The Intervention Role Of Ground Beetle On Steroid-induced BMSCs Adipogenic Differentiation

Purpose:Steroid-induced avascular necrosis of femoral head may be related to steroid-induced bone mesenchymal stem cells(BMSCs) of adipogenic differentiation,the prevention and treatment of this disease by using Ground Beetle have significant effect.This research project is to observe the Steroid-induced rat BMSCs adipogenic differentiation,as well as using Ground Beetle serum containing the role of its intervention,to explore the Ground Beetle steroid-induced bone BMSCs adipogenic differentiation,clinical prevention and treatment for steroid avascular necrosis of the theoretical foundation laid,and activating blood and resolving stasis Ground Beetle treatment of this disease and provide a theoretical basis for such research.Methods:(1) Selection of high quality Chinese Medicine Ground Beetle,and using 10 days method to prepare Ground Beetle containing serum.(2)Use one-month-old SD rats' tibia and femur bone marrow to culture and passage BMSCs.Measure the third generation of BMSCs surface antigen CD45,CD90 expression positive rate to identify the cells.(3) The third generation of BMSCs were randomly divided into blank group,control group and Chinese Medicine low,medium and high dose group.Using large doses of steroids to induce adipocyte differentiation in control group;giving Ground Beetle containing serum and steroid to interfere in Traditional Chinese Medicine groups.(4) After 6 days intervention,to test osteogenic and adipogenic target markers in each groups,including the inverted microscope, alkaline phosphatase(ALP),triglyceride(TG),osteocalcin(BGP) of the detection,RT-PCR of typeⅠcollagen(ColⅠ),fat cells,peroxisome proliferator-activated receptor(PPARγ) and fatty acid binding protein(aP2) mRNA expression were determined.Results:1.According to BMSCs adherent growth characteristics can separate BMSCs and hematopoietic cells.BMSCs isolated and cultured through this method,the surface antigen CD45,CD90 positive expression rates of which were 1.9%and 99.1%respectively. 2.After interfering cells for 6 days,the triglyceride content in control group cells is higher than the blank group,and the difference was significant(P＜0.01).Chinese Medicine groups were all lower than the control group,the differences were very significant(P＜0.01). With the increased concentration of traditional Chinese medicine and the triglyceride content of cells decreased.3.Intervention cells for 6 days,osteocalcin content in the control group was lower than the blank group,the difference was very significant(P＜0.01).Chinese Medicine groups were higher than the control group in varying degrees,and with the increased concentration of Traditional Chinese Medicine the osteocalcin content trend was increased.The dose group and high dose group compared with the control group differences were significant(P＜0.01).4.Intervention cells for 6 days,the alkaline phosphatase(ALP) content in control group cells was significantly lower than the blank group,and the difference was significant(P＜0.01).Compared with control group,the ALP was increased in varying degrees in Chinese Medicine groups,and the differences were very significant(P＜0.01).With the increased concentration of traditional Chinese medicine the intracellular ALP content are increasing trend.5.Genes expression:cells cultured 6 days after intervention by gel scanning imaging system to do a product of semi-quantitative PCR analysis showed that collagen typeⅠ,fat cells,peroxisome proliferator-activated receptor(PPARγ) and fatty acid binding protein(aP2) mRNA expression in the control group,blank control group and Chinese medicine groups were significantly different.Conclusions:1.The cells cultured in vitro this experiments identified by morphology,and surface markers by flow cytometry belong to BMSCs,and contain quite high purity.2.High dose steroid induce can promote BMSCs adipogenic differentiation,and so reduce the osteogenic differentiation.3.After interfering by Ground Beetle containing serum,triglyceride content, PPARγmRNA and aP2 mRNA expression in cells than the control group were significantly increased,suggesting that Ground Beetle can reverse steroid-induced adipogenic differentiation of BMSCs.4.Interfered by Ground Beetle containing serum,ALP,BGP contented and the expression of ColⅠmRNA were increased,suggesting that Ground Beetle can promote BMSCs osteogenic differentiation.5.The mechanism of Ground Beetle to combat femoral head avascular necrosis is not only to improve the microcirculation of the femoral head,may also be related to the inhibition of adipogenic differentiation of BMSCs,and increase of osteoblast differentiation. Fund Project:plans to focus on science and technology projects of Fujian Province number:2007F5060...