An immortalized human liver cancer vascular endothelial cell line and its preparation method and application

A vascular endothelium and immortalization technology, applied in the biological field

Active Publication Date: 2018-06-29
杨俐萍 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no literature reports about the method and identification of SV40-related genes introduced into primary human tumor vascular endothelial cells or immortalized human tumor vascular endothelial cell lines established by other means.

Method used

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  • An immortalized human liver cancer vascular endothelial cell line and its preparation method and application
  • An immortalized human liver cancer vascular endothelial cell line and its preparation method and application
  • An immortalized human liver cancer vascular endothelial cell line and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Establishment and passage of human liver cancer vascular endothelial cell line ECDHCC-1

[0064] 1. Separation and extraction of human liver cancer (moderately differentiated) tissue vascular endothelial cells and primary culture:

[0065] 1.1. After informed consent and medical ethics approval, take fresh specimens of human liver cancer (moderately differentiated) surgical resection, and prepare single-cell suspension: wash the tissue fluid, red blood cells and ice-based basal medium, and cut the tissue into 1-2mm 3 The fragments were transferred into 10 mL of 0.5% collagenase type I and type IV mixed with Hank's solution, incubated at 37°C for 25 min, 10% BSA / PBS was used to stop the digestion, and the cell suspension was aspirated repeatedly with needles and syringes of different specifications, through 70 μm, 40 μm Filter the cell suspension, centrifuge at 1000r / min at 4°C for 10min, discard the supernatant and then make a single cell suspension, and add ...

Embodiment 2

[0082] Example 2: Identification of biological characteristics of human liver cancer vascular endothelial cell line ECDHCC-1

[0083] 1. The ultrastructure of W-P bodies in the cytoplasm of ECDHCC-1 was observed by transmission electron microscopy (see Figure 4 Arrow), which is one of the morphological characteristics of vascular endothelial cells. The 64-generation ECDHCC-1 cells were collected, and the electron microscope examination sections were made according to the conventional transmission electron microscope method, and the electron microscope was magnified 10,000 times to take pictures.

[0084] 2. Laser confocal microscopy to detect the expression of CD31 and CD105, etc.: Prepare cultured cell slides in a six-well plate, fix with 4% paraformaldehyde for 15min, wash with PBS for 3x5min, 0.3% Triton X-100 / PBS for 10min, wash with PBS for 1x5min, wash with Mark the edge with nail polish to prevent leakage of blocking solution or antibody, drop 100 μL of 10% BSA / PBS on...

Embodiment 3

[0101] Example 3: Application experiment of immortalized human liver cancer vascular endothelial cell line ECDHCC-1

[0102] Apply the cell line ECDHCC-1 provided by the present invention to basic research: vascular regulation-related molecules Dicer (GENBANK NW-177438), miRNA-210 (Accession: MIMAT0000267), FGFRL-1 (GENBANK NW-021923) on ECDHCC-1 functional impact.

[0103] Use lentiviral vectors to carry these target genes or silence these genes to transfect ECDHCC-1 cells, verify their effective transfection, and detect changes in cell function. Experimental method: see Xiao F, Qiu H, Zhou L, Shen X, Yang L, and Ding K. WSS25 inhibits Dicer, downregulating microRNA-210 which targets Ephrin-A3, to suppress human micro-vascular endothelial cell (HMEC-1) tube formation. Glycobiology , 2013; 23 (5): 524-535, will carry Dicer gene virus, miRNA-210 gene virus, silence (sh) FGFRL-1 gene virus (all viruses are purchased from Shanghai Jikai Biological Co., Ltd.) into the present inv...

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Abstract

The invention belongs to the field of biotechnology. The present invention provides an immortalized human liver cancer vascular endothelial cell line ECDHCC‑1, the preservation number of which is CCTCC NO: C2015172, which was preserved in China Center for Type Culture Collection on October 15, 2015. The invention also provides the construction method and identification of ECDHCC-1. The cell line obtained by the present invention has been subcultured by monoclonal subculture, and the subculture lasted for 18 months. There are 119 generations in total until the date of storage, and its biological characteristics remain consistent; The cumbersome procedures of "use now and take now" and the lack of resources for human liver cancer surgical specimens provide relevant researchers with a commercially stable tumor-derived vascular endothelial cell line, which is truly time-saving, labor-saving, efficient and convenient.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction, in vitro culture system, identification and application of an immortalized human liver cancer vascular endothelial cell line. Background technique [0002] Cancer is a major disease that seriously endangers human health, and the task of overcoming cancer and curing it is still very arduous. So far, although anticancer drugs have sprung up in the market, the problem of radical cure of cancer has not yet been solved. Studies have shown that tumor growth and metastasis are closely related to tumor blood vessels. In recent years, "starvation therapy" combined with chemotherapy has been used clinically to treat cancer, which is to block the neovascularization of tumors so that the supply of nutrients and oxygen required by tumor cells is lacking, resulting in starvation of cancer cells. However, anti-cancer drugs targeting blood vessels such as bevacizumab ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12Q1/02
Inventor 杨俐萍赵文静钱红燕陈旭东张一心强福林
Owner 杨俐萍
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