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Preparation method and kit of human stem cell with prolonged service life and enhanced angiogenesis

A life extension, stem cell technology, applied in medical preparations containing active ingredients, cardiovascular system diseases, cells modified by introducing foreign genetic material, etc. The ability of differentiation potential to homing to the lesion area is lost, so as to achieve the effect of prolonging cell lifespan, ensuring safety, and enhancing the ability of angiogenesis.

Active Publication Date: 2015-11-04
广州赛琅生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, adult stem cells, like most adult cells, will undergo senescence, which is mainly manifested in two aspects: ① With age, the quantity and quality of BMSCs are significantly reduced (and the elderly are suffering from ischemic cardiovascular and cerebrovascular diseases The high-incidence population is the subject that may need cell transplantation); ②During the in vitro expansion process, the proliferation ability, differentiation potential, and even the ability to homing to the lesion area of ​​the cells will gradually lose
This severely limits the clinical application of adult stem cell autologous transplantation

Method used

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  • Preparation method and kit of human stem cell with prolonged service life and enhanced angiogenesis
  • Preparation method and kit of human stem cell with prolonged service life and enhanced angiogenesis
  • Preparation method and kit of human stem cell with prolonged service life and enhanced angiogenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of recombinant T-STAR and Angl lentiviral vectors and preparation of human stromal stem cells

[0054] The T-STAR and Angl sequences were amplified from normal human peripheral blood genomic DNA by PCR, and the target fragments of T-STAR and Angl and the pLenti6.3_MCS vector PacI / AscI were double-enzymatically digested respectively, and T4DNA ligase (Japan TAKARA Company) under the action of the double-stranded T-STAR and Angl at 4 ° C for 12 hours to prepare the clone connection solution, and transform the Escherichia coli competent cell DH5a (Invitrogen, USA) to carry out positive clone PCR identification and sequencing identification.

[0055] Take 293T cells in good condition and in the logarithmic growth phase. After counting the cells, 6×10 6 The number of cells was seeded in a culture dish at 37°C, 5% CO 2 cultured overnight in an incubator. Remove the culture medium before transfection the next day, and replace with 5ml Opti-MEM culture ...

Embodiment 2

[0057] Example 2 Analysis of the proliferation ability of human stromal stem cells transfected with recombinant T-STAR and Angl lentivirus

[0058] The hBMSCs transfected with the recombinant T-STAR and Angl double lentiviral vectors in Example 1 were subcultured in vitro by complete culture medium DMEM (containing 10% fetal bovine serum). % or more, it needs to be digested and passaged with 0.25% (mass volume ratio) trypsin (containing 0.05% EDTA, mass volume ratio). Calculate the cell population doubling level based on one per cell passage, figure 2 Among them, untransfected hBMSCs and hMSCs transfected with Angl alone (hBMSC-A) could no longer expand when subcultured to 120 days, and the growth stopped. However, the hBMSCs (hBMSC-T&A) transfected with T-STAR (hBMSC-T) and T-STAR and Angl alone can be expanded to more than 210 days, indicating that the hBMSCs transfected with T-STAR and Angl ( hBMSC-T&A) have good proliferation ability.

[0059] image 3 In the middle i...

Embodiment 3

[0060] Example 3 Detection of telomerase activity in human stromal stem cells transfected with recombinant T-STAR and Angl lentivirus

[0061] Telomerase PCR ELISA by Roche TeloTAGGG Telomerase PLUS Kit detection, take the hBMSC of the 1st generation obtained by subculture in Example 2, the hBMSC (hBMSC-A) of the 10th generation that was transfected with T-STAR (hBMSC-T) alone, and the 10th generation that was transfected with Angl alone The 10th generation of hBMSCs transfected with T-STAR and Angl (hBMSC-T&A) was tested for relative telomerase activity according to the instructions.

[0062] Figure 4 It is a comparison chart for detecting the relative telomerase activity of hBMSC. hBMSCs transfected with T-STAR alone (hBMSC-T) and hBMSCs transfected with T-STAR and Angl (hBMSC-T&A) had high telomerase activity, compared with untransfected hBMSCs and Angl-transfected hBMSCs alone The comparison of hMSCs (hBMSC-A) has obvious differences, in which the relative telomerase a...

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Abstract

The invention provides a preparation method of a human stem cell with prolonged service life and enhanced angiogenesis. The preparation method comprises the following steps of establishing gene segments of a RNA (ribonucleic acid) testis signal transduction and activating body and angiogenin-1; and recombining the gene segments to a virus expression vector. The service life of the cell can be prolonged by transfection of the human stem cell, the angiogenesis of the human stem cell is improved, and tumorigenesis is avoided. The invention also provides a kit of the human stem cell with the prolonged service life and the enhanced angiogenesis, and the human stem cell is expected to treat ischemic cardiovascular and cerebrovascular diseases well.

Description

technical field [0001] The present invention relates to a method for human stem cells with extended cell life and enhanced angiogenesis ability and the method for obtaining human stem cells with extended cell life and enhanced angiogenesis ability. In particular, the present invention relates to the construction of RNA testis signal transduction and activation The double-gene recombinant lentiviral vector of angiopoietin-1 and angiopoietin-1 enables human stem cells to overexpress RNA testis signal transducer and activator and angiopoietin-1; the human with extended cell life and enhanced angiogenesis ability of the present invention Stem cell proliferation activity and telomerase activity increase, prolong the life of human stem cells, promote the formation of new blood vessels, without tumorigenicity, it is expected to be used in the clinical treatment of ischemic cardiovascular and cerebrovascular diseases, improve the blood supply of damaged parts, Promotes the repair of d...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N5/10A61K35/12A61K35/30A61K35/545A61P9/00
Inventor 陈振洲汤浩
Owner 广州赛琅生物技术有限公司
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