) Dimethylcholanthrene (MC)-induced nasopharyngeal carcinoma in rats

1 Take a rigid plastic rice tube with a diameter of 2 to 3 mm and draw a cone on a small lamp on an alcohol lamp. Each section is about 3.5 cm long and the tube is filled with crystal MC.
2 One end of the small tube is closed with fire to prevent the drug from overflowing. The tip uses a needle to punch a few holes, so that the MC can overflow from the small pot.
3 Take the rats with a body weight of about 120 g, male or female. After anesthesia with ether, insert the MC-containing plastic tubule into the nasal cavity from the anterior nares, and use the small end of the small nostril of the anterior nares to make a small force. All enter the nasal cavity, and its tip can reach the nasopharyngeal cavity. The tubules can be left in the nasal cavity for a long time without additional fixation.
4 After the scheduled time (more than half a year), or when the animal dies by itself, it is fixed to the nasopharynx, 10% formalin, decalcified, embedded in paraffin, and serially sliced. The cancer rate can reach more than 60%.

(2) Diethylnitrosamine nasal inducing nasopharyngeal carcinoma

1 Take about 120 g of white rats, male or female. After anesthesia with ether, use a No. 8 needle that smoothes the tip of the needle, gently insert it from the front nostril, and the tip of the needle can reach the nasopharyngeal cavity;
2 Injected with syringe 1% Tween-80 33.3% DEN suspension 0.0 2 ml (containing DEN 6.7 mg) once a week for 15-20 times, can induce nasopharyngeal carcinoma.

6, cervical cancer

Dimethylcholanthrene (MC) induced cervical

Female mice were taken and the cotton yarn with 0.1 mg MC was attached to the cervix with the aid of a vaginal dilator and a purely curved needle in a state where the animal was not anesthetized. Pass through the back of the right uterine horn to secure the knot to the cervix. The other end of the line was fixed to the back muscles, the skin was sutured, and after the thread was hung, the penicillin was continuously injected for 2 to 3 days on the same day. In case of postoperative infection. The animals were sacrificed at a certain time (about half a year), and the cervical tissue was fixed with 10% formalin, embedded in paraffin, and serially sectioned.

7, colon cancer

Dimethlhydrazine (DMH) induced nasopharyngeal carcinoma 1 to male rats of four weeks old, subcutaneous injection of Dimethlhydrazine (DMH) once a week for 21 weeks, each DMH 21 mg / kg;
2 1 to 4 weeks after the last administration, the animals were sacrificed;
3 The descending colon was fixed with Bouin solution, dehydrated, embedded in paraffin, and sectioned. The DMH used was first formulated to contain 400 mg of mother liquor per 100 ml, and EDTA 37 mg was added, and the pH was adjusted to 6.5 with sodium hydroxide (0.1 N).

Precautions:

1. To design different experimental groups and control groups, the number of animals in each group is generally 5-10;
2, after 6-8 weeks of general tumor inoculation, the tumor of the mouse can grow to a diameter of 15-20mm (that is, the mouse will be dying), blood can be taken from the eyeball, serum is separated and serum is stored, the mice are sacrificed, and the tumor is taken. Tissue photography, part of the tumor tissue was taken for frozen tissue section (or - 80 ° C preservation), the corresponding immunohistochemical staining, part of the tumor tissue was fixed with 3% neutral formaldehyde, stone wax embedded, for routine HE staining.

other:

1, the characteristics of aromatic amines and azo dyes carcinogens (1) usually require long-term, large doses to cause cancer;
(2) Tumors often occur in organs at distant sites such as the bladder and liver;
(3) There are obvious species differences;
(4) It is not a direct carcinogen itself, and carcinogenesis is due to its role as a metabolite;
(5) Its carcinogenic effect is often affected by nutrition or hormones. For example, milk yellow only causes liver cancer when fed to rats with protein and riboflavin-free feed, and male rats are more sensitive, ortho-amino azo Toluene is easy to cause liver cancer in female rats.

2, carcinogenic characteristics of nitrosamines (1) carcinogenic, small doses can cause cancer;
(2) Many organs (including animals that are not susceptible to cancer, such as monkeys, guinea pigs, etc.) can cause cancer, and can even cause cancer through the placenta, such as pregnant rats. Diethyl nitrosamine (DEN) can cause gliomas in mice in a relatively quick manner;
(3) Nitrosamines with different structures have obvious organ affinity. Symmetrical derivatives such as dimethyl nitrosamine often cause liver cancer, and asymmetric nitrosamines such as methylbenzyl nitrosamine are often induced. Esophageal cancer; in rats, dibutyl nitrosamine can cause bladder cancer, diamyl nitrosamine can induce lung cancer, and N-methyl-N-nitro-N-1-nitrosoguanidine can cause Gastrointestinal cancer.

3, carcinogenic characteristics of aflatoxin (1) aflatoxin is very toxic, a small dose (1mg / kg body weight) can cause dogs, young rats, turkeys or ducklings to die;
(2) Its carcinogenicity is also extremely strong, and the minimum carcinogenic dose of bifidoamine is 10 times smaller, which is the most powerful chemical carcinogen;
(3) It can induce liver cancer of various animals (from fish to monkey), and can also cause adenocarcinoma of kidney, stomach and colon. Dropping into the trachea can cause lung squamous cell carcinoma;
(4) Injecting into the skin can cause local sarcoma, and it is reported that it can cause tumors in other parts of the lacrimal gland, breast, ovary and the like.

4, other in animal experiments have long been determined: ethionine can induce liver cancer in rats; urethane (urethane, C2H5O-CONH2), aminoethylamine can cause lung cancer in mice; some halogenated hydrocarbons such as tetrachloro Carbon, chloroform, etc. can cause liver cancer in rats or mice.

Third, self-hair tumor animal model

Principles of experimental methods: The spontaneous tumor animal model refers to the non-human treatment; the naturally occurring tumor animal model, mainly the spontaneous breast cancer model and the spontaneous leukemia model.

Experimental materials: C3H mice, mice, A-line mice, CGA mice

Reagents, kits: test drugs

Instruments, consumables: marker, foot gauge

Experimental steps:

1. Spontaneous breast cancer mouse model type

1 C3H mice: the incidence of spontaneous breast cancer in breeding females is 85% to 100%

2 A-line mice: The incidence of breast tumors in females is 30%~80%

3 CBA mice: the incidence of spontaneous breast cancer in females is 60% to 65%
2. Tumors can occur on both the left and right sides of the breast and the nipple . More than one tumor is common in each mouse. Animals with similar tumor numbers and sizes were selected, paired into groups, and the test drugs were administered to observe the effects of the test drugs on tumor development.

3. The dosage regimen and route of administration can be determined based on the characteristics of the test drug . Because of the slow development of spontaneous breast cancer, it is appropriate to use intermittent administration or continuous administration in small doses.
4, the result is judged

1 observe the tumor growth daily after administration, record the time and location of occurrence;

2 The tumor nodules grew to a certain extent, and the subcutaneous tumor was measured twice a week with a foot gauge. The maximum diameter (a) and minimum diameter (b) of the mass were measured. Tumor volume (cm ³ ) = ab ² /2;
3 The growth curve of the tumor volume change versus time can be judged by the slope change of the curve to determine the effect of the drug on inhibiting tumor growth, especially whether the tumor can completely disappear.

5, AKR leukemia model

AKR mice are high-risk strains of leukemia. The incidence of lymphocytic leukemia is 76%-90% for males and 68%-90% for females.

6. The experiment used AKR mice of 6-12 months old. At this time, the spleen and lymph nodes of the rats were swollen and the blood was abnormal.

7. The next day after the diagnosis of leukemia by blood test, pairing the group and starting to give the test drug, observe the result.

8, the result is judged

Observation indicators: peripheral blood, white blood cell count, lymph node and spleen size, animal survival time. The effective survival period was longer than that of the control group, and the induction of remission and maintenance of the drug was evaluated according to the blood image.

Precautions:

1 In the same strain, the incidence rate is relatively stable, and the differences between the different strains are very large.

2 Animal breast cancer is associated with genetics, breast cancer factors (ie, breast cancer virus) and hormones.

3 Changes in feed have a significant impact, using fixed-price nutrient feeds.

4 Pairing the grouping.
5 The course of each mouse leukemia is different. After the diagnosis of spontaneous tumors, the mice should be paired in the experiment.

Xiaobian Message

About Tumor Animal Experiment, Nanjing Ouji has a complete set of technical service management system

Drug screening and pharmacological evaluation services

I. Anti-tumor drug screening services and pharmacological evaluation

1. In vitro screening services for anti-tumor drugs ( more than 200 cell lines are available)

2. Anti-tumor drugs in nude mice screening services

2.1. Human tumor xenograft model in nude mice

2.2. Human tumor in situ transplantation model in nude mice

2.3. Mouse tumor model

3. Animal transfer model

4. Anti-angiogenic drugs in vitro screening services

5. Anti-angiogenic drug in vivo activity screening service

6. Chemotherapy drug adverse drug treatment screening service

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