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Human tumor stem cell line and application thereof

A tumor stem cell line and tumor stem cell technology, which can be used in the determination/examination of microorganisms, microorganisms, biochemical equipment and methods, etc. Effect

Active Publication Date: 2010-03-03
SUZHOU BOJUHUA BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of drugs targeting CSCs first requires a stable tumor stem cell line, which can be used as a tool to easily develop drugs that specifically target CSCs, but such cell lines have not been reported so far

Method used

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  • Human tumor stem cell line and application thereof
  • Human tumor stem cell line and application thereof
  • Human tumor stem cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Establishment of human tumor stem cell lines

[0030] Primary isolation: the human tumor stem cell line described in the present invention is obtained during the process of isolating and culturing microvascular endothelial cells from human liver cancer tissue. The tumor tissue was obtained from a surgical specimen of an inpatient with liver cancer in the Cancer Hospital of the Chinese Academy of Medical Sciences, and the pathological diagnosis was hepatocellular carcinoma. Initially, microvessels were isolated and cultured from human liver cancer tissue resected by clinical surgery according to the method described in the literature (Wu LQ, et al. Phenotypic and functional differences between human liver cancer endothelial cells and liver sinusoidal endothelial cells. J Vasc Res 2008; 45:78-86) Endothelial cells, a group of rapidly proliferating cell clones appeared when the microvascular endothelial cells were passed to the 8th passage. Cells are small in s...

Embodiment 2

[0036] Example 2 RT-PCR detection of stem cell-related gene expression in T3A-A3 cells

[0037] Use stem cell medium to culture T3A-A3 in T25 culture flasks, grow to 90% confluence, digest with 0.1% trypsin-0.1% EDTA to collect cells, add 10ml sterile PBS, 1200rpm / min, centrifuge at room temperature for 8min, cells Total RNA was extracted with Qiagen Total RNA Extraction Kit. Then 2ug of total RNA was taken and reverse-transcribed using Qiagen's reverse transcription kit. Then PCR method was used to detect the expression of genes related to stem cell proliferation, self-renewal and differentiation. The amplified target gene and its primer sequences are as follows, and the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.:

[0038] Table 1. Gene-specific primer sequences in PCR amplification reactions

[0039] target gene

[0040]

[0041] The PCR reaction conditions are pre-denaturation at 94°C for 2min, and cycle reaction 40 times. T...

Embodiment 3

[0042] Example 3. Evaluation of self-renewal ability of T3A-A3 cells

[0043] Colony formation experiment and secondary colony formation experiment Prepare 10g / L poly-HEMA (P3932, sigma) in absolute ethanol and coat 96-well plate to inhibit cell adhesion. T3A-A3 cells were seeded in a 96-well plate at 20 cells / well, with 9 replicate wells. The medium is 0.9% methylcellulose (H4100, stemcell company) (the final concentration of each additive in the medium is 5% fetal bovine serum for stem cells, 10ng / ml LIF, 100μg / ml FGF, 40u / ml heparin) , the culture volume is 0.2ml / well. Place at 37°C, 5% CO 2 , cultured in saturated humidity. Observed under an inverted microscope, more than 50 cells were counted as a colony. T3A-A3 cells cultured in 0.9% methylcellulose, colony formation can be seen after 14 days; pick out the colonies, wash the basal medium twice, make a single cell suspension, and then carry out the second colony according to the above steps In the formation test, col...

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Abstract

The invention provides a human tumor stem cell line T3A-A3, which is obtained by separation of liver cancer tissue excised in an operation of a primary hepatic carcinoma patient. The separated cells can be in long-term passage culture in vitro, can grow rapidly and retain stem cell property after more than 100 times of passage. The cell can express markers of multiple stem cells, has self-updatingcapability of stem cells and directional differentiation potentials for different tumor cells, and also has tumor property, tumorigenicity ability and metastatic ability. The invention is a powerfultool for preparing stem cell drugs for targeting tumor, due to the facts that the cells can be in long-term passage culture in vitro and retain unchanged stem cell property, and the tumorigenicity ability and metastatic ability thereof are strong in immunodeficient mice.

Description

technical field [0001] The invention belongs to the field of tumor biology, and in particular relates to a human tumor stem cell line and its application. Background technique [0002] Malignant tumors are one of the major diseases that threaten human health. At present, the global cancer situation is becoming more and more serious, and its morbidity and mortality are increasing year by year. Therefore, it is very important to develop more effective tumor treatment schemes and therapeutic drugs. [0003] In recent years, more and more studies have shown that in the same tumor tissue, tumor cells are heterogeneous in tumorigenesis ability, differentiation degree and metastasis ability, and only a small number of cells have the ability to initiate tumors. Such cells are called cancer stem cells (cancer stem cells, CSCs) or tumorigenic cancer cells (Tumogenic cancer cells). Cancer stem cells are a cell population with both stem cell and tumor properties. Like stem cells, the...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12Q1/02C12R1/91
Inventor 刘虹麟张文健叶丽亚娄晋宁
Owner SUZHOU BOJUHUA BIOMEDICAL TECH CO LTD
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