| The topic of " kidney governs the bones and engenders marrow "as the theoretical basis, in previous studies, based on the bone marrow from ovariectomized rats were isolated mesenchymal stem cells were cultured in vitro systems, combined with cell morphology, molecular biology techniques, observation Chinese medicine GuKang contained serum directed differentiation of bone marrow mesenchymal stem cell osteogenic differentiation capacity, immunohistochemistry, Western blot determination of osteogenic differentiation markers expression, detected using RT-PCR determination of bone specific transcription factor Cbfal expression, and further analysis of serum containing bone health to induce osteogenic differentiation of MSCs may signal mechanism to clarify P38 signal pathway in ovariectomized rats (osteoporosis, pathologic state model) MSCs into osteoblasts important role in. This will further clarify the quality of bone health in the prevention of bone disease in the reduction of value and mechanism for the regulation of stem cells in medical treatment of osteoporosis and provide experimental basis for the mechanism for the provision of traditional Chinese medicine into the world of experimental dataMethods1. To get thirty-four SPF Sprague-Dawley rats, ten of which are for the preparation of pastille serum of Gukang. Fourteen of them are ovariectomized to build osteoporotic models. Others are the normal control group.2. By the psoralen, the system Epimedium, Rehmannia, Astragalus, dodder, etc. composed of ten Chinese herbs brewed GuKang decoction of Chinese medicine, by human and rat body surface area than the calculated dose of GuKang side to give medicine GuKang gavage, in the last 2 hours after administration cardiac blood, prepared the pastille serum.3. Were taken 4 weeks old SPF SD rats with normal levels 10 and osteoporosis 12 rats, under anesthesia injection, sterile conditions, the isolated femur and tibia double, with D-Hank's solution were repeatedly washed and DMEM Femur and tibia bone marrow cavity, extraction and separation of MSCs, the concentration of 0.25% trypsin (0. 1ml/cm2) 37℃under the conditions of digestion, the L-DMEM complete medium, cultured and passaged MSCs, MSCs established in vitro model.4. Experimental group:No1:normal control group; No2:control osteogenic agent group(Control group+50μg/ml Vitamin C+10mmol/Lβ-glycerophosphate +10-8mol/L dexamethasone); No3:normal Gukang group (pastille serum of Gukang medicine); No4:ovariectomized control group; No5:ovariectomized osteogenic agent group; No6:ovariectomized Gukang group.5. ALIZARIN RED staining for calcification of nodules in each group, identifying mineralized extracellular matrix; determined by radioimmunoassay in the culture medium osteocalcin (BGP) content; to immunohistochemistry to detect bone formation markers ALP, OPN expression, to determine the osteogenic differentiation capacity.6. Western blot technique to detect P38 protein, ATF-2 expression; with Real-Time PCR Detection of osteogenic markers Runx2, P38 protein, ATF-2 expression.7. Statistical MethodsData were analyzed with SPSS13.0 package, was used to compare each group t test and x2 test was used for statistical analysis. Probability p<0.05 indicates statistically significant difference. Images by computer image analysis system strip gray value analysis.Results1. Radioimmunoassay method using to detect osteocalcin (BGP) of the cells after cultured 7d,14d determination, N04 (castrated rats group) 7d,14d detection of osteocalcin (BGP) were 3.98ug/L,4.31 ug/L, NO1 (control group) and osteocalcin (BGP) was 4.67 ug/L,4.85 ug/L, ovariectomized group than in the control group rats were significantly reduced, with significant differences; After cultured 7d,14d the cells ALIZARIN RED staining, detection of calcified nodules, to determine the extracellular matrix mineralization, N04 (castrated rats group) 7d,14d detection of calcified nodules was 1.16,2.01, NO1 (control group) in the number of calcified nodules were 1.67,2.52, ovariectomized group than in the control group rats significantly reduced, with significant differences; after 7 days the bone formation markers ALP, OPN immunohistochemical staining, detection of osteogenic markers ALP, OPN expression, to determine the osteogenic differentiation capacity of adult ovariectomized rats showed that bone differentiation marker ALP, OPN positive cells decreased.2. Analysis by radioimmunoassay in the cells after cultured 7d,14d to detect osteocalcin (BGP), N06 (OVX GuKang group) 7d,14d detection of osteocalcin (BGP) were 6.12ug/L,6.66 ug/L, N05 (castration bone induction group) osteocalcin (BGP) was 5.97 ug/L,6.94 ug/L, N04 (OVX control group) and osteocalcin (BGP) was 3.98 ug/L,4.31 ug/L, OVX induced GuKang group and castrated group compared with OVX control group increased significantly, with significant differences; ALIZARIN RED staining on the cells cultured after 7d,14d to detect Calcified nodules, to determine the extracellular matrix mineralization, N06 (OVX GuKang group) 7d,14d number of calcified nodules detected were 3.12,5.11, N05 (castration bone induction group) in the calcified nodules Respectively 4.01,5.79, N04 (OVX control group) 7d, 14d number of calcified nodules detected were 1.16,2.01, OVX induced GuKang group and the ovariectomized group than in the control group, castrated A significant increase, with significant differences; after 7 days the bone markers ALP, OPN immunohistochemical staining to detect osteogenic markers ALP, OPN expression, to determine the osteogenic differentiation capacity of GuKang showed that castration Group, ovariectomized group osteogenic osteogenic differentiation markers ALP, OPN positive cells were significantly increased. RT-PCR, expression of Runx2:castration GuKang group, castration Runx2 osteogenic induction group than the OVX control group, the expression of a significant increase, a significant difference.3. After the detection of the expression of osteogenic markers P38 and ATF-2 with Western blot, low expression of P38 normal rats, castrated rats group expressed slightly increased, but not obvious, but the pastille serum of Gukang can increase the expression of P38. ATF-2 is a downstream molecule P38, the main regulation of cell proliferation and apoptosis, this experiment the same as the change in trends and P38; RT-PCR quantitative analysis showed that castration GuKang group P38, ATF-2 expression OVX control group, there were significant differences.Conclusions1. Osteoporosis in the rat animal model, and the preparation of drug-containing serum, the separation of MSCs, pure culture and osteogenic induced differentiation of bone biology to establish in vitro cell model, build up an experimental model to study the control of mechanism Gukang on the dynamic process of bone marrow mesench-ymal stem cells'differentiation to osteoblast on the basis of building osteoporotic models.2. It is found in the experiment that the osteoblast differentiation of MSCs in the ovariectomized rats decline, which is embodied in the decrease of calcified nodules, BGP content, the numbers of positive cells in osteogenic markers ALP and OPN at different times.3. It is found in the experiment that the osteoblst differentiation culture fluid can successfully induce the differentiation of MSCs in ovariectomized rats, which may be accelerated by the Chinese medicine Gukang through transfering the expression of Cbfal.4. Gukang induced osteogenic differentiation of MSCs in ovariectomized rats may activate the p38 signaling pathway, mediated through the activity and expression of Cbfal on the key regulatory role to play.5. From the above results suggested the possible regulatory mechanism: Gukang→activate the P38 signaling pathway→MAPK moves into nucleus→transfer the osteoblast-specific transcription factor Cbfal→combine the osteoblast-specific cis-acting element 2 OSE2→activate alkaline phosphatase, Type I Collagen, osteopontin and BGP, etc.→biological effect→induce MSCs osteoblast differentiation.
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