Fetus liver stroma cell strain expressing alkaline fibroblast growth factor and uses thereof

A technology of fibroblasts and growth factors, applied in the application, cells modified by introducing foreign genetic material, genetic engineering, etc., can solve the problems of high safety and high cost of hES cell culture, and achieve the effect of low cost and broad application prospects.

Inactive Publication Date: 2008-06-25
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the safety and high cost of hES cell culture have always been the bottleneck restricting its application, especially the key issues that need to be solved in the final application of hES cells in cell therapy

Method used

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  • Fetus liver stroma cell strain expressing alkaline fibroblast growth factor and uses thereof
  • Fetus liver stroma cell strain expressing alkaline fibroblast growth factor and uses thereof
  • Fetus liver stroma cell strain expressing alkaline fibroblast growth factor and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1. Construction of fetal liver stromal cell line expressing 18kDa subtype basic fibroblast growth factor and identification of bFGF expression level

[0033] 1. Construction of fetal liver stromal cell line expressing 18kDa subtype basic fibroblast growth factor

[0034] Taking the fetal liver stromal cell line stably expressing the 18kDa subtype of basic fibroblast growth factor as an example, the method for constructing a fetal liver stromal cell line stably expressing basic fibroblast growth factor using a lentiviral vector system in the present invention is illustrated, specifically The method includes the following steps:

[0035] 1. Construction of recombinant lentiviral vector

[0036] Design RT-PCR primers according to the nucleotide sequence of the 18kDa subtype bFGF gene (GenBank number: NM_002006), and the primer sequence is as follows: P1 (forward primer): 5'-AA CTGCAG ATGGCAGCCGGGAGCATC-3' (underlined base is the recognition site of restriction e...

Embodiment 2

[0057] Example 2. Detection of PKU-1 and Specific Antigen Expression of Human ES Cells Cultured with bFGF / FLSC as Feeder Layer

[0058] 1. bFGF / FLSC as feeder layer to culture human ES cell PKU-1

[0059] For human ES cell PKU-1 (purchased from PLA General Hospital, Peng H M. Neural precursors derived from human embryonic stem cells. Science in China Ser. C, 2005, 48(3): 295-299) at 37°C, 5% CO 2 Routine subculture was carried out in the cell culture box, and the medium was 4 ng / mL of recombinant bFGF (purchased from R&D Company) added on the basis of DF-12 medium, 20% serum substitute (purchased from HyClone Company), 0.1 mmol / L non-essential amino acids (purchased from HyClone), 2mmol / L L-glutamine, 0.1mmol / L β-mercaptoethanol and 1% penicillin-streptomycin. When the strongly fluorescently expressed bFGF-FLSC CGMCCNo.1804 cells obtained in Example 1 and the weakly fluorescently expressed bFGF / FLSC cells grown to 90% confluency were treated with 6 μg / mL mitomycin-C for two...

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Abstract

The invention discloses a fetus liver matrix cell strain for expressing alkaline fibroblastic growth factors. The fetus liver matrix cell strain of the invention is a recombinant fetus liver matrix cell strain carried with alkaline fibroblastic growth factor genes. By experimental verification, recombinant fetus liver matrix cells of the invention can highly efficiently produce secretory alkaline fibroblastic growth factors, and embryonic stem cell proliferation of human beings and embryonic stem cell drying can be maintained under the condition of no exogenous alkaline fibroblastic growth factors by taking the cells as feeder layer cells for in vitro culture of embryonic stem cells of human beings. The recombinant fetus liver matrix cells of the invention and a construction method thereof provide appropriate microenvironment for researches in the aspects of construction of a low-cost (reduction of introduction of exogenous alkaline fibroblastic growth factors), safe and highly efficient (capable of replacing odontogenic zooblast as the feeder layer cells) in vitro culture system of the embryonic stem cells of human beings and development and differentiation of hematopoietic cells, and the invention has wide application prospect.

Description

technical field [0001] The present invention relates to a recombinant fetal liver stromal cell line and its construction method and application, in particular to a fetal liver stromal cell line capable of stably expressing basic fibroblast growth factor and its construction method and its use in culturing human embryonic stem cells (human embryonic stem cells) , application in hES). Background technique [0002] Basic fibroblast growth factor (basic fibroblast growth factor, referred to as bFGF or FGF-2) is a basic member of the fibroblast growth factor family. Play an important supporting role in the proliferation, migration, differentiation and survival of cells (BikfalviA, et al. Biological roles of fibroblast growth factor-2. Endocr Rev, 1997, 18(1): 26-45), especially in hES cells Plays a fundamental role that cannot be ignored in the in vitro culture system (Dvorak P, et al.Expression and Potential Role of Fibroblast Growth Factor2 and Its Receptors in Human Embryonic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/12C12N15/86
Inventor 裴雪涛习佳飞王韫芳张鹏南雪白慈贤
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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