阅读说明：本技术 维生素C在抑制prelamin A表达以及促进细胞增殖与延缓细胞衰老中的应用 (Application of vitamin C in inhibiting prelamin A expression, promoting cell proliferation and delaying cell aging ) 是由 谢晓华 曲延钕 江小霞 于 2019-11-22 设计创作，主要内容包括：本发明公开了维生素C在抑制prelamin A表达以及促进细胞增殖与延缓细胞衰老中的应用。本发明的实验发现,维生素C可以抑制细胞中的prelamin A蛋白,还可以促进表达prelamin A的间充质干细胞的增殖、延缓表达prelamin A的细胞衰老,也可以抑制表达prelamin A的细胞中衰老相关转录蛋白的表达、促进表达prelamin A的细胞中端粒遮蔽复合物的表达、抑制衰老相关炎性因子的表达、提高细胞外基质I/III型胶原表达,促进细胞周期相关因子的表达。表明,维生素C可以应用于延缓细胞的衰老与促进细胞的增殖。(The invention discloses an application of vitamin C in inhibiting prelamin A expression, promoting cell proliferation and delaying cell senescence. Experiments show that vitamin C can inhibit prelamin A protein in cells, can promote proliferation of mesenchymal stem cells expressing prelamin A, delay senescence of cells expressing prelamin A, can inhibit expression of senescence-related transcription protein in cells expressing prelamin A, promote expression of telomere shielding compounds in cells expressing prelamin A, inhibit expression of senescence-related inflammatory factors, improve expression of extracellular matrix I/III collagen, and promote expression of cell cycle-related factors. The vitamin C can be applied to delaying the aging of cells and promoting the proliferation of the cells.)

1. The application of vitamin C in preparing the products for inhibiting the expression of prelamin A protein.

2. The use of vitamin C in inhibiting the expression of prelamin A protein.

3. Application of vitamin C in preparing product for promoting cell proliferation is provided.

4. Use of vitamin C for promoting cell proliferation.

5. Application of vitamin C in preparing product for delaying cell aging is provided.

6. The application of vitamin C in delaying cell aging is provided.

7. Use according to any one of claims 3 to 6, characterized in that: the cells are cells expressing prelamin A protein.

8. Any one of the following uses of vitamin C:

x1, inhibiting the expression of senescence-associated transcription factors in cells;

x2, promoting telomere masking complex expression in cells;

x3, promoting extracellular matrix type I/III collagen expression;

x4, inhibiting the expression of senescence-associated inflammatory factors in cells;

x5, promoting cell cycle-associated factor expression in a cell;

x6, preparing an expression product for inhibiting the aging-related transcription factor in cells;

x7, preparing a product for promoting the expression of the telomere shielding compound in the cells;

x8, preparing a product for promoting the expression of extracellular matrix I/III collagen;

x9, preparing products for inhibiting the expression of the inflammatory factors related to the aging in the cells;

x10, preparing the product for promoting the expression of cell cycle related factors in cells.

9. Use according to claim 8, characterized in that: the cells are cells expressing prelamin A protein.

10. A product having any one of the functions Y1-Y3, comprising vitamin C:

y1, inhibiting the expression of prelamin A protein;

y2, promoting cell proliferation;

y3, delaying cell aging.

Technical Field

The invention relates to the application of vitamin C in inhibiting prelamin A expression, promoting cell proliferation and delaying cell senescence in the field of biotechnology.

Background

With the increase of the life of human beings and the proportion of the aged population, how to delay aging, inhibit the occurrence and development of aged diseases, improve the quality of life and reduce the burden of the national people becomes the key point to be solved urgently in the field of scientific research at present.

Research shows that abnormal deposition of aging-related molecules often causes cell function reduction and tissue repair capacity reduction, and aging-related factors can also start peripheral cell inflammatory reaction and distant cell tissue dysfunction to aggravate aging. The deposition of presenilin and prelamin a in the nucleus leads to cellular senescence. The presenilin and prelamin A are mainly distributed in the body, especially blood vessel smooth muscle and mesenchymal stem cell. It has been confirmed that abnormal genetic changes are one of the important factors for inducing senescence. The presenilin and prelamin A are also expressed in the aged cells of normal people, and the cell structure function metabolism is recovered after the expression is eliminated. Therefore, how to effectively reduce the expression of presenilin and prelamin A has important significance for delaying senility and reducing disease occurrence.

Vitamin C, as an antioxidant, can prevent excessive oxidation of unsaturated fatty acids and scavenge free radicals, and is widely used in the fields of foods, medicines and cosmetics. Meanwhile, the vitamin C can play a role in killing tumors in the treatment of cancers.

Disclosure of Invention

The technical problem to be solved by the invention is how to inhibit the expression of prelamin A protein.

In order to solve the technical problem, the invention provides any one of the following applications:

1. the application of vitamin C in the preparation of a product for inhibiting the expression of prelamin A protein;

2. the application of vitamin C in inhibiting the expression of prelamin A protein;

3. the application of vitamin C in preparing products for promoting cell proliferation;

4. the application of vitamin C in promoting cell proliferation;

5. the application of vitamin C in preparing products for delaying cell aging;

6. the application of vitamin C in delaying cell aging is provided.

In the above application, the cell may be a cell expressing prelamin A protein.

The cell may be specifically a pluripotent stem cell expressing a prelamin a protein. In one embodiment of the invention, the cell is a mesenchymal stem cell expressing a prelamin a protein.

Any of the following uses of vitamin C are also within the scope of the present invention:

x1, inhibiting the expression of senescence-associated transcription factors in cells;

x2, promoting telomere masking complex expression in cells;

x3, promoting extracellular matrix type I/III collagen expression;

x4, inhibiting the expression of senescence-associated inflammatory factors in cells;

x5, promoting cell cycle-associated factor expression in a cell;

x6, preparing an expression product for inhibiting the aging-related transcription factor in cells;

x7, preparing a product for promoting the expression of the telomere shielding compound in the cells;

x8, preparing a product for promoting the expression of extracellular matrix I/III collagen;

x9, preparing products for inhibiting the expression of the inflammatory factors related to the aging in the cells;

x10, preparing the product for promoting the expression of cell cycle related factors in cells.

In the above application, the senescence-associated transcription factor may be p 21.

The telomere masking complex can be POT1, RAP1A, or TERF 1.

The senescence-associated inflammatory factor can be TNF α or IL1 β.

The cell cycle related factor may be CCND1, CCNE, CDK6 or CDK 2.

In the above application, the cell may be a cell expressing prelamin A protein.

The cell may be specifically a pluripotent stem cell expressing a prelamin a protein. In one embodiment of the invention, the cell is a mesenchymal stem cell expressing a prelamin a protein.

The invention also provides a product with any one of the functions of Y1-Y3, wherein the product contains vitamin C:

y1, inhibiting the expression of prelamin A protein;

y2, promoting cell proliferation;

y3, delaying cell aging.

The active ingredient of the product may be vitamin C.

In the above product, the cell may be a cell expressing prelamin A protein. The cell may be specifically a pluripotent stem cell expressing a prelamin a protein. In one embodiment of the invention, the cell is a mesenchymal stem cell expressing a prelaminA protein.

In the present invention, the concentration of vitamin C in the system may be 50 to 5000. mu.M in practical use. Further, the concentration of vitamin C in the system may be 100-800. mu.M. Further, the concentration of vitamin C in the system may be 200-400. mu.M.

Experiments prove that the vitamin C can inhibit the prelamin A protein in cells, can also promote the proliferation of mesenchymal stem cells expressing prelamin A, delay the aging of cells expressing prelamin A, can also inhibit the expression of an aging transcription protein p21 in the cells expressing prelamin A, promote the expression of telomere masking complex POT1, RAP1A, TERF1 and TERF2 genes in the cells expressing prelamin A, inhibit the expression of aging-related inflammatory factors TNF α and IL1 β genes, promote the expression of extracellular matrix I/III type collagen NE, promote the expression of cell cycle-related factors CCND1, CCK, CDK6 and CDK2 genes.

Drawings

FIG. 1 shows the fluorescent expression of a virus expressing prelamin A and a control virus. Bar is 500 μm.

FIG. 2 shows the isolation of mesenchymal stem cells and the identification of stem cell surface antigens. The left panel shows primary cells crawled out of the bone fragments. The right panel shows the dry markers identifying positive expression of CD73, CD90, CD105 and CD44, and no expression of hematopoietic markers CD45 and CD 31.

FIG. 3 is a graph showing a significant increase in the deposition of prelamin A in MSCs following viral transfection. The upper left and middle panels are GFP fluorescence signals of cells transfected with negative control viruses and positive results detected by a flow cytometer; the lower left and middle panels are GFP fluorescence signals of cells transfected with a virus expressing prelamin A and positive results of flow cytometry detection, Bar is 200 μm; the right panel shows the results of western blot assays, with MSC/GFP showing cells transfected with negative control virus and MSC/PLA showing cells transfected with a virus expressing prelamin A.

Figure 4 shows that the intracellular deposition of prelamin a protein is significantly improved after vitamin C treatment. The left graph is the detection result of the western blot; the right panel shows the quantitative results of the expression level of prelamin A protein. MSC/PLA indicated no vitamin C addition, and MSC/PLA + VC indicated the medium vitamin C concentration of 200 u M results. P < 0.01.

FIG. 5 shows that the mesenchymal stem cell proliferation potency was improved at vitamin C concentrations of 200 and 400. mu.M. PLA shows no vitamin C, 100, 200, 400 show the concentration of vitamin C in the culture medium is 100, 200, 400. mu.M, p is less than 0.01.

FIG. 6 shows that Vitamin C (VC) significantly reduced the positive rate of X-GAL staining in the 50-800. mu.M interval. The upper panel shows the staining results and the lower panel shows the quantification results. PLA means no vitamin C added, 0, 25, 50, 100, 200, 400, 800, 5000 means the concentration of vitamin C in the medium is 0, 25, 50, 100, 200, 400, 800, 5000 μ M, × P <0.01, × P <0.001, × P <0.0001, respectively.

FIG. 7 shows that the expression level of the cell aging-related transcription factor p21 is significantly improved after vitamin C treatment. MSC/PLA means no vitamin C was added, MSC/PLA + VC means a concentration of vitamin C in the medium of 200. mu.M, p < 0.05.

FIG. 8 shows the amount of expression of telomeric masking complex genes in the marked cells after vitamin C treatment. MSC/PLA indicated no vitamin C addition, MSC/PLA + VC indicated medium vitamin C concentration of 200. mu.M,. about.p <0.0001.

Fig. 9 shows that vitamin C treatment significantly reduced the expression of senescence-associated inflammatory factors in cells. MSC/PLA indicated no vitamin C addition, MSC/PLA + VC indicated medium vitamin C concentration of 200. mu.M, p <0.01, p < 0.001.

FIG. 10 shows that the expression level of extracellular matrix is significantly increased after vitamin C treatment. MSC/PLA means no vitamin C was added, and MSC/PLA + VC means that the concentration of vitamin C in the medium was 200. mu.M,. p.ltoreq.0.05,. p.ltoreq.0.01.

FIG. 11 shows that the expression level of cell cycle-related factors is significantly improved after vitamin C treatment. MSC/PLA indicated no vitamin C addition, MSC/PLA + VC indicated the concentration of vitamin C in the medium was 200. mu.M, p < 0.05, p <0.01, p < 0.001.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

AD 293 cells: beijing Hesheng Gene science and technology, Inc.

The MSC culture medium is obtained by adding fetal bovine serum, penicillin and streptomycin into α -MEM (Gibco), wherein the concentration of the fetal bovine serum in the MSC culture medium is 10% (volume percentage content), and the concentrations of the penicillin and the streptomycin are both 1g/100 ml.

Vitamin C (L-ascorbic acid): sigma, A8960.