The Character Of Proliferation, Osteogenic And Adipogenic Activity In Bone Mesenchymal Stem Cells (BMSCs) Of Osteoporotic Mice

Estrogen deficiency, which is important in the pathogenesis of postmenopausal osteoporosis, has received increasing attention in the studies related to the osteoporosis diseases in postmenopausal women. It has been reported that defects in the number and proliferative potential of MSCs may underlie age-related defects in osteoblast number and function[1]. It is known that marrow fat content increases in a variety of osteoporoses and there was a reciprocal relationship between marrow fat content and bone formation rate.But the molecule mechanism is still unknown[2].Other studies reported that in vitro the proliferation and osteogenic activity in BMSCs would be increased with the stimulus of Estrogen[3].But the changes in vivo are still unknown.In this study, we aim to isolate and culture the bone marrow stromal cells(BMSCs) of osteoporotic Sprague-Dawley (SD) rats . Then identified them by flow cytometer and multi- differentiation cultivation.The cells were screened by inverted microscope each day and MTT assay was perfomed to determine the cell proliferative activity.At last, the BMSCs to differentiate into osteoblast and adipogenesis in vitro were induced and the mRNA level of osteogenic and adipogenic gene was examined by RT-PCR.The study consists of three parts:1.Establishment of animal experimental model on ovariectomized rats Methods:Three-month-old SD rats were used in this study. 12 female rats were randomly divided into two groups.six rats were ovariectomied as experimental group I, while other rats were received sham-operation as groupⅡ.Results:All of the animals were alive successfully after the operation.Two groups have significant difference in weight,index of BMD ,the level of Estrogen.2.Cultivation and identification in bone mesenchymal stem cells (BMSCs) of osteoporotic SD rats in vitro and the character of proliferation.Methods:Three months later BMSCs of 12 rats were isolated by gradient centrifugation as primary culture.Then identified them by flow cytometer and multi- differentiation cultivation.The cells were screened by inverted microscope each day and MTT assay was perfomed to determine the cell proliferative activity. Results:Von Kossa′s staining demonstrated positive osteoblast phenotypes of mineralized nods by osteogenic inducer,while Oil Red O staining identified BMSCs treated with adipogenic medium resulted in adipocyte formation. Flow cytometer showed the positive expression of CD29 and CD44 while the negative expression of CD45 and CD133. MTT method detected that during seven days the cells of experimental group keep higher proliferation activity than those of control group every day.3.osteogenic and adipogenic activity in bone mesenchymal stem cells of osteoporotic miceMethods: After the BMSCs to differentiate into osteoblast and adipogenesis in vitro were induced for 15 days,we examined the mRNA level of osteogenic and adipogenic gene by RT-PCR.ALP and BSP were used for the test of osteogenic gene. PPARγ2 was used for the test of adipogenic gene. Results:The expression of osteogenic gene(ALP, BSP) was decreased while The expression of adipogenic gene(PPARγ2) was increased in experimental group.