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TPO gene modifying human marrow stroma stem cell and its preparation method and use

A technology of stem cells and gene modification, applied in the field of gene modification of bone marrow mesenchymal stem cells, can solve the problem of undetectable TPO protein expression and other problems

Inactive Publication Date: 2008-08-13
CENT SOUTH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

IL-6, IL-11, LIF, SCF, and TPO are expressed at the transcriptional level in MSCs, but the expression of IL-6, IL-11, LIF, and SCF protein can be detected by ELISA method, but TPO protein cannot be detected expression

Method used

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  • TPO gene modifying human marrow stroma stem cell and its preparation method and use
  • TPO gene modifying human marrow stroma stem cell and its preparation method and use
  • TPO gene modifying human marrow stroma stem cell and its preparation method and use

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Embodiment Construction

[0024] The concrete implementation of the present invention is described in detail below in conjunction with accompanying drawing:

[0025] (1) Construction of pAAV-TPO-IRES-hrGFP: The full-length TPO cDNA was obtained from human fetal liver total RNA by RT-PCR, and the TPO gene was cloned into the multiple cloning site of the pAAV-IRES-hrGFP vector to construct pAAV -TPO-IRES-hrGFP (pAAV-TPO), its structure is attached figure 1 shown.

[0026] The β-globin intron (β-globin intron) sequence in the pAAV-IRES-hrGFP plasmid is located upstream of the EcoRI site, and the TPO gene fragment is inserted into pAAV-GFP after digestion with EcoRI, so it is in the β-globin intron sequence Synthesize a primer as a sequencing primer. The sequencing primer sequence is: ATTCTGAGTCCAAGCTAGGC. In order to continue to detect the full-length sequence of the TPO gene, sequencing primers were synthesized at the 480bp position and 858bp position of the TPO gene, respectively: 5'-GCGTTTCCTGATGCTT...

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Abstract

The invention discloses human marrow-interstitial stem cell modified by TPO gene, its preparing process and use. The human marrow-interstitial stem cell is obtained by recombination gland relevant virus meso-guide TPO gene decoration. The preparation method comprises the following steps consequently cloning human tyre liver TPO gene, constructing carrier plasmid Paav-tpo-ires-hrGFP, preparing Raav-TPO viral vectors, culturing human marrow-interstitial stem cell, transfecting Raav-TPO on human marrow-interstitial stem cell. The human marrow-interstitial stem cell modified by TPO gene or human marrow-interstitial stem cell exudate modified by TPO gene can be used for preparing medicine of urging blood platelet-generating. The MSCs modified by TPO gene generates internal source TPO through cell secreting for treating blood platelet reduction so the side-effect generated from injection human recombination TPO is not generated.

Description

technical field [0001] The invention relates to the field of genetically modified bone marrow mesenchymal stem cells. Background technique [0002] Thrombopoietin (TPO) is a cytokine that regulates the formation of megakaryocytes and platelets. TPO has the whole process of initiating the formation of megakaryocyte clones, stimulating the production of hyperploidy megakaryocytes and mature megakaryocytes, and supporting functional platelet production. . [0003] Animal experiments have shown that recombinant human TPO is more effective than other hematopoietic factors in restoring platelet levels and promoting the activity of other hematopoietic factors; clinical experiments have also shown that recombinant human TPO can effectively alleviate chemotherapy-induced thrombocytopenia. There are two main forms of TPO currently used in clinical research: PEG-modified half-length molecule TPO expressed by bacteria (PEG-rHuMGDF) and full-length, glycosylated TPO expressed by mammali...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/861A61K35/28A61P7/00
Inventor 谭孟群周小莹
Owner CENT SOUTH UNIV
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