Based On Aging Related Genes To Explore The Mechanism Of Bushen Huayu Shengxin Decoction On The Regulation Of The Senescence Of BMSCs

Objective:Based on aging related genes p16^INK4a,p53,p21 to explore the mechanism of Bushen Huayu Shengxin Decoction on the regulation of the senescence of Bone Mesenchymal Stem Cells（BMSCs）.Methods:60 SD rats were randomly divided into two groups,Bushen Huayu Shengxin Decoction group and saline group,for the preparation of drug containing serum;SD rats（3 weeks old）were extracted,cultured,purified and identified by BMSCs,taking good state of the third generation CD105⁺-BMSCs,and senescence model was established with 6g/L D-gal induced.Cells for natural aging group were randomly divided into blank group,blank TCM group,blank saline group,aging model group were randomly divided into model group,model TCM group,model saline group;the morphological changes of BMSCs in each group were observed under light microscope;MTT method was used to determine the optimum concentration of serum to promote the differentiation and proliferation of BMSCs;Real-time fluores cence quantitative PCR technology to detect the expression of CD105⁺-BMSCs p16^INK4a,p21,p53 mRNA in different generations.Results:（1）Under the light microscope,the young BMSCs form a spindle or triangle,vortex like distribution,rapid growth,good refraction;aging BMSCs present flat triangle,oval,and slow growth,the refractive index difference;（2）According to the detection of MTT method can be known,The best concentration of promoting the differentiation and proliferation of BMSCs was 10%;（3）Real time fluorescent quantitative PCR results display: In the third generation of BMSCs,compared with each other among the blank group,model group,model TCM group,model saline group,p16^INK4a,p53,p21 mRNA expression levels was not statistically significant（P>0.05）;in the sixth generation of BMSCs,compared with the blank group,model saline group and blank saline group′s p16^INK4a,p53,p21 mRNA expression level differences were of statistical significance（P<0.05）,the expression level differences of blank TCM group and model TCM group was not significant（P>0.05）;Compared with model group,there was significant difference in the expression of the model TCM group（p<0.01）,but no statistical significance in the expression of model saline group（P>0.05）;and compared with each other among the model TCM group and model saline group,blank TCM group and blank saline group,the differences were statistically significant.In the seventh generation of BMSCs,compared with the blank group,model group,model TCM group,model saline group and blank saline group p16^INK4a,p53,p21 mRNA expression level differences were of statistical significance（P <0.05）,the expression level differences of blank TCM group was not significant（P>0.05）;Compared with model group,there was significant difference in the expression of the model TCM group（p<0.05）,but no statistical significance in the expression of model saline group（P>0.05）;and compared with each other among the model TCM group and model saline group,blank TCM group and blank saline group,the differences were statistically significant（P<0.05）.In the ninth generation of BMSCs,compared with the blank group,the expression level differences of blank TCM group and was not significant（P>0.05）,model group,model TCM group,model saline group and blank saline group′s p16^INK4a,p53,p21 mRNA expression level differences were of statistical significance（P<0.01）;Compared with model group,the expression level of p16^INK4a mRNA in model TCM group was no statistical significance（P>0.05）,the expression level of p53,p21 mRNA in the same group were of statistical significance（P<0.05）;and compared with the model TCM group,there was no tatistical significance in the expression of model saline group’s p16^INK4a mRNA（P>0.05）,the expression of p53,p21 mRNA was opposite（P<0.05）.Conclusion:（1）Under the condition of natural aging and successful aging model,Bushen Huayu Shengxin Decoction can delay the senescence of BMSCs,but it is not obvious for young BMSCs;（2）Bushen Huayu Shengxin Decoction can decrease the expression of aging related genes p16^INK4a,p53,p21,thus delaying the senescence of BMSCs,but with the extension of aging time,the acting force gradually weakened,and to a certain extent,this kind of aging is irreversible;（3）Regulating the aging by Bushen Huayu Shengxin Decoction is closely related to p16/pRb and p53/p21 cell signaling pathways.