Study On The Biological Role Of SEMA3B In Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells In Elderly Patients With Osteoporosis

Objective: In order to observe the osteogenic differentiation effect of SEMA3 B on human bone marrow mesenchymal stem cells(h-BMMSC)in elderly patients with osteoporosis(SOP).Methods: We separate senile patients who were diagnosed with proximal femoral fractures into two groups,namely osteoporotic and non-osteoporotic group based on QCT bone mineral density(BMD).Bone marrow samples were taken from 10 people in each group.The whole bone marrow adherent method was used for cell culture,the cells were purified by limited dilution method,and the cell apparent identification of the obtained h-BMMSC was performed by flow cytometry.CCK-8 method was used to detect the proliferation ability of h-BMMSC in the two groups.We detected the osteogenic differentiation capability of h-BMMSC by the methods of alkaline phosphatase activity and ARS staining.RT-PCR was further introduced to detect the relationship between h-BMMSC and SEMA3 B expression levels in the two groups,the changes of SEMA3 B expression levels during bone differentiation(day 3,7,10 and 14)in the two groups.Then,SEMA3 B was overexpressed by lentivirus transfection,to observe the proliferation and osteogenic differentiation of h-BMMSC.Further in vitro experiments,the cells were divided into blank group(h-BMMSC group),control group(h-BMMSC+lentivirus infection group)and experimental group(h-BMMSC+lentivirus transfection group).SEMA3 B m RNA was extracted from the three groups,and the expression level of SEMA3 B in h-BMMSC was detected by RT-PCR.The transfection efficiency of SEMA3 B was detected.Then cell culture was conducted,and CCK-8assay was used to detect the proliferation ability of h-BMMSC among the three groups.We measure the osteogenic differentiation capability of h-BMMSC by the methods of alkaline phosphatase activity and ARS staining.We detected the expression of osteogenic genes(Runx2,ALP,OCN)among the three groups by RT-PCR.Results:(1)Homogenous primary h-BMMSC could be obtained from human bone marrow tissue after culture of whole bone marrow adherent cells and purification by limited dilution.Flow cytometry showed that over 90% of h-BMMSC turned out to be positive for CD29,CD90 and CD105,and the results were negative for CD34 and CD45.The experimental results were consistent with the biological phenotypic characteristics of h-BMMSC.(2)The results of CCK-8 test showed that compared with the non-osteoporosis group,the proliferation ability of the osteoporosis group was not significantly different in the first 5 days.Versus the osteoporosis group,after day 5,the proliferation ability of non-osteoporosis group was significantly improved.(3)The results of alkaline phosphatase assay showed that the activity of alkaline phosphatase in h-BMMSC during osteogenic differentiation in non-osteoporosis group was significantly higher than that in osteoporosis group.Alizarin red assay showed that the mineralization capacity of h-BMMSC in osteoporotic group was weaker than that in non-osteoporotic group at the 21 st day of osteogenic differentiation.The number of calcium nodules in osteoporotic group was significantly lower than that in non-osteoporotic group.(4)SEMA3B expression was detected by RT-PCR,and the expression of SEMA3 B in non-osteoporosis group was 1.27 times that of osteoporosis group.The expression level of SEMA3 B in both osteoporosis group and non-osteoporosis group increased with the time of osteogenic differentiation.The expression level of SEMA3 B in the non-osteoporosis group was lower than that in the osteoporosis group at 3d,7d,10 d and 14 d.(5)After transfection,the transfection rate of SEMA3 B in h-BMMSC was detected by RT-PCR.The results showed that there was no difference between the blank group and the control group,but the expression level of SEMA3 B in the experimental group increased significantly compared with the control group.(6)The results of CCK-8 transfection showed that there was no difference in the proliferation of h-BMMSC between the control group and the blank group.In the first 5days,there was no difference in the proliferation of h-BMMSC between the control group and the experimental group.The proliferation of h-BMMSC in the experimental group was significantly higher than that in the control group after the 5th day(7)Results of alkaline phosphatase after transfection: There was no difference in the osteogenic differentiation of h-BMMSC cells between the control group and the blank group.The activity of h-BMMSC alkaline phosphatase in the experimental group was significantly higher than that in the control group.(8)Results of Alizarin red assay after transfection: there was no difference between blank group and control group on day 21 of osteogenic differentiation of h-BMMSC cells in the three groups.The mineralized ability and the number of calcium nodules of h-BMMSC in the experimental group were significantly higher than those in the control group.(9)After transfection,the expression of osteogenic genes in h-BMMSC cells showed no difference between the control group and the blank group in the process of osteogenic differentiation.Compared with the control group,the expression of osteogenic marker genes in h-BMMSC was increased in the experimental group.Conclusion: The proliferation ability,osteogenic differentiation activity and SEMA3 B expression of h-BMMSC in senile osteoporosis patients are lower than those in non-osteoporosis patients.SEMA3 B can significantly promote cell proliferation and osteogenic differentiation of h-BMMSC in senile osteoporosis patients.