Growth Regulation of Human tenocytes by Transforming Growth Factor-β1
|School||First Military Medical University|
|Keywords||Transforming growth factor - beta1 Tendon Cell Proliferation Collagen Smad Flow cytometry Reverse transcription - polymerase chain reaction|
The study of tissue-engineered tendon, tendon cells is a high degree of differentiation of the cells, and how to regulate the proliferation and matrix synthesis in vitro culture conditions which must be studied by tendon cells as seed cells. Recent studies show that transforming growth factor-beta 1 (TGF-beta in 1 ) in the tendon of the formation, growth and repair process plays an important role. Order to explore TGF-beta in 1 and its mechanism of human embryonic tendon cell proliferation and collagen metabolism and its downstream signaling molecules Smad. First, we used immunohistochemistry and flow cytometry analysis on cultured of tendon cells TbetaR I, II, III, of expression and density were analyzed in vitro tendon cells TbetaR < The sub> Ⅰ, Ⅱ, Ⅲ each maintain a certain density; analysis by flow cytometry of PI and FITC double labeling of the same cell cycle phase TbetaR I, II, III difference between the number of receptors, TβR Ⅰ , TβR Ⅱ and TβR Ⅲ , in the cell cycle G the 2 M cells surface receptor density is higher than G 1 of cell receptor density. In addition 3 sup> H-TdR and 3 sup> H--proline incorporation was observed TGF-β 1 DNA and human embryonic tendon cells collagen synthesis, confirmed in 0.5% fetal bovine serum conditions TGF-beta in 1 can stimulate the tendon cell proliferation in a dose-dependent enhancement of its role in the 0.5-10μg / L (P <0.05 ), saturated dose of 10 microg / L increase in DNA synthesis 73% role in 10μg/LTGF-β 36 hours beginning proliferation significantly (P <0.05), and continued until 72 hours. Cell cycle analysis showed that: the the TGF 1 role of the G / G 1 % is decreasing, while the S% showed an increasing trend, reflecting the proliferation activity PI values ??also tended to increase, especially in the concentration of 5 ug / L significantly (P <0.05). TGF-β 1 each dose group both collagen synthesis promoting effect (P <0.05), the maximum effective dose of 5 ug / L (107%). Smad2 gene cloning and sequence of the MH2 domain identification confirmed tendon cells memory of SMAD2 signal transduction pathways. Finally, using RT-PCR reaction for detection of TGF-beta 1 role 24h tendon cells Smad2, 3 and 7mRNA changes in expression levels. TGF-beta 1 role after 12 hours can be significantly lowered the level of gene expression of Smad2 and Smad3, induced by TGF-beta 1 signal antagonistic factor of Smad7 is generated. Therefore, our conclusion is that the presence of TGF-beta receptor system and its intracellular signaling molecules tendon cells. TGF-β 1 by feedback regulation of intracellular signaling molecules within a certain range can effectively promote tendon cell proliferation and collagen synthesis. Provide a basis for the regulation of seed tissue-engineered tendon structure tendon cell proliferation and differentiation.