| Object: To optimize the seeding cells in cartilage tissue engineering and improve the quality of constructing tissue engineered cartilage , we investigated the effect of pilose antler polypeptides(PAP) on the chondrogenic phenotype differentiation of rabbit bone marrow-derived mysenchymal stem cells (MSCs) cultured in vitro by the sensitive molecular and celluar technique. Methods: 1.The MSCs were isolated from the nucleated cells fraction of autologus bone marrow by density gradient centrifuge and cultured in vitro. 2.Growth and ultromicrostructure of the MSCs were observed by light-microscope and electron-microscope. Specific antigens on the surface of the MSCs were detected by flow cytometry. The grow curve of the MSCs was described by MTT assay. 3.The harvested MSCs were randomly divided into different groups and treated with drugs. The MSCs were observed for proliferation by MTT assay, immunohistochemistry for detection PCNA, flow cytometry for analyses of cell cycle distribution after 48 hours. 4. The third passage MSCs were divided into control group, induced group and PAP group, which were cultured in centrifuge tubes respectively in ordinary medium, induced medium and induced medium containing 10ug/ml PAP. The cellular morphological and functional characteristics were observed after cultured for 1, 2, 3 weeks in centrifuge tubes by histological, biochemical and RT-PCR technique. 5. The MSCs differentiated into chondrogenic phenotype were divided into control group, induced group and PAP group, which were induced into apoptosis cells by IL-1β and treated with PAP. Morphology was observed by transmission electron microscope, apoptosis was detected by flow cytometry analysis, the expression of caspase-3 was determined by RT-PCRand caspase-3 protein activity was determined by ELISA. Results: 1. Primary MSCs proliferated in visible symmetric colonies with long-sindle shape. The morohological characteristics of MSCs had no change during passaging, and its homogenicity rose, its function was active, its capacity enlarged with passaging. A large amount of endoplasmic reticulum, Golgi complex and mitochondria was observed in passaging MSCs. It was confirmed that there was expression of CD29 and CD90 , no expression of CD34 and CD45 on the surface of MSCs. These cells didn't belong to haemale system. They proliferated showly from 1 to 3 day, proliferated speedly from 3 to 5 day, proliferated gently from 5 to 7 day and proliferated descendly by contact inhibition from 7 to 9 day. 2. The best promotion of proliferation of MSCs was achieved when the amount of PAP was lOug/ml and PAP was able to eliminate the generation of heteroploid of MSCs cultured in vitro continually. PAP could increase the secret of GAG and mRNA expression of type II collagen when MSCs were cultured with induced medium. PAP could decrease the mRNA expression of caspase-3, inhibit caspase-3 protein activity and reduce the apoptosis of MSCs. Conclution: 1. A perfect culture system of MSCs was established when MSCs were cultured in vitro. 2. PAP can significantly improve proliferation of MSCs and prevent from the heterology of MSCs cultured continually in vitro. PAP can enhance chondrogenic phenotype differentiation of MSCs. 3. Caspase-3 is involved in aoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs. 4. PAP can optimize the seeding cells in cartilage tissue engineering and improve the quality of constructing tissue engineered cartilage, because it affects the bioactivity of chondrogenic phenotype differentiation of MSCs in vitro. This could be the mechanism of pilose antler strengthening bong and musculature.
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