| Alzheimer’s disease(AD)is a progressive cognitive dysfunction and behavioral impairment as the main clinical manifestations of central nervous system degenerative diseases, which embodies memory ability and executive capability to reduce such behavior changes. With the increase of age, the incidence of AD is also increasing year by year. Our country is facing increasingly serious aging at present. There is 210 million over 60. Therefore, to seek effective prevention and treatment of AD therapy is a very important problem to be solved. Human umbilical cord mesenchymal stem cells(h UC- MSCs) is a kind of adult stem cells which has highly self-renewal and differentiation potential. It derived from umbilical cord Wharton glue(Wharton ’s Jelly) organizations with extensive sources, low immune response, safe and reliable using, no ethical issues and easy to cultivate in vitro, etc. h UC – MSCs is the seed cells of engineering and stem cell replacement therapy and brings the hope for the treatment of AD. Research group in early stage found h UC- MSCs transplantation to treat AD can improve the learning and memory function of AD and delay senescence. But with the increase of number of cell batches, stem cells is not only in form which shows characteristics of aging, but also whose proliferation and differentiation ability weakens later. Therefore, clinical and basic research more likely to choose 3 to 5 generations of stem cells for transplantation, limited number of cells greatly limit the use of stem cells. Therefore, the research in key genes which regulate stem cell aging and improve the stem cell microenvironment is the key to improve the activity of stem cells and the curative effect.p16 gene is a tumor suppressor gene which was discovered by Kamb, etc. in American cold spring laboratory in 1993. Meanwhile, it is also the key regulatory genes of the cell cycle. p16 can inhibit cycle D in combination with 4/6 cycle dependent protein kinase protein(CDK4 / CDK6), inhibit retinoblastoma(RB) phosphorylation, while not phosphorylation of RB can’t combine with E2 F start the replication of DNA, which will block the cell cycle at the G1 / S checkpoint, and results in the stagnation of the cell cycle. our research group in early stage found overexpression of p16 gene could lead to 293 cell aging. But the relationship between p16 and h UC-MSCs and AD is not clear. Therefore, this subject studies down-regulated expression of p16 in control of h UC-MSCs and APP+ rats aging through the vitro and vivo experiments. ObjectivesThis study put p16 gene as the breakthrough point. LV is a carrier. Using RNAi technology down-regulated expresses p16 gene in P15 h UC-MSCs, and then observe down-regulated expression of p16 gene to the influence of h UC-MSCs aging. At the same time, in the vivo experiment, we put APP+ rats as the research object and study P15 h UC-MSCs of down-regulated expression of p16 gene to the nerve function repair and the anti-aging effect of APP+ rats. Methods 1. Screening si RNA of targeted down-regulated expression of p16 gene and building LV and LV-sh RNA.Get cesarean and healthy neonatal umbilical cord, and cultivate and separate h UC-MSCs in vitro. Flow cytometry detected the expression of h UC-MSCs cell surface markers CD44, CD90, CD34 and CD45. Collect 3, 10, 15 generations of cells, q RT –PCR detected the expression of p16, and β-galactosidase staining detected the aging degree of h UC-MSCs of different generations. Screen the si RNA(sip16) which could interference the expression of p16 effectively and compound LV containing sip16(LV-shp16). Observe and detect the expression of green fluorescence and p16 gene infected by LV-shp16 in h UC-MSCs. 2. Down-regulated expression of p16 gene to the influence of h UC-MSCs agingThis vitro experiment was divided into four groups: P3 group, P15 group, LV group and LV-shp16 group respectively. CCK-8 detected cell proliferation of every group. PI dyeing and FITC dyeing detected cell cycle and cell proliferation of every group. β-galactosidase staining detected the cell aging state of every group. q RT-PCR and Western blot detected the expression of aging related genes(p16, p53, p21,p CNA, sirt1, sirt2) and cell cycle key genes(CDK4,CDK6, RB1, p- RB1). 3. P15 h UC-MSCs of down-regulated expression of p16 gene to the nerve function repair effect of AD rats.In the vivo experiment, take 40 APP+ male rats of 4 months which were divided into 5 groups: P3 h UC- MSCs transplantation group(P3 group), blank group(AD group), P15 h UC-MSCs transplantation group(P15 group), P15 h UC – MSCs infected by LV transplantation group(LV group) and P15 h UC-MSCs infected by LV-shp16 transplantation group(LV- shp16 group) respectively. Meantime, set up the same genetic background of non-genetically modified wild rats as normal control group(WT group). The rats of P3 group, P15 group, LV group and LV- shp16 group are treated by the means of tail vein injection of h UC-MSCs. The cell quantity of Every rat was injected was 1X106 cell/200 ul, the rats were injected once a week and 4 weeks continuously, and the other groups were injected physiological saline. Cell transplantation after 28 days, Morris water maze experiment detected mice behavior change. Blood samples were collected from mice heart, and TAB and hydroxylamine method was used to detect the activity of SOD and MDA in the mice serum. Immunohistochemical was used to detect the expression of MAB1281, DCX, βⅢ-Tubulin and APP in mice brain tissue. Extract RNA and protein of mice hippocampal tissue. q RT PCR and Western blot were used to detect the expression of cell cycle key genes(CDK4, CDK6, RB1 and p- RB1), aging related genes(p16, p53, p21, p CNA, sirt1 and sirt2), apoptosis related genes(Caspase 3 and Bcl- 2) and Tau, p-Tau. Results1. with the increase of number of batches, h UC-MSCs gradually showed the characteristics of flat form and taking off the wall easily.β- galactose glucoside enzyme positive cell rate increased significantly(P < 0.05), q RT-PCR results showed the expression of p16 m RNA increased gradually in P3, P10, P15 h UC-MSCs(P <0.05).2. The si RNA which could down-regulated express p16 effectively has been screened successfully. Compound LV containing sip16(LV-shp16) and infect h UC-MSCs successfully. Compared to P15 group, LV-shp16 could promote P15 h UC-MSCs proliferation, promote cell cycle into S phase, inhibit the late apoptosis of P15 h UC –MSCs, reduce the aging degree of h UC-MSCs(P < 0.05). Meanwhile, The expression of p16, p53, p21 m RNA and protein decreased in LV-shp16 group, The expression of RB1 was unchanged, and The expression of p CNA, sirt1, sirt2, CDK4,CDK6, p-RB1 m RNA and protein was increased(P < 0.05).3. During cells transplantation, the growth state of AD mouse was good which had no adverse reaction. Compared to AD group, the treatment groups had certain anti-aging effect. Compared to P15 group, Lantency and lantency distance of the rats reduce and cross platform and the first quadrant residence of the rats time increased in LV-shp16 group(P < 0.05). The activity of SOD elevated and the activity of MDA reduced in serum of LV-shp16 group(P < 0.05). The expression of p16, p53, p21, Caspase-3, p- Tau protein decreased, the expression of p CNA, sirt1, sirt2, CDK4, CDK6, p-RB1, Bcl-2 protein increased, the expression change of RB1, Tau protein was not obvious in the hippocampus tissue of LV-shp16 group(P < 0.05). In addition, the positive cells which expressed MAB1281 was obviously increased, the expression of DCX,βⅢ-Tubulin was increased, but the expression of APP was decreased in brain tissue of LV-shp16 group(P < 0.05). Conclusion1. With the increase of cell batches, h UC-MSCs started to appear aging, and the expression of p16 was positively associated with aging.2. Down-regulated expression of p16 in P15 h UC-MSCs could regulate cell cycle key genes(CDK4, CDK6, p- RB1) and aging related genes(p53, p21,p CNA, sirt1, sirt2). It could delay and improve h UC-MSCs senescence and promote stem cell proliferation.3. LV-shp16 could promote migration and Neural differentiation of h UC-MSCs, alleviate nerve cells apoptosis, and then improve the therapeutic effect of h UC-MSCs on APP+ rats through regulating the expression of CDK4, CDK6, p-RB1, p53, p21, p CNA,sirt1 and sirt2. |
PDF Full Text Request