The Effect Of Melatonin To Adipogenic Differentiated Of Rat's Bone Marrow Mesenchymal Stem Cells

Study background There are 2 hundred millions osteoporosis(OP) patients all over the world now,which disease rate is stand at the 7th position among common and frequently-occurring diseases.Especially,no safe and effective method to cure this desease radically at the present.More and more studies have found that there is a close relationship between osteoporosis and increasing fatty tissue in the bone marrow.If bone marrow mesenchymal stem cells(BMSCs) differentiate to lipoblast redundantly,accordingly,it will reduce the cell differentiation to osteoblast,and then to cause OP with ossification defect.Melatonin(MT),a hormone,which is synthetized and excreted by pineal gland(PG) and bone marrow in physiological condition,it plays an important role in the all internal humoral regulation directly or indirectly.The secretory volume of MT is degrading by the human being's aging.According to the newest studies,MT could promote bone fracture's healing and refrain the aggregation of fatty acid.These newfound effect of MT and secretory volume with age dependence,will make new way to study and research OP possible.Objectives To isolate and cultivate rBMSCs in vitro,and to study and research the effects of MT on the differentiation of Rat's BMSCs into adipocytes and the mechanism of MT's effect.moreover,to discuss the pathogenesy of OP.Methods1. rBMSCs isolation,culture and identificationTo take marrow from six month aged SD Rat in axenic condition,the primary BMSCs were purified from the marrow by density gradient centrifugation and cultured in Eagle medium(DMEM) with 10% fetal bovine serum(FBS).To digest cells by using trypase in 0.25g/L concentration to get passage cells when their growth reaches 90% confluence.To observe BMSCs' morphous below the inverted microscope,to detect 3th generation BMSCs CD29,CD34 and CD 44's expression by flow cytometry.2. rBMSCs adipogenic differentiation and intervention with different groups Rat's BMSCs was isolated and cultured, divided into three groups and interfered in after adipogenic differentiated. The control group didn't received any intervention except adipogenic differentiation,the experiment group A was interfered in MT, the experiment group B was interfered in MT and it's receptor antagonist luzindole (LZD). We detected the proportion of adipose cells to compare the adipogenesis levels in different groups, and maked use of Rt-PCR to detect the expression of adipogenic transcription factor PPARγmRNA .Results1. We can get BMSCs through gradient centrifugation and the adherent layer nurture.2. The experiment group A(add MT to intervente),compared with control group(adipogenic differentiated simply),its adipogenesis level was obviously lowe(rp<0.01).The experiment group B(add LZD and MT to intervent),compared with the control group,their adipogenesis levels had no significant difference(p>0.05);while compared with the experiment group A,its adipogenesis level was significantly higher(p<0.01).Conclusions1. The BMSCs can be purified and nurtured in vitro through the way of gradient centrifugation and adherent layer culture. And BMSCs could possess a differentiation capacity towards adipocytes.As a result,BMSCs can differentiate into the same mesodermal cells.2. MT could inhibit BMSCs differentiating to adipogenesis pathway and decrease the PPARγmRNA level of adipocytes that it effects on the role of its receptor mechanism. This maybe relate to the nosetiology of OP.