Experiments on the establishment of a nude mouse model of human pancreatic cancer
Experiments on the establishment of a nude mouse model of human pancreatic cancer
Human pancreatic cancer nude mouse models were established for (1) diagnosis of pancreatic cancer, (2) study of pancreatic cancer treatment, and (3) observation of whether pancreatic cancer cell lines changed morphologically before and after transplantation.
Operation method
Nude mouse model of human pancreatic cancer
Principle
Human pancreatic cancer cell line 8988 was inoculated subcutaneously in the axilla of nude mice, and the tumor size was measured weekly, and the mice were executed on the 42nd day. Tumor tissues and related organs were sent for pathological and electron microscopic examination, and related antigens were detected by radioimmunoassay. Subcutaneous tumor tissue cells and cell lines were cultured and HE stained.
Materials and Instruments
Nude Mouse BALB c nu-nu Pancreatic Adenocarcinoma Cell Line 8988 Move I. Preparation of experimental materials Caveat 1. Different experimental groups and control groups should be designed, and the number of animals in each group is usually 5-10. 2;2. Generally, after 6-8 weeks of tumor inoculation, when the tumor of mice can grow to 15-20mm in diameter (i.e. the mice will be on the verge of death), blood can be taken from the eyeball, serum can be separated and preserved, the mice can be executed, and tumor tissues can be photographed, and part of the tumor tissues can be used for frozen tissue section (or preserved at -80℃), and immunohistochemical staining accordingly, and some of the tumor tissues can be fixed with 3% neutral formaldehyde, and embedded in paraffin for conventional HE staining. Some tumor tissues were fixed with 3% neutral formaldehyde, embedded in paraffin, and stained with conventional HE staining. Common Problems I. Experimental discussion Pancreatic cancer is a tumor with high degree of malignancy, rapid development and extremely poor prognosis. Early symptoms are atypical, and by the time obvious symptoms appear, it has already entered the advanced stage and lost the chance of radical treatment. It is not sensitive to traditional radiotherapy and chemotherapy, and even for patients who have successfully undergone surgery, the 5-year survival rate is only close to 20% due to easy metastasis after surgery. For more product details, please visit Aladdin Scientific website.
RPMI-1640 complete medium trypsin glutaraldehyde
CO2 incubator Test tubes
Nude mice BALB/ c nu- nu, 4-6 weeks old, weighing 16-20 g, both male and female, were housed in an SPF-grade environment with a constant temperature of 25-27 ℃ and a constant humidity of 45%-50%, and the drinking water and food were sterilized.
Human pancreatic cancer cell line 8988 was cultured in vitro.
1. Put the human pancreatic cancer cell line into RPMI-1640 complete medium containing 10 % of RPMI-1640, put it in 37 ℃, 5 % CO2 incubator , change the liquid once every 2d, 2-3d, 0.25 % trypsin digestion, one passaging 3 passaging culture. 2.
2. When the cells filled up the bottom of the bottle, collect the single cell suspension, each 5x106 tumor cells, 0.2ml, injected into the axilla of nude mice subcutaneously, to establish the tumor model. 3.
3. Measure the tumor size with vernier caliper every week, calculate the tumor size according to the formula V= π/6 x length x width x height, and draw the growth curve.
III. Serologic Tests
At the 6th week, mice were executed by pulling out the eyeballs, and blood was collected in test tubes, and serum was taken at rest to detect tumor-associated antigens and amylase.
Giant test
1. After executing the nude mice, peel off the tumor subcutaneously, record the texture, infiltration and blood supply, observe whether there are abdominal adhesions and metastatic status of each organ.
2. Place the specimens of tumor tissue, lung, liver, spleen, mesentery, duodenum, stomach and brain in 10 % formalin fixative for use.
V. Pathological testingThe above specimens were routinely paraffin-embedded sectioned, stained with HE staining and observed by light microscope.
Ultrastructure observation
Tumor tissues were double-fixed with 2.5% glutaraldehyde and 1 % osmium acid, embedded, ultrathin sectioned, double-stained with uranium and lead, and observed by transmission electron microscope.VII. Microscopic examination
1. Tumor tissue cell culture and live cell observation: take fresh subcutaneous tumor tissue from nude mice, rinse it with PBS 3 times, cut it into 1 m3 size, digest it with 0.25% pancreatic enzyme, remove the coarse tissue and then make a single-cell suspension, place it in 37 ℃, 5% CO2 incubator for cultivation, and then observe its morphology and growth under the inverted microscope and compare it with cell line 8988. 2.
2. HE staining and morphological observation of cells: Tumor cells and cell lines were cultured in the same amount in six-well plates containing coverslips. After growth to sub-fusion, the medium was discarded, washed three times with PBS, fixed with 2.5% glutaraldehyde and washed three times with PBS, then stained with HE staining, and observed and compared with those of cell line 8988 under the light microscope.
In order to study the biological characteristics of pancreatic cancer and explore the effective measures for its diagnosis and treatment, it is necessary to establish animal models. There are three general sources of animal tumor models: spontaneous tumors, induced tumors and transplanted tumors. Due to the uncertainty and instability of spontaneous tumors, they are rarely used as tumor models. The success rate of induced tumors is low when the triggering factors of tumors are not very clear, and the induced tumors exist multiple histological types at the same time, which is not conducive to a more detailed study of the tumors. mice are congenitally deficient in the thymus, and the T cells can not be differentiated normally, and they do not have any rejection of tissues from allogeneic bodies, and they have been widely used in the transplantation of human tumors, but the nude mice are not immune blank animals, and the adult N K cells of them have higher activity. However, nude mice are not immune blank animals and their adult N K cells have higher activity, so subcutaneous implantation has a higher success rate than in situ transplantation.
In this experiment, subcutaneous implantation was used, the biological characteristics of tumor cells were kept intact, and the light and electron microscopic observation of the tumor tissues conformed to the original structural characteristics of cell line 8988, and there was no obvious difference in the morphology of the tumor tissues. The subcutaneous implantation was considered to be caused by the intact tumor envelope, local infiltration and no remote metastasis.
In general, the factors affecting the results of animal experiments include species, age, weight, gender, growth environment, etc. In this experiment, BALB/c nu- nu healthy nude mice were used, 4-6 weeks old, with a weight control of 16-20 g, and were kept in an SPF environment, and the nude mice consumed the drinking water and food freely after sterilizing them, which basically avoided the influence of these factors, and the nude mice were of both genders. The nude mice were both male and female, and there was no significant effect of sex on the results in the experiment.