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Immortalized rat marrow stroma cell system and preparation method thereof

A bone marrow stromal cell, immortalization technology, applied in biochemical equipment and methods, botanical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve problems such as difficult in vitro serial passage and large-scale expansion, etc., Achieve strong proliferative ability, cell line stability, induction and differentiation promotion in vivo and in vitro

Inactive Publication Date: 2010-11-17
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although bMSCs have stem cell characteristics, it is difficult to achieve continuous passage and large-scale expansion in vitro, which limits their in vitro induction and differentiation, in vivo migration and differentiation potential, and research on cell and gene therapy.

Method used

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  • Immortalized rat marrow stroma cell system and preparation method thereof
  • Immortalized rat marrow stroma cell system and preparation method thereof
  • Immortalized rat marrow stroma cell system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Isolation and culture of rat bone marrow stromal cells and detection of biological characteristics

[0025] 1. Isolation, culture and passage of rat bone marrow stromal cells

[0026] Adult SD rats (Sprague-Dawley closed group rats) were executed by decapitation. After routine disinfection of the limbs and chest skin, the limb bones and sternum were quickly taken out, and pre-cooled D-hank's solution (containing: 8.00 g of NaCl per liter, KCl0 .40g, Na 2 HPO 4 0.06g, KH 2 PO 4 0.06g, NaHCO 3 0.35g, phenol red 0.02g, containing 10 5 u / L Penicillin (North China Pharmaceutical) and 0.1g / L Streptomycin (North China Pharmaceutical)) soaked for 3-5 minutes, use a 10-gauge injection needle to draw serum-free pre-cooled α-MEM (Gibco) to flush the bone marrow cavity to obtain Flush for bone marrow cells. Take 1 portion of cell washing solution, carefully add 3 times the volume of lymphocyte separation solution (Tianjin Haoyang Biological Products Technology ...

Embodiment 2

[0042] Example 2, the acquisition of immortalized rat bone marrow stromal cell line and the detection of its biological characteristics

[0043] 1. Obtainment of Immortalized Rat Bone Marrow Stromal Cell Line

[0044] 1. Acquisition of human telomerase reverse transcriptase catalytic subunit gene (hTERT)

[0045] Extract mRNA from human Jurkat T-cell lymphoma (human Jurkat T-cell lymphoma, ATCC), and obtain cDNA by reverse transcription. Using the cDNA as a template and 5'-ctgctgcgcacgtgggaag-3' as an upstream primer, 5'-cgggtggccatcagtccag- 3' is the downstream primer, the 3.4Kb target product is obtained by PCR, and the target product is purified and recovered and directly connected to pTARGET TM The 3'T end (overhangs) of the vector (Promega) was used to obtain pTARGET-hTERT, and positive clones were selected for sequencing to obtain the sequence of the human telomerase reverse transcriptase catalytic subunit gene (hTERT), whose sequence is as in the sequence listing 1, t...

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Abstract

The invention provides a immortal stromal cell systme of rat's marrow and preparation thereof. Immortal stromal cell of rat's marrow can be obtained in short time by the inventive method. The inventive hTERT immortal stromal cell systme of rat's marrow is stable, can be sequentially passed through generations and keep diploid karyotype and characteristics of normal stromal cell of rat's marrow, has extremely strong proliferation ability, and realize immortalization. Immortal stromal cell system of rat's marrow established by the inventive method can conveniently and quickly obtain large amount of stromal cell of rat's marrow with diploid karyotype. This no doubt has great promotion to research of introduction and bone marrow stromal cell in vitro and in vivo.

Description

technical field [0001] The invention relates to an immortalized rat bone marrow stromal cell line and a preparation method thereof. Background technique [0002] Bone marrow stromal cells (bMSCs) are a kind of stem cell population with multilineage differentiation potential existing in bone marrow. Under certain conditions, bMSCs can differentiate into bone cells, chondrocytes, cardiomyocytes, hepatocytes, neurons and glial cells in vitro or in vivo. Bone marrow stromal cells have the advantages of rich sources, convenient collection, weak immunogenicity, low bacterial contamination rate, and no ethical issues, which provide a new method for cell transplantation or replacement therapy of Parkinson's disease (PD) and other degenerative diseases. A new source of seed cells. It is precisely because the cells have multi-directional differentiation potential, and can realize autologous materials and autologous transplantation, thereby avoiding immune rejection, so bMSCs have be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/52C12N15/867A61K35/28A61P25/00A61P25/16
Inventor 段春礼杨慧赵春礼孙晓红鲁玲玲
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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