Growth Regulation Of Human Tenocytes By Transforming Growth Factor-β₁

ABSTRACTTo date, little is known about the regulatory signah involved tendon repair ,atid this lack of understanding has hindered attempts to develop biological based therapies for tendon repair. Transforming Growth factor- P 1(TGF- P ~) is a cytokine that play a fundamental role in soft-tissue repair. Members of the SMADfamily of intracellular proteins are phosphoryiated by TGF- P receptors arid convey signals to specific TGFP ,-inducible genes. Ligand bindii~ initiates signaling through the SMAD pathway, but it is unkonwn howsignaling is terminated. Since cellular proliferation and matrix synthesis are critical elements of healing.We investigated the expression of transforming growth factor- P receptor I ,II and III (T P R .11,111) in human tenocytes in vitro.The response of tenocytes to TGF- P as a function of titus and doses and the physiological regulation of endogenous Smads by TGF- P in tenocytes was also investigated. The expression of T P R1,11, ~ were all found in tenocytes using immunocytochemistry. The dottle staining of P1 and FITC was using to assay the density of T P R1 .11,111 in phases of cell cycle measured by flowcytometry. The mean fluorescence intensity ofTP R,,TP R11 and TP R111associated with theGl phase was 7.93?.20,8.76?.16,8.46?.14 despite it was 17.7 ?.36,18.9?.35,18.3?.67 associated with the G2M phase.The difference was significantly (P <0.01) .Tenocytes cultured for different hours(2-4,36, 48,60,72) or at different concentrations (0.5.. 1.. 5.. 10.. 20 ~tg/L) with TGF- P were radiolabeled for the final 6 hours with 113H]thymidine, and DNA synthesis was quantified as trichloroacetic acid-precipitable radioactivity. (3H]proline incorparation was used to detect collagen synthesis of tenocytes treated with TGF- P at different concentrations. Cell cycle affected TGF- P ,by was quantified by flowcytometry(FCM) analysis. MH2 domain of Smad2 and Smad4 cDNA from human fetus tenocytes was cloned and seqt~nced.The levels of Smads and Procoiagen a 1(1) mRNA were assessed by semi-quantitative reverse tran~ription polymerase chain reaction (RT-PCR). The expressiones of T P R1?~ ~ were all found in tenocytes. It indicate tlut the ratio of type 1,11 and III of T P R levels is cell cycle dependent.Statistical analyses(analysis of variance) showed that there was dose-effect relationship between TGF- P 1i and tenocyle roliferation at concentration of 0.5-20~tg/L, tenocytes responded to TGF- P doses of I 0~tg/L by increased DNA synthesis by 73%. From 36 hour, thesignificant proliferation of tenocytes were observed and lasted to the 72 hour. TGF- P at different concentrations could all stimulate collagen synthesis. Procollagen mRNA expression of group treated with TGF- P at S~.ig/L was significantly higher than that of the control group(O.266監.061J vs O.162監.05U, P