| Osteoporosis is a key program attracted attention from the entire world. The methods to prevent or cure osteoporosis such as cinesiateics, dietotherapy and pharmacotherapy can reduce the loss of bone, and decrease the fracture rate., But these methods can not increase the bone mass, and they can not restore the normal bone mass of a patient, so their action to reduce fracture risk, even highly significant, is rarely more than 50%. Especially, drug therapy was far from satisfactory effect in clinical work, because until now all the antiosteoporosis drugs such as hormone, selective hormone receptor regulator, calcitonin, and diphosphate are all antiresorptives. On strict oppinion, they are just prevention drugs, and can not really use for "cure". Therefore, find new "cure" drugs is an important thesis in the study of osteoporosis. Our former work identified that PDGF-AA can enhancde the proliferation and differetiation of osteoblasts, accelerate bone fracture healing; PDGF-AA has positive effect on bone formation. This implied that PDGF-AA may provide a new tool for osteoporosis "cure". So, it is necessary go on work to find more accurate indentifications and provide sufficient conditions for PDGF-AA's next experiment.Objectives1. Establish osteoblasts-osteoclasts co-culture system, to provide a new in vitro model for bone metabolism research.2. Investigate PDGF-AA effect on bone turnove, to find sufficient identifications for PDGF-AA entering antiosteoporosis research.3. Clone PDGF-AA coding gene and construct high expressed recombinant adenovirus, to settle groudwork for PDGF-AA's massive produce and in vivo tranfection test, and provide sufficient condition for PDGF-AA's next eaperiment.Methods1. The osteoblasts and osteoclasts were isolated from cancellous bone in illium. Osteoblasts-osteoclasts co-culture system was built to prevent these two kinds of cells from contact but their medium can exchange.2. The osteogenety of osteoblasts was evaluated by MTT, ALPase activity and Cell Cycle; the resorption activity of osteoclasts was investigated by the activity of TACPase, TRAPase, ATPase and the area of absorption lacuna. The co-culture's effect on the functions of osteoblasts and osteoclasts were investigated.3. By the parameter issued in method 2, PDGF-AA effect on the two kinds of cells and the information exchange between them were investigated (mono-culture and co-culture).6. Total RNA was extracted from human normal osteoblasts, and PDGF-AA cDNA were cloned by RT-PCR. Then the gene were cloned into Adeno-X, and after packaged by HEK293 cells, the complete recombinant adenovirus were harvested. PDGF-AA gene containing in recombinant adenovirus were identified by PCR, and its expression were detected by Western Blot. After infected by recombinant adenovirus, the expression of PDGF-AA was showed by immunohistochemistry method. Human osteoblasts' proliferation and ALPase activities were investigated.7. Statistics Analysis: SPSS 10.0 softwire used for statistics analysis. During compare two samples, Mest used if homoscedasticity and rank sum test for heteroscedasticity. While compare multiple samples, One-way ANOVA apllied firstly and then LSD method for the differences between each groups.Results1. Osteoblasts are full fusiform, ALPase stain positive cells, while osteoclasts are multinuclear and TRAPase stain positive, with the ability of forming bone lacuna; while, in the co-culture system, the proliferation of osteoblasts in co-culture (0.60\0.08) OD is quicker(P=0.000) than that in mono-culture (0.36±0.03) OD. And the ALPase activity increased significantly ( P=0.000 ) from (222.63±6.36)u/mg to (270.24±28.61) u/mg; while the average area of bone resorption formed by osteoclasts (6.55±0.34) x10 -2μm2 was also increased obviously (P=0.000) from (5.15±0.17) x10μ2um2.2. Co-culture and PDGF-AA can elevate MTT OD value of osteoblasts from (0.38±0.05) to (0.46±0.06, P<0.05) and (0.62±0.03, P<0.01) respectively. Whi...
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