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Application of human sirt3 gene in stem cell differentiation inducing

A technology for inducing differentiation and stem cells, which is applied in the field of protein research and application mechanism, and can solve problems such as expression reduction

Pending Publication Date: 2020-02-14
广州溯原生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Similarly, exercise, diet and energy restriction can enhance the expression activity of sirt3; while a long-term high-fat diet will reduce its expression

Method used

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  • Application of human sirt3 gene in stem cell differentiation inducing
  • Application of human sirt3 gene in stem cell differentiation inducing
  • Application of human sirt3 gene in stem cell differentiation inducing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of embodiment 1 sirt3 gene and plasmid construction

[0033] 1. Experimental materials and methods

[0034] 1.1 Cloning of sirt3 gene

[0035] Use primers to amplify the human sirt3 target gene, and purify and recover the PCR product. The specific operation steps are as follows:

[0036] sirt3 primer sequence: F: 5′-ATCGATGGGCTTGAGAGAGT-3′

[0037] R: 3′-AGGTTCCATGAGCTTCAACC-5′

[0038] 10×PCR Buffer 5.00 μl DNA polymerase 0.25 μl dNTPs (2.5mM) 2.00 μl MgCl 2

1.50 μl UL43-for (10μM) 1.00 μl UL43-rev (10 μM) 1.00 μl template 4.00 μl wxya 2 o

12.75 μl total 25.00 μl

[0039] PCR condition setting: 94°C for 5 min, 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 30 s, 30 cycles; 72°C for 10 min. After PCR, the PCR products were stored at 4°C and separated and identified by 1% agarose gel electrophoresis.

[0040] 1.2. Recovery and purification of PCR products

[0041] PCR p...

Embodiment 2

[0074] Embodiment two transfection and expression of sirt3 gene

[0075] 1. Experimental materials and methods

[0076] 1.1. Lipofectamine transfection

[0077] Take 6-8 week male SD rats, take femoral bone marrow on both sides, prepare it into a single cell suspension, and inoculate the isolated cells into L-DMEM medium, the composition of which is fetal bovine serum (FBS), volume fraction is 0.1. Cultured in an incubator under the following conditions: 37°C, 5% CO 2 . Cells can be purified by the differential attachment method, and subcultured when 90% confluent. Take the third generation MSC S For transfection, refer to the optimized transfection conditions after screening: cell density 80%-90% confluence, DNA concentration 6.0 μm / ml, DNA:Lipofeetamine 2000 (trg:trL)=3:4. Six hours after transfection, the L-DMEM medium containing FBS (volume fraction 0.1) was replaced and the incubation was continued. Cells were collected 48 h after transfection.

[0078] 1.2. Weste...

Embodiment 3

[0085] Example 3 Effect of sirt3 on cells

[0086] 1. Experimental materials and methods

[0087] 1.1. Mitochondrial fluorescence staining

[0088] After the plasmid pc-DNA3.1-sirt3 was transfected into MSCs cells, they were cultured in a CO2 incubator at 37 °C for 4 h, and the anti-serum-free medium was replaced with normal medium to continue the culture. Mitochondria were stained with MitoTracker Green FM (Invitrogen) around 48 h, and the green fluorescence was observed with a fluorescence microscope and photographed.

[0089] 1.2. Determination of ATP content in cells

[0090] Intracellular ATP content was measured using a luciferin-luciferase reaction with the ATP Bioluminescence Somatic Assay Kit (Sigma). Cells were harvested at 0, 12, 24, 36, 48 h and suspended in KRH buffer containing 0.1 mM glucose and 0.2% BSA. and incubated at 37°C in a shaking water bath. Luminescence was measured immediately after adding the assay mix containing luciferin and luciferase in a b...

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Abstract

The invention discloses application of a human sirt3 gene or a sirt3-like gene or a product thereof in stem cell differentiation inducing, and provides a method for promoting stem cell differentiationor mitochondrial biogenesis increase. The high-expression sirt3 protein can significantly enhance aerobic metabolism of stem cells and promote conversion of stem cell metabolism from glycolysis to oxidative phosphorylation; the sirt3 improves the biogenesis of stem cell mitochondria, and is beneficial to inducing stem cell differentiation. After the stem cells are induced and differentiated by the sirt3, the stem cells can be developed into various tissues and used for artificial organ regeneration, for example, bone marrow mesenchymal stem cells are differentiated into osteoblasts used for bone tissues for 3D printing.

Description

technical field [0001] The invention belongs to the field of protein research and application mechanism, more specifically, the invention relates to the application of human sirt3 gene or sirt3-like gene or its product in stem cell induced differentiation. Background technique [0002] Stem cells are primitive cells that play the role of "backbone" in the growth and development of biological individuals, and are cell populations with self-renewal, high proliferation and multidirectional differentiation potential. On the one hand, stem cells differentiate in an orderly manner with time as the main axis to form different types of cells; on the other hand, they proliferate, migrate, arrange and combine with other types of cells strictly according to the overall structural blueprint predetermined by morphogenesis to form various types of cells. tissues and organs until they develop into complete individuals. [0003] According to the stage of development, stem cells can be divi...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0654C12N2501/73C12N2506/1346
Inventor 孙晗笑利时雨
Owner 广州溯原生物科技股份有限公司
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