| Objective To investigate the effect of theaflavin-contained serum on osteoblastic cell differentiation of rabbit bone marrow stromal cells(BMSCs) in vitro .Methods①preparation of theaflavin-contained serum 3～4 months old Chinese rabbits provided by Animal Center of Anhui Medical University were erandomly divided into experimental group which were fed with theaflavin -5% sodium carboxymethy–cellulose solution by means of a gastric tube once a day with the dosage of 5 times as high as that of a adult man, and blank control group which received no treatment. 1015ml cardiac blood was taken from every rabbit after 7 days treatment. After standing, centrifugation, deactivation and sterile filteration, the sample serum was preserved in a -20℃refrigerator. Simultaneously,1ml sample serum was taken from each group into High Performance Liquid Chromatograph Instrument(HPLC) for detecting whether theaflavin components can be found in serum of two groups .②culture and separation of BMSCs in vitro 4ml bone marrow drawn from superior part of both femoral condyle of 3～4 months old Chinese rabbits following general anesthesia was isolated by means of density gradient centrifugation at sterile condition. Then it was inculated into DMEM culture medium containing 10% fetal bovine serum . When the cells grew to the fusion of 80%～90%, digesting and passage with pancreatin/ EDTA.③Induction of osteogenesis the second passage cells were randomly divided into three groups, group 1 were added with theaflavin-contained serum , group 2 with 10 mmol/L ?-glyceraldehyde -3-phosphate,50mg/L L-ascorbic acid and 10-8mmol/L dexamethasone. group 3 was control group.④Identification of osteoblast The osteogenic characteristics were identified through Von Kossa staining,ALP and type I collagen staining with the second passage cells collected at 6,12,18,24 days respectively and assay of ALP quantity of cells from the third passage.Results Absorption peak was found at 39 min with ordinary temperature, flow-rate of 1.2mL·min-1, wavelength of 280nm in group of theaflavin -contained serum by HPLC, negative was in blank control group. At the 3rd day, huge amounts of cell adhered, grew like colony and shaped as fusiform, polygon. At the 9th day, Cells gradually covered with the bottom, lined as whirlpool. At 10～15 days, round or ellipse nodule emerged after inducing culture. Calcium deposition occurred in 2 weeks and increased significantly at the 16th day. Calcium nodule began to form at the 15th day, grew in number at the 18th day in group theaflavin-contained serum. But the number of calcium nodule of group of theaflavin-contained serum was less than that of inducing osteogenesis group. Both groups were found to be positive for Von Kossa staining. Positive ALP staining in both groups were found at the 6th day, The maximum positive rate came to 90% on days 12 in group of inducing osteogenesis and 80% on days 18 in group of theaflavin -contained serum. Both groups were positive for type I collagen staining on days 12. Feminine was found in control group for Von Kossa staining ,ALP staining and type I collagen staining. Assay of ALP quantity of cells of the third passage shown significant difference between the three groups according to q test in SNK of ANOVA.conclusion Theaflavin-contained serum can induce rabbit BMSCs to differentiate into osteoblasts, but its osteogenic ability was lower than that ofβ-glyceraldehyde-3-phosphate, L-ascorbic acid and dexameth -asone. |
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