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Construction method and application of hascs for overexpressing metallothionein

A metallothionein and construction method technology, applied in the field of hASCs construction, can solve problems such as unsolved convenience, cell content and expansion ability, and achieve the effects of stable biological characteristics, safe and reliable method, and advanced technology

Active Publication Date: 2015-12-30
SHENZHEN WINGOR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology does not solve the defects in the convenience of drawing materials, cell content and amplification ability.

Method used

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  • Construction method and application of hascs for overexpressing metallothionein
  • Construction method and application of hascs for overexpressing metallothionein
  • Construction method and application of hascs for overexpressing metallothionein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The acquisition, purification and amplification of embodiment 1 hASCs

[0027] (1) Isolation and primary culture of hASCs

[0028] Fat samples were obtained from healthy donors who underwent liposuction in cosmetic plastic surgery clinics. 20ml of adipose tissue obtained by liposuction was taken, hASCs were separated by collagenase digestion, and further purified by adherent culture.

[0029] The index standards for hASCs flow detection indicators and expression are:

[0030] CD105 must not be lower than 85%

[0031] CD73 must not be lower than 70%

[0032] CD90 must not be lower than 95%

[0033] CD45 must not exceed 2%

[0034] CD79a or CD19 must not exceed 2%

[0035] HLA-DR must not exceed 2%

[0036] CD34 must not exceed 2%

[0037] CD14 must not exceed 2%

[0038] The prepared cells fully complied with the standards of the above eight indicators, and reached a very high purity (above 95%) and good stability in the second generation. The lower the number ...

Embodiment 2

[0044] Example 2 Construction of an adeno-associated virus system carrying the MT gene

[0045] The MT sequence was introduced into the adeno-associated virus vector by DNA recombination technology, and the recombinant adeno-associated virus vector rAAV-hMT, packaging plasmid, and helper plasmid were co-transfected into host cells by calcium phosphate co-precipitation method to complete the packaging and expansion of the adeno-associated virus. increase. Determine the appropriate titer for AAV infection, the interval between multiple infections, the duration of infection, and the number of infections. Finally, the identification and gene sequencing of the adeno-associated virus were completed.

[0046] Both AV (adenovirus) and AAV (adeno-associated virus) can be used as vectors for gene therapy. At present, there are technologies that use AV to integrate MT to remove lead. AV vectors are easy to prepare and operate, have stable physical and chemical properties, and low genot...

Embodiment 3

[0047] Example 3 Recombinant adeno-associated virus rAAV-hMT infects hASCs to obtain MT-hASCs

[0048]RT-PCR technique was used to identify the expression of MTmRNA in MSCs. After the cells grow to 80% density, wash 3 times with PBS, add 1ml TRIzol reagent to extract the total RNA of the cells. A nucleic acid detector was used to detect the quality and concentration of RNA, and 2 μg of total RNA was taken and reverse-transcribed using a reverse transcription kit to obtain cDNA. Real-time quantitative PCR was used to identify hMTmRNA levels. At the same time, set 6 concentration gradients to make a standard curve. According to the standard curve, the relative contents of hMTmRNA and internal reference gene β-actinmRNA in cell line 1, cell line 2 and cell line 3 were calculated. Repeat the detection 3 times.

[0049] The results showed that the expression level of hMTmRNA in cell line 3 was about 8 times higher than that in cell line 1 and cell line 2 ( Figure 4 ).

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Abstract

The invention discloses a hASCs construction method of overexpressed metallothionein and an application thereof. The hASCs construction method of overexpressed metallothionein comprises the following steps: A) acquisition, purification and amplification of hASCs; B) construction of an adeno-associated virus vector carrying an MT gene; C) infection of hASCs with recombinant adeno-associated virus to obtain MT-hASCs. According to the method disclosed by the invention, the stem cell technology is combined with the advanced gene vector treatment means, a new method for improving the functions is explored, revolutionary change is brought to the traditional clinical medical technology, and the method plays an important role and is of profound significance.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a method for constructing hASCs capable of overexpressing metallothionein and its application Background technique [0002] Aging is an inevitable law of the human life process, but since ancient times, long life and eternal youth have been the beautiful wishes of people's dreams, and they are also issues of common concern to human beings today. Anti-aging research has also become a hot topic in physiological science in the 21st century. With the aging of the human body, the degradation of various functions is inevitable. How to comprehensively and effectively improve the functions of the human body and delay the aging process is a hot spot in current research. [0003] The application of stem cells in the field of anti-aging is in full swing. Scientists have confirmed that there are dozens of human cell metabolism promoting differentiation enzymes in the cells, which can imp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N5/10
Inventor 姜舒罗朝霞刘颖斐曹娜黄文婷
Owner SHENZHEN WINGOR BIO TECH
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