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Animal models for in vivo screening biological macromolecules having influences on tumor cell biology

A biological macromolecule and cell biology technology, applied in the field of animal model preparation, can solve the problems that are not easy to master, and sponges are easy to cause inflammatory reactions, etc.

Active Publication Date: 2017-11-24
SUINING CENT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used in vivo experiments for tumor angiogenesis include chicken chorioallantoic membrane experiment, corneal microcyst experiment, sponge implantation experiment, disk angiogenesis system, matrigel plug experiment, sponge-matrigel experiment, chicken chorioallantoic membrane experiment Chicken embryos are used as the experimental body. Chickens belong to the class of Aves, which is different from mammals. The corneal microcyst experiment requires surgery under a microscope. Use a blade to make a 1.5-2mm long incision on the cornea near the pupil without cutting through the cornea. Use a self-made iris shovel to advance from the incision to the corneoscleral limbus along the arc of the eyeball to form a microcapsule with a size of 1.5mm×0.8mm, which requires very delicate operation and is not easy to master. Implantation of the sponge under the skin may easily cause inflammation, etc.

Method used

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  • Animal models for in vivo screening biological macromolecules having influences on tumor cell biology
  • Animal models for in vivo screening biological macromolecules having influences on tumor cell biology
  • Animal models for in vivo screening biological macromolecules having influences on tumor cell biology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Primary culture, isolation, purification and identification of salivary gland MEC cells

[0050] Cultivation, separation and purification: The direct culture method of tissue blocks is used for primary culture of sterile tissue samples. Under aseptic conditions, the tumor tissues treated with double antibodies were taken out, and gently rinsed with sterile RPMI-1640 complete medium (containing 10% fetal bovine serum) for 3 times; the tumor tissues of each group were cut into pieces with sterile ophthalmic scissors to about 2mm 3 Disperse the chopped tumor tissue into the culture bottle, slowly turn the culture bottle over, first add 2ml of complete medium, and then in 5% CO 2 , 37 ℃ incubator for tissue block culture; after 4 hours, turn over to normal state and add a small amount of culture medium to continue the culture. The culture medium was changed routinely until the cells reached 70%-80% confluence, the cells were digested with 0.25% trypsin and reatt...

Embodiment 2

[0053] The selection of embodiment 2 tumor cells

[0054] The expressions of TSP-1, CD36 and CD47 were detected in highly, medium and poorly differentiated salivary gland MECs by immunofluorescence and Western Blot. Immunofluorescence also adopts the method of cell crawling, 2ml per well at a concentration of 2×104 / ml is inoculated into a 6-well plate previously placed on a cover glass, and incubated with 1:100 mouse anti-human TSP-1 monoclonal antibody at 4°C Overnight, in the dark, add FITC fluorescently labeled rabbit anti-mouse IgG secondary antibody (1:200), biotin-labeled goat anti-rabbit IgG secondary antibody (1:200), and DAPI for fluorescent staining of the nucleus for 2-3min, and seal The slices were protected from light and observed under a fluorescence microscope. The specific method of immunofluorescence double staining of CD36 and CD47 is roughly the same as that of TSP-1. Among them, the primary antibodies of CD36 and CD47 are both (1:50), human biotinylated-a...

Embodiment 3

[0057] Embodiment 3 selects suitable inoculum concentration and TSP-1 experimental action concentration and detection time point

[0058] The selected tumor cells were divided into 5×10 3 / ml, 1×10 4 / ml, 2×10 4 / ml, 5×10 4 / ml initial concentration, inoculate 96-well plate. Cells of each concentration were divided into 7 groups, each group was inoculated into 6 multiple wells, 200 μl of single cell suspension was added dropwise to each well, and placed in 5% CO 2 Incubate at 37°C. After 24 hours, use the MTT method to detect the MTT value in each well of the first group in each concentration group, and measure continuously for 7 days, and record the results with the horizontal axis as time and the vertical axis as MTT value to draw the cell growth of each concentration inoculation group curve. Select the appropriate cell inoculation concentration according to the results of each group, the results are shown in Figure 4 .

[0059] Inoculate the selected tumor cells i...

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Abstract

The invention relates to animal models for in vivo screening biological macromolecules having influences on tumor cell biology, in particular to genes or protein which have influences on tumor angiogenesis. It cannot be proved that the genes or protein have the influences on functions of the tumor cell biology through in-vivo experiments, but an interference factor that new blood vessels affect the growth of tumors can be effectively shielded by using the models. According to the animal models for in vivo screening the biological macromolecules having the influences on the tumor cell biology, the reliable models are provided for studying the further functions of the biological macromolecules on an angiogenic factor and an inhibiting factor of the existing tumor vessels, a foundation is provided for the clinical application of the models, and the models are of great significance.

Description

technical field [0001] The invention belongs to the field of animal model preparation, and specifically relates to an animal model for in vivo screening of biological macromolecules that have an influence on tumor cell biology and its application, and more specifically relates to some genes or proteins that have an influence on tumor angiogenesis, which cannot It has been proved by in vivo experiments that it has an impact on the biological function of tumors, and this bottleneck can be solved by using this model. Background technique [0002] The notion that tumor growth is dependent on angiogenesis began in the early 1970s, but its significance was underappreciated. In the past ten years, due to the discovery of the role of angiogenesis factors on angiogenesis, and the important impact of angiogenesis on tumor growth, invasion and metastasis, especially on the early occurrence of tumors, tumor angiogenesis has become one of the hot spots in tumor research in recent years. ...

Claims

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Application Information

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IPC IPC(8): A01K67/02A61K35/13
Inventor 杨森郭丽娟
Owner SUINING CENT HOSPITAL
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