| OBJECTIVE:Adult stem cell senescence is the newest theory of human aging and senile disease and development,so it is very important to find the key points to regulate the senescence of stem cells.More than 30 years of research has shown that traditional Chinese medicine "qi and blood" theory is closely related to stem cell function.Mesenchymal stem cells(MSCs)can proliferate and differentiate into a variety of tissue cells derived from mesoderm,and MSCs play an extremely important role in the study of modern medical tissue engineering.Team early have shown that ginseng is the doctor of traditional Chinese medicine clinical "tonifying qi to medicine,ginseng saponin Rg1 is its important anti-aging ingredients,it can delay the MSCs aging,antagonism to failure agent of oxidative damage caused by it.But what signal pathway does Rg1 regulate MSCs senescence? The mechanism is not clear,it needs to be further discussed.Recent studies have shown that the Wnt/?-catenin signaling pathway plays an important role in regulating stem cell senescence.Is the activation level of this signaling pathway related to the aging of human bone marrow mesenchymal stem cells(HBMSCS)? Is ginsenoside Rg1 antagonistic anti-aging agent to delay the aging of hBMMSCs and regulation of this pathway? To elucidate these problems,we studied the new mechanism of Rg1 regulating the aging of hBMMSCs in the communication with Wnt/?-catenin signaling pathway by hBMMSCs.The new theory of modern stem cell aging and the new mechanism of "supplementing qi" to delay the senescence of hBMMSCs will be explained in theory.It provides experimental basis for the application of effective components of traditional chinese medicine to regulate the aging of stem cells.To provide new ideas and technologies for stem cell senescence in technology;To promote multidisciplinary cooperation and young talents in discipline construction.METHODS:1.Human bone marrow biopsy and tissue morphometry: collecting normal bone marrow biopsy specimens from different age groups,excluding patients with hematopoietic system disease and other serious diseases.Bone marrow,Wright's.The morphology of bone marrow cells was observed under the microscope,and the morphology was determined by using SPSS.2.Human bone marrow mesenchymal stem cells(hBMMSCs)were isolated,purified and identified: the bone marrow was taken at different ages,and the individual nucleus cells(MNCS)were isolated and cultured in vitro,purified hBMMSCs.Flow cytometry was used to determine the expression of cell surface antigen markers CD34,CD45,CD90,CD73,CD11 b,CD19,CD105,CD14 and HLA-DR for cell identification.3.The hBMMSCs collected are divided into: < 25 years old group;25 to 35 age group;35 to 45 years old group;45 to 55 years old group;55-65 age group;> 65 years old group;> 65 years old+ Rg1 group.The following tests are conducted:(1)determination of the biological indicators of senescence: the proliferation ability of CCK-8 was detected.The ability of cell proliferation was detected by the.edu method.The ability of cell multidirectional differentiation was detected by induction culture,lipid-induced culture and chondrogenic induction culture.The degree of senescence of the cells was detected by the staining method of SA-?-galactosidase.(2)the Wnt/?-catenin signaling pathway related proteins and genetic testing: including ?-catenin protein localization and detection of expression level of GSK-3?,p-GSK-3?,TCF-4,LEF protein expression detection,?-catenin,GSK-3?,TCF-4,LEF,C-myc,Cyclin D1 mRNA expression detection.RESULTS:1.With the increase of age,the hematopoietic tissue in bone marrow decreased,the adipose tissue increased,and the bone trabecula showed no significant difference.2.Low expression of CD14,CD45,CD19,CD34 and HLA-DR in vitro adherent culture of h BMMSCs.CD11 b expression was expressed.High expression of CD73,CD105 and CD90.The expression level of the surface antigen markers of hBMMSCs in the international community;3.The proliferation of h BMMSCs gradually decreased with age,and the ability of osteogenesis and chondrogenic differentiation decreased,and the lipid capacity increased gradually.The number of positive cells was increased in SA-?-Gal.4.The Wnt/beta-catenin signaling pathway related protein expression level and mRNA level changes: hBMMSCs intracellular total beta catenin protein expression level increased,?-catenin protein to nuclear transfer,nuclear inside/outside the nuclear ?-catenin expression level increased with age;gsk-3? protein and mRNA levels declined;p-gsk-3?,TCF-4,LEF protein expression increased,TCF-4,LEF,c-myc,and CyclinD1 mRNA expression increased.5.Ginseng saponin Rg1 elderly hBMMSCs senescence detection: ginseng saponin Rg1 can significantly improve hBMMSCs proliferation,promote osteogenesis,and a concomitant differentiation into cartilage,SA-?-Gal staining positive percentage reduction cell,cell total ?-catenin protein expression levels drop,nuclear inside/outside the nuclear ?-catenin expression levels drop;Gsk-3? protein and mRNA levels increased;P-gsk-3?,TCF-4,LEF protein expression decreased,TCF-4,LEF,c-myc,and Cyclin D1 mRNA expression decreased.CONCLUSIONS:1.With the increase of age,hBMMSCs will gradually show the biological expression of senescence and increase the activation degree of Wnt/?-catenin signaling pathway;2.Ginsenoside Rg1 can significantly delay the aging of hBMMSCs.3.Ginsenoside Rg1 can be activated by inhibiting Wnt/?-catenin signaling pathway,thus regulating the aging of hBMMSCs. |
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