Study On The Influence Of Adipogenic And Ossific Differentiation Of Bmscs Induced By Eupolyphaga Drug-containing Serum With Proteomics

ObjectiveTo explore the molecular mechanisms of the adipogenic and ossific differentiation of BMSCs induced by eupolyphaga, analyse the proteinum which inhibit the adipogenic and ossific differentiation of BMSCs, provide a theoretical basis in treating SANFH with eupolyphaga.Methods:1.Prepare the serum with high quality eupolyphaga by ten days method.2. Bone marrow were taken out of tibia and femur from 4 weeks old SD rats of clean grade, adherent BMSCs were cultured and passaged, detect surface antigen CD45, CD90 from the third generations of BMSCs to identify the cells, the differentiated adipocyte were identified by oil red "O".3. The third generation of BMSCs were randomly divided into blank group, control group and eupolyphaga group. Large doses of steroids were used to induce adipocyte differentiation in control group; Both eupolyphaga containing serum and steroid were given to interfer eupolyphaga groups.4. The osteogenic marker and adipogenic marker:alkaline phosphatase (ALP), triglyceride (TG) were tested in every group after 6 days.5. The structure and ultrastructural features of BMSCs cells in three groups were observed by light microscopy and electron microscopy.6. The proteins of there groups were marked ITHAQ technique, The proteins of there groups were indentified and relative quantitative analyzed by 2D LC-MS/MS mass spectrometry.Results:1. The cultured cells were proved to be BMSCs with the positive rate of CD45 and CD90 surface antigen; According to the rusult of oil red "O" staining, the cell were proved to be adipocyte induced by BMSCs.2. Triglyceride test results:the contral group was significantly higher than the blank group (P< 0.05), Eupolyphaga group was significantly lower than the control group (P<0.01).3. Alkaline phosphatase test result:control group was significantly lower than blank group (P< 0.05), Eupolyphaga group was significantly higher than the contral group, (P<0.01).4. The ultrastructural observed by transmission electron microscope:there were plenty of microvilli-like protrusions in the BMSCs surface of control group. More cytoplasmic organelles and few fat droplets were visible. Contral group:no microvilli-like protrusions in the BMSCs surface. A great quantity of fat droplets and few cytoplasmic organelles were visible. Some nucleus were dissolved, broken. Eupolyphaga group:there were a small amount of microvilli-like protrusions in the BMSCs surface. The number of organelles were in general, no fat droplets.5.520 kinds of proteins were successfully identified by mass spectrometry.217 kinds of proteins have quantitative information. To the contral group, There were 15 kinds of proteins appeared significantly upregulated, and 12 kinds of proteins were down regulated in blank group. There were 16 kinds of proteins appeared significantly upregulated,14 kinds of proteins were significant down regulated in Eupolyphaga group.Conclusion:1. Eupolyphaga containing serum can inhibit the adipogenic differentiation of BMSCs and promote osteogenic differentiation of BMSCs.2. 520 kinds of proteins were successfully detected By mass spectrometry, and 217 kinds of proteins have quantitative information.3. Hspal 1 and Prx V may be two targets of anti-apoptosis in eupolyphaga4. Serpinhl and ADP/ATP translocase 1 may be the target in promoting osteogenic differentiation in Eupolyphaga.