The Study On The Biological Characteristics Of MSCs Islated From Ovariectomied Rats And The Intervention Research Of Drugs On The Osteoblast-differentiation Of MSCs From Normal SD Rats

Objective: The research was to investigate the effects of ovariectomy on SD rats by compairing the biological characteristics of the mesenchymal stem cells(MSCs) derived from the osteoporosis (OP) rats and normal rats. To study the effects of traditional chinese medicine such as Drynaria Rhizome, epimedium, Benefiting-bone capsule (BBC) and cytokines such as transforming growth factor-betal (TGF-β₁),bone morphogenetic protein-2 (BMP-2) on the proliferation and differentiation into osteoblast of MSCs to further confirm the best proliferation and differentiation concentration of them on MSCs. To observe the expression of TGF-β₁ and BMP-2 in the induced systems of the herbs above which were confirmed to have the accurate effects of preventing and treating OP to further explain the mechanism of them in inducing MSCs differenting into osteoblast, to explain the mechanism of them in preventing and treating OP and to provide the experimental exploration in establish a platform by TGF-β₁,BMP-2 to screen the medicine of preventing and treating OEMethods:1. MSCs was isolated and purified by differential time adherent method;2. MSCs was identified by morphologic characteristics, surface antigens detected by flow cytometer and differentiation into osteoblasts, adipocytes and chondrocytes;3. The osteoporotic animal model was established by ovariectomy on female Sprague-Dawley (SD) rats aged 10 month;4. To investigate the effects of ovariectomy by compairing the morphology, growth curve and cell cycle and differentiation capacity into osteoblasts, adipocytes and chondrocytes of the MSCs derived from the OP rats and normal rats;5. MTT method was used to confirm the most effective concentration of the traditional chinese medicine and cytokines on the proliferation of MSCs;6. The most effective concentration of the traditional chinese medicine and cytokines on the differentiation of MSCs into osteoblast was jurged by alkaline phosphatase (ALP);7. According to the different induced condition, MSCs were divided into 4 groups: control group, classic group (induced by the classic osteoblast-induced system), drugs group (induced by the most effective concentration of drugs on differentiation into osteoblast) and drugs+classic group (induced by the combination of classic osteoblast-induced system and the most effective concentration of drugs on differentiation into osteoblast). ALP,typeâ… collagen (Colâ… ),bone gla protein (BGP)and calcium nodes in each group were detected and compaired to indicate the osteoblast-formation function of different group;8. ELISA was used to detect the expression of TGF-β₁,BMP-2 of each induced group in the progress of MSCs differenting into osteoblast to explain the mechanism of the drugs in inducing MSCs differenting into osteoblast;9. Real-time PCR was used to detect the expression of Smad4 and core binding factor alphal (Cbfα1) mRNA in the induced groups of TGF-β₁ and BMP-2 to further explain the mechanism of the drugs in inducing MSCs differenting into osteoblast.Results:1. The results of the surface antigens of MSCs detected by flow cytometer showed the positive expressions of CD29 and CD44 and the negative expressions of CD34 and CD45; MSCs isolated from SD rats could be induced successfully into osteoblasts, adipocytes and chondrocytes;2. The proliferation in vitro of MSCs from OP rats decreased compaired with that form normal rats; the ability of differentiation into osteoblast and the reaction to the classic osteoblast-induced agent of MSCs from OP rats was lower than that form normal rats; the ability of differentiation into adipocytes and the reaction to the classic adipocytes-induced agent of MSCs from OP rats was higher than that form normal rats; the reaction to the classic chondrocytes-induced agent of MSCs from OP rats decreased compaired with that form normal rats; 3. The most effective concentration of the Rhizoma Drynariae and epimedium water-extraction on the proliferation of MSCs both was 5μg/ml; and the most effective concentration of them on the differentiations of MSCs into osteoblast were 50μg/ml and 500μg/ml; the Rhizoma Drynariae and epimedium water-extraction could both increase the expression of osteoblast-indices which follwed the increase of the secretion of TGF-β₁ and BMP-2;4. The 25% concentration of the drug-containing sera of BBC collected form SD rats one hour later after intragastric infusion with low dose BBC was proved to be the most effective concentration on the proliferation of MSCs; the 25%-30% concentration of the drug-containing sera of BBC collected form SD rats one hour later after intragastric infusion with high dose BBC was proved to be the most effective concentration on the differentiations of MSCs into osteoblast; the induced groups of BBC were confirmed to increase the expression of osteoblast-indices which follwed the increase of the secretion of TGF-β₁ and BMP-2;5. Naringin and Icariin which had been proved to possess the ability to induce MSCs differenting into osteoblast in former study could both increase the expression of TGF-β₁ and BMP-2;6. The most effective concentrations of rhTGF-β₁ and rhBMP-2 water-extraction on the proliferation of MSCs were 10ng/ml and 80ng/ml; and the most effective concentration of them on the differentiations of MSCs into osteoblast were 5 ng/ml and 40ng/ml; rhTGF-β₁ and rhBMP-2 could both increase the expression of osteoblast-indices which follwed the increase of the secretion of TGF-β₁ and BMP-2;7. The induced groups of rhTGF-β₁ and rhBMP-2 could improve the expression of Smad4 and Cbfα1 mRNA.Conclusion:1. The stable and uniformal MSCs could be obtained by differential time adherent method;2. The MSCs from OP rats' ability of differentiation into adipocytes and chondrocytes decreased and the ability of differentiation into adipocytes increased;3. The Chinese herbs of Rhizoma Drynariae, epimedium and BBC and the cytokines of rhTGF-β₁ and rhBMP-2 could all promote the differentiation of MSCs into osteoblast;4. In the osteoblast-differentiation progress, the induced groups of the drugs in this expriment all could increase the expression of TGF-β₁ and BMP-2, which may he the mechanism of them on improving the differentiation of MSC into osteoblast;5. The increase of the expression of Smad4 and Cbfα1 mRNA may be the further mechanism of the drugs on improving the differentiation of MSC into osteoblast after they stimulated the secretion of TGF-β₁ and BMP-2.