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Application of SIRT3 to promotion of rejuvenation of adipose-derived mesenchymal stem cells of old people

A mesenchymal stem cell and elderly technology, applied in the application field of SIRT3 in promoting the rejuvenation of adipose-derived mesenchymal stem cells in the elderly, can solve the problems of low survival rate of ADSC and limit the treatment effect, and achieve the increase of expression level and protein expression level , the effect of reducing the apoptosis rate

Pending Publication Date: 2018-06-22
FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also certain problems. The low survival rate of ADSCs implanted in the ischemic area severely limits the therapeutic effect.

Method used

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  • Application of SIRT3 to promotion of rejuvenation of adipose-derived mesenchymal stem cells of old people
  • Application of SIRT3 to promotion of rejuvenation of adipose-derived mesenchymal stem cells of old people
  • Application of SIRT3 to promotion of rejuvenation of adipose-derived mesenchymal stem cells of old people

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 lentiviral LV5-SIRT3 plasmid construction

[0031] 1. Experimental materials

[0032] The amino acid sequence of the SIRT3 protein and the base sequence encoded by it can be referred to or selected from the prior art. In the present invention, the SIRT3 target gene is selected, as shown in SEQ ID NO: 1, but it is not a limiting condition of the present invention. The SIRT3 target gene can be commissioned to be synthesized by a commercial company.

[0033] The lentiviral vector can refer to or be selected from the prior art, and can also be commissioned or purchased from a commercial company. In the present invention, the lentiviral LV5 shuttle plasmid is selected, and the plasmid structure is as follows figure 1 shown. The LV5 shuttle plasmid contains the reporter gene GFP. Lentivirus LV5 is not a limitation of the present invention.

[0034] 2. Construction method

[0035] The SIRT3 target gene was introduced into the lentiviral LV5 shuttle plasmid ...

Embodiment 2

[0039] Example 2 Preparation of lentivirus LV5-SIRT3

[0040] 1. Lentiviral packaging

[0041] 1) Culture 293T cells in a 10cm culture dish until 80-90% confluent. Digest with Trypsin-EDTA solution, inoculate the cell suspension into a 15 cm culture dish, add 18 mL of DMEM (containing 10% FBS) culture solution, mix well and incubate overnight at 37°C.

[0042] 2) Add 1.5mL serum-free DMEM to a 5mL centrifuge tube, add lentiviral LV5-SIRT3 shuttle plasmid and packaging plasmid (pGag / Pol, pRev, pVSV-G) in proportion, mix well, take another 5mL centrifuge tube, add 1.5mL serum-free DMEM, then add 300μL RNAi-Mate, mix well, place at room temperature for 5min, mix the two tubes, and place at room temperature for 20-25min.

[0043] 3) Remove the culture medium in the 15cm culture dish, and add 8mL of serum-free DMEM culture medium. Add the transfection mixture dropwise to the petri dish, shake and mix the complex, and incubate at 37°C for 4-6h. The transfection solution was remo...

Embodiment 3

[0053] Example 3 Preparation of Aged Adipose-derived Mesenchymal Stem Cells (OhASC)

[0054] 1. OhASC isolation and culture

[0055] 1) Adipose tissue from the greater omentum of elderly patients (over 60 years old) was aseptically collected, soft tissues and small blood vessels were removed, and blood cells and tissue fragments were removed by washing with PBS. Cut into small pieces with ophthalmic scissors, digest with 1.0 g / L type IV collagenase, and stop digestion by adding DMEM (containing 10% FBS) culture solution.

[0056] 2) Centrifuge the digestive juice at 3000 rpm for 10 min. Discard the supernatant, add DMEM (containing 10% FBS) culture solution, centrifuge again, and repeat 3 times. Mix the cells with culture medium by blowing and blowing, and press the density of 1×10 5 / cm 2 Inoculate the culture flask and incubate at 37°C for 48h.

[0057] 3) When the cells reach 80% confluence, they are digested with 2.5g / L trypsin (EDTA) and subcultured, and can be used ...

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Abstract

The invention relates to application of SIRT3 to promotion of rejuvenation of adipose-derived mesenchymal stem cells of old people. An SIRT3 gene is induced into the adipose-derived mesenchymal stem cells of the old people through a lentiviral vector; over-expression of the SIRT3 is adopted so that the adipose-derived mesenchymal stem cells of the old people are improved very well in the aspects of stress oxidization, oxidative damages, cell apoptosis and the like. A regulation and control mechanism can be used for promoting the rejuvenation of the adipose-derived mesenchymal stem cells of theold people and a thought is provided for treating diseases including ageing, cardiovascular diseases, cancers and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of SIRT3 in promoting the rejuvenation of adipose mesenchymal stem cells in the elderly. Background technique [0002] Sirtuin (SIRT) protein is a mitochondrial NAD-dependent class III sirtuin with sirtuin activity. The mammalian SIRT protein family includes 7 members (SIRT1-SIRT7), and they are located in different organelles. SIRT1, SIRT6, and SIRT7 are localized in the nucleus, SIRT2 is localized in the cytoplasm, and SIRT3, SIRT4, and SIRT5 are localized in the mitochondria. SIRT proteins are involved in biological processes such as cell proliferation, aging, apoptosis, oxidative stress, metabolism regulation, and cell life extension. SIRT3 is highly expressed in metabolically active tissues, such as heart, muscle, liver, and brown adipose tissue. [0003] The lentiviral expression vector contains the genetic information required for packaging, transfection, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867A61K48/00A61P9/10A61P35/00A61P39/06
CPCA61K48/005C12N15/86C12N2740/15043
Inventor 李良夏云峰
Owner FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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