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Method for inhibiting gastric cancer angiogenesis by using micro vesicles as miRNA (Micro Ribonucleic Acid) transport carriers

An angiogenesis and microvesicle technology, applied in the field of molecular biology, can solve the problems of complex interspecies immune response, time-consuming, and heavy financial burden on patients

Inactive Publication Date: 2017-12-01
TIANJIN TUMOR HOSPITAL
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Problems solved by technology

In addition, the preparation process of monoclonal antibody is costly and time-consuming, with an average of about 3-6 months, and the financial burden on patients is heavy; and bevacizumab is produced by the expression system of Chinese hamster ovary cells, and the immune response between species is more severe than that within species. complex

Method used

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  • Method for inhibiting gastric cancer angiogenesis by using micro vesicles as miRNA (Micro Ribonucleic Acid) transport carriers
  • Method for inhibiting gastric cancer angiogenesis by using micro vesicles as miRNA (Micro Ribonucleic Acid) transport carriers
  • Method for inhibiting gastric cancer angiogenesis by using micro vesicles as miRNA (Micro Ribonucleic Acid) transport carriers

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Embodiment 1

[0037] Example 1: Extraction and Identification of Microvesicle Cells

[0038] Human embryonic kidney cells HEK293T were untreated or transfected with miR-29a / c mimics / inhibitor, NCmimics / inhibitor 24-48 hours after cell culture medium was collected; room temperature 3000 rpm, centrifuged for 20 minutes, 4 ℃ 10,000 g centrifugation for 30 minutes, the purpose is to remove cells and cell debris; after centrifugation, take the supernatant and ultracentrifuge at 110,000g at 4°C for 60 minutes; then discard the supernatant, resuspend the pellet with an appropriate amount of 1×PBS, and repeat for 4 Ultracentrifuge at 110,000g for 60 minutes at ℃; discard the supernatant, resuspend the pellet with an appropriate amount of 1×PBS and store at -80℃ for later use, or add it directly to the cells to be incubated or perform mouse tail vein injection experiments.

[0039] Immediately transfer the microvesicles obtained by ultracentrifugation to a centrifuge tube and add 2.5% glutaraldehyde...

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Abstract

The invention discloses a method for inhibiting gastric cancer angiogenesis by using micro vesicles as miRNA (Micro Ribonucleic Acid) transport carriers. Through extraction and identification of micro vesicles, quantitative analysis of miRNA, protein extraction, Western blot, ELISA (Enzyme-linked Immuno Sorbent Assay) and EdU (5-Ethynyl-2'-deoxyuridine) proliferation assays, an in-vitro angiogenesis assay, a tumorigenesis assay and a tail intravenous injection micro vesicle assay, the situation that the quantitative analysis of the content of corresponding miRNA in artificially modified micro vesicles shows that the content of miR-29 is obviously increased by being compared with a negative control is discovered, the VEGF (Vascular Endothelial Growth Factor) protein secretion level of gastric cancer cells is obviously inhibited after the gastric cancer cells are hatched by the micro vesicles containing the miR-29, the proliferation of vascular endothelial cells co-cultured by the gastric cancer cells treated by the micro vesicles secreted by HEK (Human Embryonic Kidney)-293 cells treated by high-expression miR-29a and miR-29c is obviously inhibited, ring formation of the vascular endothelial cells co-cultured by the gastric cancer cells treated by the micro vesicles secreted by the HEK-293 cells treated by the high-expression miR-29a and the miR-29c is obviously inhibited, and the tumor volume in a mouse containing the micro vesicles which are subjected to the tail intravenous injection and high-expression miR-29a / c treatment is obviously smaller than the tumor volume in a mouse treated by the corresponding negative control.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for inhibiting gastric cancer angiogenesis. Background technique [0002] Gastric cancer is one of the most common malignant tumors in the world. The latest epidemiological survey shows that the incidence of gastric cancer in China ranks second, and the death rate ranks fourth. Like most malignant tumors, the multiple biological behaviors of gastric cancer are not only related to the characteristics of cancer cells themselves, but also closely related to their microenvironment, especially the need to continuously form new blood vessels to achieve the supply of nutrients they need. Therefore, if the process of tumor angiogenesis can be blocked, it is possible to inhibit tumor growth, invasion and metastasis to a certain extent. Many factors related to tumor angiogenesis have been discovered so far, among which vascular endothelial growth factor (vascular endo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50A61K49/00A61K31/7088
CPCA61K31/7088A61K49/0008G01N33/5011
Inventor 张海洋巴一王欣怡邓婷刘锐
Owner TIANJIN TUMOR HOSPITAL
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