Effects Of Angiopoietin-like Protein 7(ANGPTL7)on Proliferation And Differentiation Of Mouse Preosteoblasts MC3T3-E1 And Its Mechanism

[Background] ANGPTL7 is one of the newly discovered angiopoietin like protein family members.At present,various researches of ANGPTL7 mainly focus on the regulation of angiogenesis,which can regulate angiogenesis by promoting the proliferation,migration and differentiation of vascular endothelial cells.In addition,more and more studies have revealed that ANGPTL7 plays an important role in the proliferation and differentiation of hematopoietic stem cells and smooth muscle cells.Recently,it has been found that there is a new capillary endothelial cell subtype in bone tissue,and it is believed that the occurrence of osteoporosis is related to the formation of blood vessels.So,can ANGPTL7 promote the formation of bone by regulating the formation of blood vessels in the bone tissue,and play a related role in the prevention and treatment of osteoporosis?This experiment will focus on this issue.Part ? Expression of ANGPTL7 in mouse preosteoblasts MC3T3-E1Objective: To study the expression of ANGPTL7 in MC3T3-E1 cells,and to provide experimental basis for further study of the effect of ANGPTL7 on the proliferation and osteogenic differentiation of MC3T3-E1 cells.Methods: MC3T3-E1 cells were cultured in osteogenic medium for osteogenic differentiation.The growth of cells at different time points of induced differentiation was observed under microscope,and the expression levels of ANGPTL7 were detected by RTPCR and Western blot assays.Results:(1).Microscopically,the cells grew close to the wall at 7 d and 3 d,and showed fibroblast like growth.The cells were plump,polygonal or fusiform.At 7 d,they were almost full,and the cells became slender and fusiform,and began to grow in multiple layers,with angular processes and cluster growth trends.At 14 d,the cells gathered into a multi-layered structure with multiple nodules and protuberances,and some calcified nodules were observed.At 7 d and 14 d,cell aggregation and mass growth were more obvious than at 0 d and 3 d.(2).The results of RT-PCR showed that in osteogenesis induced MC3T3-E1 cells,with the increase of induction time,the expression of ANGPTL7 m RNA increased gradually.The expression of ANGPTL7 m RNA reached the peak at 7 d,and continued to be highly expressed until the 14 d.The expression of ANGPTL7 m RNA at7 d and 14 d of induction was statistically significant different from that at 0 d and 3 d of induction(P<0.05).Western blot assays showed the similar results to those of RT-PCR.Conclusion: In this study,we found that the expression level of ANGPTL7 in osteogenic differentiated MC3T3-E1 cells was significantly increased,and it was timedependent.Part ? Effects of ANGPTL7 on proliferation and differentiation of MC3T3-E1 cellsObjective: To study the effect of ANGPTL7 on the proliferation and differentiation of MC3T3-E1 cells,and to provide experimental basis for further study on the effect of ANGPTL7 on osteoporosis.Methods: MC3T3-E1 cells were respectively transfected by the overexpressed plasmid with puromycin resistance of ANGPTL7 p MSCV-ANGPTL7 and the negative control p MSCV.The following groups were set up as follows:(1)blank control group;(2)p MSCV negative transfection control group;(3)p MSCV-ANGPTL7 overexpression transfection group.At 48 h post-transfection,the expression level of ANGPTL7 was detected by RT-PCR and western blot respectively,and the transfection efficiency was evaluated.CCK-8 was used to evaluate cell proliferation.Alkaline phosphatase(ALP)activity and alizarin red staining were used to detect the effect of ANGPTL7 on the differentiation and mineralization of osteoblasts.Finally,western blot was used to detect the expression levels of ALP,runt-related transcription factor 2(Runx2),Osteocalcin(OCN)and collagen I(Col I).Results:(1).The overexpressed plasmid of ANGPTL7 were transfected into MC3T3-E1 cells.RT-PCR and western blot were used to detect the expression of ANGPTL7 protein and m RNA in MC3T3-E1 cells.The results of RT-PCR and western blot showed that ANGPTL7 expression was significantly increased in both protein and m RNA levels in p MSCV-ANGPTL7 transfected MC3T3-E1 cells.(P<0.05).(2).To determine the effect of ANGPTL7 on the proliferation of MC3T3-E1 cells,CCK-8 assay was performed at 48 h post-transfection.The results showed that the proliferation ability of the three groups increased gradually with the prolongation of culture time;and ANGPTL7 overexpression markedly increased cell proliferation of MC3T3-E1 cells at 24,48 and 72 h.(P<0.05).(3).At 7 d post-transfection,the ALP activity was assessed.The results showed that compared with blank control group and negative transfection control group,the number of positive cells in ANGPTL7 overexpression group increased significantly(P<0.05),suggesting that ANGPTL7 significantly increased ALP activity.Moreover,the expression levels of osteoblast differentiation marks ALP,Runx2,OCN and Col I were measured by western blot.And the results suggested that overexpression of ANGPTL7 significantly increased ALP,Runx2,OCN and Col I expression(P<0.05).(4).The effect of ANGPTL7 on mineralization was determined using alizarin red-S staining at 21 d posttransfection.Compared with the negative transfection group,much more calcium deposition was found in p MSCV-ANGPTL7 transfected MC3T3-E1 cells.The activity of mineralization osteogenesis in the ANGPTL7 overexpressed group was increased compared with the control group and the negative transfection control group.Conclusion: After ANGPTL7 was transfected into MC3T3-E1 cells,the expression level of ANGPTL7 increased significantly,indicating that the transfection was successful.The overexpression of ANGPTL7 in MC3T3-E1 cells significantly increased the expression of ALP,Runx2,OCN and Col I as well as the formation of mineralized bone nodules,which are important markers of osteogenic differentiation and mineralization.Part ? The role of BMP signaling pathway in the promotion of proliferation and differentiation of MC3T3-E1 cells by ANGPTL7Objective: To explore the mechanism of ANGPTL7 promoting the proliferation and differentiation of MC3T3-E1 cells,and to provide theoretical basis for further study on the application of ANGPTL7 in the prevention and treatment of osteoporosis.Methods:(1).MC3T3-E1 cells were used as the research object,and the groups in the second part of this experiment were still used:(1)blank control group;(2)p MSCV negative transfection control group;(3)p MSCV-ANGPTL7 overexpression transfection group.Western blot was used to detect the expression of BMP proteins in BMP signalingpathway.(2).In order to further study the mechanism of BMP signaling pathway in promoting the proliferation,differentiation and mineralization of MC3T3-E1 cells,we added the BMP signaling pathway inhibitor LDN-193189,and reset the following groups:(1)blank control group;(2)ANGPTL7 overexpression group(Angptl7 group);(3)ANGPTL7overexpression + LDN-193189 group(Angptl7 + inhibitor).Two weeks after osteogenic differentiation,ALP staining and alizarin red staining were used to detect ALP activity and cell mineralization.Western blot was used to detect the expression levels of ALP,Runx2,OCN and Col I.Results:(1).At 7 d post-transfection,Western blot was used to detect the expression level of BMP proteins in BMP signaling pathway.The results showed that the expression level of BMP 2,4,6 and 7 in ANGPTL7 overexpression group was significantly higher than that in blank control group and negative transfection control group(P<0.05).And the results suggested that ANGPTL7 could promote the expression of BMPs in MC3T3-E1 cells.(2).The osteogenic differentiation ability of cells in the three groups were detected by ALP staining.The results showed that the number of staining positive cells in Angptl7 group was significantly higher than that in blank control group and Angptl7+inhibitor group(P<0.05),suggesting that the osteogenic differentiation ability of Angptl7+inhibitor group was significantly weaker than that of Angptl7 group.(3).At 21 d post-transfection,the effect of mineralization of cells was determined by alizarin red staining.The results showed that: compared with the control group,the calcium deposition in ANGPTL7 overexpression group increased significantly(P<0.05);after adding BMP signal pathway inhibitor LDN-193189,the number and depth of calcium nodule staining in Angptl7 +inhibitor group decreased significantly(P < 0.05).(4).Western blot was used to detect the expression of osteogenic differentiation related proteins in the three groups.Compared with the blank control group,the expression levels of ALP,Col I,OCN and Runx2 in Angptl7 group were significantly increased(P<0.05);after the BMP signal pathway inhibitor was added,the expression levels of ALP,Col I,OCN and Runx2 in the Angptl7+inhibitor were all significantly decreased(P<0.05).Conclusion: ANGPTL7 could directly promote proliferation,differentiation and mineralization of osteoblasts by activating the expression of BMP signal pathway.