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Method for preparing person stem cells with improved neural restoration function and application of person stem cells

A neural stem cell and nerve repair technology, applied in the field of constructing recombinant virus vectors containing multiple neurotrophic factor fusion genes, can solve the problems of limiting the clinical effect of adult stem cell autologous transplantation and limited stem cells

Pending Publication Date: 2016-01-20
ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0003] However, what is confusing about adult stem cell transplantation is that the number of stem cells that enter the lesion to perform repair functions after adult stem cell transplantation is limited, and some stem cells may have died or died before migrating to the lesion. Even more limited, which severely limits the clinical efficacy of autologous adult stem cell transplantation

Method used

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  • Method for preparing person stem cells with improved neural restoration function and application of person stem cells
  • Method for preparing person stem cells with improved neural restoration function and application of person stem cells
  • Method for preparing person stem cells with improved neural restoration function and application of person stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction and packaging of recombinant BDNF-L-NT3-L-GDNF lentiviral vector

[0055]Human BDNF, NT3 and GDNF sequences were amplified from normal human peripheral blood genomic DNA by PCR, primers were designed on the human BDNFcDNA reverse strand containing the connecting DNA sequence (CCGCCGCCGCCGTCA) 3, and NT3 was designed on the positive strand containing the connecting DNA sequence (GGCGGCGGCGGCAGT ) 3 and a pair of primers containing the linking DNA sequence (CCGCCGCCGCCGTCA) 3 in the reverse strand, GDNF designed a primer containing the linking DNA sequence (GGCGGCGGCGGCAGT) 3 in the positive strand, and the three were amplified and linked together by PCR to form a complete sequence containing The DNA of human BDNF, NT3 and GDNF is linked by the expression linker DNA, namely BDNF-L-NT3-L-GDNF (bng), see figure 1 .

[0056] After double digestion of BDNF-L-NT3-L-GDNF and pUC229 vector HindIII / BamHI, under the action of T4DNA ligase (TAKARA Co., Ltd.,...

Embodiment 2

[0061] Example 2 Preparation of recombinant bng lentivirus transfected human neural stem cells

[0062] Human neural stem cells were purchased from Neuostem Company in the United States, using complete culture medium DMEM / F12 (1:1) (containing 20ng / ml recombinant human basic fibroblast growth factor, 20ng / ml recombinant human epidermal growth factor and 5% fetal bovine serum) at 75cm 2 Culture in a culture flask; when the cell growth confluence reaches 90%, digest with 0.25% trypsin (containing 0.05 MEDTA), transfer to two 75cm 2 Grow in culture flasks at 25cm 2 Continue to digest and subculture when the fusion in the culture flask reaches 90%.

[0063] Add 20 μl (1E+7TU / ml) recombinant bng lentivirus to the obtained six-well culture plate of human neural stem cells, the system is 2ml, mix well, and store at 37°C, 5% CO 2 After incubation for 8-12 hours in the incubator, replace the complete culture medium DMEM / F12 (1:1) (containing 20ng / ml recombinant human basic fibroblas...

Embodiment 3

[0067] Example 3 In vivo tumorigenesis analysis of recombinant bng lentivirus transfected human neural stem cells

[0068] Take the hNSCs of the first generation obtained by subculture in Example 2, subculture until the 15th generation hNSCs (hNSCs-bng) transfected with bng lentivirus alone, each 3×10 6 The cells, in which the glioma cell line U87-MG cell line was used as a positive control, were subcutaneously injected into the abdominal flank of 8-week-old nude mice (Experimental Animal Center, Peking University Health Science Center), and observed continuously for 3 months, paying attention to whether there was tumor growth at the injection site .

[0069] Figure 6 This is the result of tumor formation after subcutaneous injection of hNSCs transfected with recombinant bng lentivirus in nude mice. After subcutaneous injection in nude mice, no tumor formation was found in all hNSCs (Figures A and B), while the inoculated U87-MG positive control appeared Tumor formation was...

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Abstract

The invention provides a method for preparing person stem cells with the improved neural restoration function. The method includes the steps that a fusion gene segment containing a plurality of neurotrophic factors is built and recombined to virus expression vectors, and the person stem cells are transfected. Fusion protein containing a plurality of neurotrophic factors can be highly expressed, and the combined effect of the neurotrophic factors can be developed to promote restoration of the neurological functions; meanwhile, the proliferation and differentiation capacity of the stem cells is used for participating in restoration of the neurological functions, and tumorigenicity is avoided; it is expected that the person stem cells can achieve the better treatment effect on nervous system diseases.

Description

technical field [0001] The present invention relates to a method for human stem cells with improved nerve repair function and the method for obtaining human stem cells that promote nerve repair. In particular, the present invention relates to the construction of recombinant virus vectors containing multiple neurotrophic factor fusion genes, so that human stem cells Fusion proteins that can stably express multiple neurotrophic factors can jointly exert the biological functions of neurotrophic factors; the human stem cells with improved nerve repair function of the present invention maintain good proliferation ability, promote the improvement of nerve repair, and have no tumorigenicity, and can It is expected to be used in the clinical treatment of nervous system diseases and promote the repair of damaged nerve cells. Background technique [0002] At present, adult stem cells have been used in the clinical treatment of nervous system diseases, including neural stem cells, mese...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867A61K35/30A61K35/28A61P25/00C12R1/91
Inventor 陈建勋
Owner ZICHENG RUISHENGHUI BEIJING BIOTECH DEV CO LTD
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