Application of reagent capable of overexpressing muscle phosphofructokinase in preparation of medicine for delaying cell senescence

A technology of phosphofructokinase and cell aging, applied in the field of genetic engineering

Pending Publication Date: 2021-11-12
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since cellular senescence is accompanied by many progressive metabolic changes and functional decline ...

Method used

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  • Application of reagent capable of overexpressing muscle phosphofructokinase in preparation of medicine for delaying cell senescence
  • Application of reagent capable of overexpressing muscle phosphofructokinase in preparation of medicine for delaying cell senescence
  • Application of reagent capable of overexpressing muscle phosphofructokinase in preparation of medicine for delaying cell senescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Establishment of rat MSCs replicative senescence model in vitro

[0062] 1.1 Separation and acquisition of primary cells

[0063] Rats were placed in the laboratory environment for 2 to 3 hours to adapt to the laboratory environment. After anesthesia, they were killed by taking off their necks. The whole body was disinfected with povidone iodine and deiodized with 75% alcohol. This was repeated 3 times, and the bilateral humerus and femur of the rat were quickly separated. And the tibia, soak the bone with PBS containing 1% penicillin-streptomycin, and quickly move it into the ultra-clean bench. Remove the muscles and fascia attached to the surface of the bone in an ultra-clean bench, place the bone in complete culture solution; remove both ends of the bone with a rongeur, use a 5ml syringe to absorb the culture solution and rinse the bone marrow cavity repeatedly until the bone marrow cavity becomes translucent. Can. Use a 5ml pipette to gently suck up the bone marro...

Embodiment 2

[0096] Detection of glucose metabolism level and PFKM expression in aging MSCs

[0097] 2.1 Detection of glucose consumption (glucose detection kit, built in Nanjing)

[0098] Divide EPMSCs and LPMSCs according to 4×10 3 The density of cells / well was cultured in 96-well plate in 5% CO 2 , 37 ℃ cell constant temperature incubator. After the cells adhered to the wall, the original culture medium was discarded, 50 μl of serum-free DMEM / F-12 was added to each well, and the culture supernatant was collected the next day. The supernatant was at room temperature, 1200prm, centrifuged for 3 minutes, discarded the precipitate, and collected the supernatant to another EP tube for testing;

[0099] According to the kit instructions, the specific steps are shown in Table 6:

[0100] Table 6 Glucose consumption detection operation flow

[0101]

[0102] Mix the liquid evenly and place it in a water bath at 37°C for 5 minutes to react, then use a spectrophotometer to detect the ...

Embodiment 3

[0130] Infect senescent MSCs with lentivirus to obtain improved senescent MSCs

[0131] Construction of improved senescent MSCs with high expression of PFKM

[0132] A lentivirus stably expressing PFKM was constructed by Shanghai Jikai Gene Medical Technology Co., Ltd. (with GV358 vector: Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin as the backbone vector, and a double enzyme digestion method (AgeI / AgeI digestion) ) construct the PFKM gene on this vector). The titer of PFKM overexpressing lentivirus was 3E+8. Determine the optimal multiplicity of infection, set the PFKM lentivirus infection gradient to infect aging MSCs according to 50, 100, 150, 200 and 250, and the MOI value with the infection efficiency greater than 80% and the least cytotoxicity after 48 hours of culture is the best MOI value, and the best MOI value is used. MOI=200 was used for follow-up experiments. After 72 hours of culture, LV-PFKM of the experimental group was obtained, and senescent MSCs of the same st...

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Abstract

The invention relates to application of a reagent capable of overexpressing muscle phosphofructokinase in preparation of a medicine for delaying cell senescence, and belongs to the technical field of gene engineering. The invention provides the application of the reagent capable of overexpressing the muscle phosphofructokinase in the preparation of the medicine for delaying the cell senescence. The invention discusses by focusing on that PFKM affects an MSCs glycolysis level to regulate and control the cell senescence, the PFKM and MSCs senescence are organically linked for the first time, an experimental basis is provided for clarification of a regulation and control mechanism of stem cell senescence, the number and the functions of stem cells are favorably maintained, and the invention has important significance in delaying body senescence and preventing and treating various aging diseases.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of a reagent for overexpressing muscle-type phosphofructokinase in the preparation of medicines for delaying cell aging. Background technique [0002] In modern society, the average life expectancy of human beings is gradually extended, and the social problem of population aging appears accordingly. [1] , the aging of the individual is one of the important factors leading to the increasingly serious aging of the population. Aging is a complex phenomenon dictated by natural rules. The aging of the body is regulated by multiple factors and genes. As time goes by, the ability of cell proliferation and differentiation and physiological functions gradually decline, and the ability to maintain homeostasis and regeneration weaken [2] . With the aging of individuals, the incidence of aging-related diseases such as diabetes and cardiovascular and cerebrovasc...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P39/00C12N5/10C12N15/867
CPCA61K45/00A61P39/00C12N5/0662C12N15/86C12N9/1205C12Y207/01011C12N2510/00C12N2740/15043C12N2800/107
Inventor 何旭孙慧孙亚楠于晓高星宇石英爱张海英
Owner JILIN UNIV
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