Study On The Effect And Mechanisms Of P16 Depltion On Osteogenesis Induced By Estrogen Deficiency

Aim:To clarify the role and mechanism of p16 gene knockout in osteoporosis induced by estrogen deficiency.To determine whether the role of p16 gene knockout in correcting the loss of bone loss caused by estrogen deficiency is related to osteoclast bone resorption changes.To determine whether the reduction of bone formation caused by p16 gene knockout and estrogen deficiency was associated with changes in the proliferation and differentiation of bone marrow mesenchymal stem cells(BM-MSC).To clarify whether p16 gene knockout increases the bone mass of estrogen-deficient mice in relation to the inhibition of oxidative stress and cell senescence.To clarify the effect of p16 gene knockout on the expression of apoptosis-related proteins in estrogen-deficient mice,Methods:We used ovariectomy(OVX)and sham operation(WT)to establish WT-Sham,WT-OVX,p16-/---Sham and p16-/--OVX mice.And mice were killed and the samples were collected at 12 weeks postoperatively.The difference of bone phenotype in the above four groups were analyzed by imaging,histopathology and molecular biology.The changes of osteoblast differentiation-related genes and protein expression were analyzed by real-time quantitative RT-PCR and Western blot.We used the anti-tartrate staining and RealtimeRT-PCR analysis to examine the osteoclast level and the regulation of osteoblast differentiation and activity variety.We cultured the BM-MSCs and examined the changes of methyl-blue-positive fibroblast colony forming units(CFU-f)and ALP-positive CFU-f area and BM-MSC proliferation..We measured the changes of ROS level and the expression level of antioxidant enzyme SOD 1/2 protein in mouse bone tissue.The changes of senescent cells were detected by histochemical staining of alactosidase(?-gal).We detected the changes of cell expression and apoptosis-related protein expression by Realtime RT-PCR and western blot expremeriments.Results:The results showed that bone mineral density,trabecular bone volume,osteoblast number,alkaline phosphatase(ALP)and type I collagen positive area in p16-/--Sham mice,as well as Runx2 Protein expression levels and Runx2,ALP,type I collagen and osteocalcin were significantly increased compared with WT-Sham mice,while the above indicators in WT-OXX mice were significantly decreased when compared to WT-Sham mice,and were no significant difference in p16-/--OVX mice.And the above-mentioned indexes were significantly lower in p16-/--OVX mice when compared with WT-OVX mice.These results demonstrate that p16 gene knockout can not only increase the bone mineral density,bone mass and total collagen deposition in normal mice,but also increase osteoblast formation and correct bone loss caused by estrogen deficiency.The results showed that the relative expression levels of RANKL and RANKL/OPG were significantly increased in WT-OVX mice compared with WT-Sham mice,and decreased in p16-/--Sham mice,while in p16-/--OVX mice have increased.Compared with WT-OVX mice,the above indicators were significantly lower in p16-/--OVX mice.The results showed that the percentage of methyl-blue-positive CFU-f area,the percentage of ALP-positive CFU-f area and the percentage of EDU-positive cells in WT-OVX mice were significantly lower than those in WT-Sham mice,and were significantly increased in p16-/--Sham mice,while no significant difference in p16-/--OVX mice.Compared with WT-OVX mice,the above indicators were significantly increased in p16-/--OVX mice.The results showed that the percentage of ROS and the percentage of p-gal positive cells in bone tissue were significantly higher in WT-OVX mice than in WT-Sham mice,and decreased significantly in p16-/--Sham mice,while no significant difference in p16-/--OVX mice.Compared with WT-OVX mice,the percentage of ROS and the percentage of ?-gal positive cells in bone tissue were significantly lower in p16-/---OVX mice.Compared with WT-Sham mice,the protein levels of SOD1 and SOD2 in bone tissue were significantly down-regulated in WT-OVX mice,and were significantly up-regulated in p 16-/--Sham mice,but no significant difference in p16/--OVX mice.Compared with WT-OVX mice,the percentage of ROS and the percentage of ?-gal positive cells in bone tissue were significantly up-regulated in p16-/--OVX mice.The results showed that the protein expression levels of p53,p19 and p16 were significantly up-regulated in WT-OVX mice compared with WT-Sham mice,while the expression of apoptotic protein Caspase3 was up-regulated and the expression of anti-apoptotic protein Bcl-2 was significantly down-regulated.In p16-/--Sham mice,the protein expression of Bcl-2,p53 and p19 were significantly up-regulated,while the expression of apoptotic protein Caspase3 was down-regulated.In p16-/--OVX mice,the expression of Bcl-2 was significantly down-regulated,while the protein expression levels of Caspase3,p53 and p19 were significantly up-regulated.Compared with WT-OVX mice,the protein expression levels of p53 and p19 were down-regulated in p16-/--OVX mice,while the expression of Bcl-2 was up-regulated,the expression levels of Caspase3 was down-regulated.Conclusion:The results of this study demonstrated that p16 gene knockout can promote osteoblast bone formation by promoting the bone marrow mesenchymal stem cells proliferation and osteoblast differentiation,At the same time,through inhibiting oxidative stress and cell senescence and osteoclast bone resorption,to play a correction Estrogen deficiency caused by the role of osteoporosis.