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Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof

A technology of neurotrophic factors and mesenchymal stem cells, which is applied in the field of mesenchymal stem cells and their preparation, can solve the problems of impossibility and difficulty of gene therapy

Inactive Publication Date: 2010-10-13
广州吉帝生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the cells treated by gene transplantation must be taken out of the patient's body for artificial culture, and then transplanted into the patient's body, gene therapy is very difficult. At present, only skin cells and bone marrow cells can accept this treatment, and the treatment that can be done It is only the introduction of foreign genes to express them to supplement the missing or non-functional enzymes, but not to replace the mutant genes with normal genes

Method used

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  • Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof
  • Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof
  • Mesenchymal stem cell for expressing related gene of neurotrophin family and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Isolation and culture of bone marrow MSCs and transfection of human bone marrow MSCs with green fluorescent protein gene

[0065] The green fluorescent protein (GFP) gene is the only new reporter gene found so far that can be expressed in cells without the participation of other exogenous substrates. In this study, human bone marrow stem cells were transfected with CMV-GFP gene, and the expression of GFP in human bone marrow stem cells was observed.

[0066] 1. Materials

[0067] 1. Equipment: ultra-clean bench, horizontal centrifuge, inverted microscope and ordinary microscope. Centrifuge tubes and balances, bone marrow puncture needles and blood collection bags. Flow cytometer, drying box, gloves and surgical gown. Hydrometer (1.000-1.100, 1.100-1.200)

[0068] 2. Reagents: Meglumine Diatrizoate (Sigma Company), Hydroxymethyl Cellulose (Sigma Company), Cesium Chloride (Sigma Company), Polysucrose (Ficoll-400, Phamacia Company), Potassium Dihydrogen Phos...

Embodiment 2

[0095] Example 2: Cloning of human GDNF and BDNF genes and construction of their N-terminals

[0096] 1. Cloning of human GDNF gene and its N-terminal construction:

[0097] Using human brain tissue information ribonucleotide (mRNA) as a template, use Poly(T) primers under the action of reverse transcriptase to synthesize complementary deoxyribonucleotide (cDNA) of human brain tissue, and then use human brain tissue cDNA As a template, use an artificially synthesized forward primer containing the nucleotides encoding the human interleukin-2 secretion leader polypeptide and the N-terminal sequence nucleotides of the human mature GDNF gene and the C-terminal sequence nucleotides of the human GDNF gene The reverse primer was used for PCR amplification, and the human GDNF gene clone and its N-terminal reconstruction were carried out, and the endonuclease NheI and NdeI sites were introduced at its N-terminus, and the endonuclease BamHI and HindIII sites were introduced at its C-ter...

Embodiment 3

[0120] Example 3, Construction of co-expression GDNF and BDNF expression cassette pAAV-MCS vector

[0121] Co-expression of GDNF and BDNF expression cassette pAAV-MCS vector construction method ( Figure 9 , 10) comprises the following steps:

[0122] 1) Using the multiple cloning site of the pAAV-MCS vector in the AAV helper-free expression system, the GDNF gene was purified with endonuclease BamHI and Xbal1 nucleotide fragments, digested with BamHI and Xbal1, and the purified pAAV- After the ligation reaction of the MCS vector, the competent bacteria were transformed, amplified, spread on a petri dish containing appropriate antibiotics, and grown overnight in a 37°C incubator;

[0123] 2) Screen multiple clonal colonies in culture medium containing appropriate antibiotics, culture overnight at 37°C with shaking, extract and purify pAAV-MCS plasmid DNA the next day, and digest with BamHI and Xbal1 enzymes to determine the presence of BamHI and Xbal1. Plasmid DNA and clone s...

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Abstract

The invention relates to a mesenchymal stem cell of marrow, umbilical cord or bleeding of the umbilicus and purpose in the field of cell transplantation, stem cell culture, expansion and oriented differentiation. The mesenchymal stem cell for expressing a related gene of a neurotrophin family is prepared by the following steps of extracting a mesenchymal stem cell from the marrow, the umbilical cord or the bleeding of the umbilicus; recombining the related gene of the neurotrophin family to an AAV (Adeno-Associated Virus) used as a carrier; preparing the AAV expressing the related gene of the neurotrophin family; and infecting separated and purified mesenchymal stem cell through AAV particles to obtain the mesenchymal stem cell expressing the related gene of the neurotrophin family. The invention is used as a culture layer cell to provide a manually controlled microenvironment for stem cell culture, expansion and oriented differentiation and promote the proliferation and the oriented differentiation of the stem cells so as to provide a new curing measure for human diseases.

Description

technical field [0001] The invention relates to a mesenchymal stem cell and a preparation method thereof, in particular to the separation, cultivation and purification of mesenchymal stem cells from bone marrow, umbilical cord or umbilical cord blood, and the transfer of exogenous genes such as nerve growth factor genes into mesenchymal stem cells The method and its application in cell transplantation, stem cell culture and directed differentiation. Background technique [0002] Stem cells are cell populations existing in the normal human body, with a high degree of self-replication and differentiation capabilities. In recent years, with the deepening of stem cell research, it has been discovered that stem cells can be used to regenerate various tissues and organs, such as liver, pancreas, nerve tissue, bones, tendons, etc. [0003] Due to the source and isolation, stem cells currently used in clinical practice mainly include adult bone marrow mesenchymal stem cells (BMSCs)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N5/08A61K35/28A61K35/48A61P9/10A61P25/00
Inventor 邹清雁王尚武陈文明李远友
Owner 广州吉帝生物科技有限公司
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