Immortalized human liver cancer blood vessel endothelial cell line and preparation method and application thereof

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

Active Publication Date: 2016-01-06
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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  • Immortalized human liver cancer blood vessel endothelial cell line and preparation method and application thereof
  • Immortalized human liver cancer blood vessel endothelial cell line and preparation method and application thereof
  • Immortalized human liver cancer blood vessel endothelial cell line and preparation method and application thereof

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. Filter the cell suspension with a 40 μm filter, centrifuge at 1000 r / min at 4°C for 10 min, discard the supernatant and make a single cell suspension, and add DNase...

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. Detect the expression of CD31 and CD105 by laser confocal microscope: Prepare cultured cell slides in a six-well plate, fix with 4% paraformaldehyde for 15min, wash with PBS for 3x5min, 0.3% TritonX-100 / PBS for 10min, wash with PBS for 1x5min, wash with fingernails Mark the edge with oil to prevent leakage of blocking solution or antibody, drop 100 μL of 10% BSA / PBS on th...

Embodiment 3

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

[0102] Applying the cell line ECDHCC-1 provided by the present invention to basic research: Effects of vascular regulation-related molecules Dicer (GENBANKNW-177438), miRNA-210 (Accession: MIMAT0000267), FGFRL-1 (GENBANKNW-021923) on the function of ECDHCC-1 .

[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 XiaoF, QiuH, ZhouL, ShenX, YangL, and DingK. 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-icer will carry Gene virus, miRNA-210 gene virus, silencing (sh) FGFRL-1 gene virus (all viruses were purchased from Shanghai Jikai Biological Co., Ltd.) were respectively tr...

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Abstract

The invention belongs to the biotechnological field and provides an immortalized human liver cancer blood vessel endothelial cell line ECDHCC-1 with the preservation number of CCTCC NO: C2015172, and the cell line is preserved in China Center for Type Culture Collection on 15 October, 2015. The invention further provides a construction method and authentication for ECDHCC-1. The cell line conducts passage by means of monocloning, subculture lasts as long as 18 months, 119 generations are produced in total until the preservation date, and biological characteristics of the generations keep consistent. The cell line thoroughly improves the tedious process that tumor vessels are not taken until needing to be used for current research, as well as the current situation that human liver cancer operation samples are short of resources; a commercialized stable tumor-derived blood vessel endothelial cell line is provided for relevant researchers, and time saving, effort saving, high efficiency and convenience are achieved in a true sense.

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 Applications(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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