INFLUENCE OF TRANSFORMING GROWTH FACTOR β_1 ON DENERVATED MOUSE MUSCLE DERIVED STEM CELL PRODUCING CONNECTIVE TISSUE GROWTH FACTOR AT DIFFERENT TIME POINTS IN VITRO

Objective To observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse muscle-derived stem cells(MDSCs) producing the connective tissue growth factor(CTGF)at different time points in vitro. Methods MDSCs from the primary culture of the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culture in vitro with TGF-β1 (10 ngml) for 24 hours. Then, MDSCs were randomly divided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ngml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivity rate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate 99%. The Western blot test showed that the ratios of CTGF to the average absorbance of β-actin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups A-D,the absorbance increased gradually, with a significant difference between the above-mentioned groups (P0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P0.05). The RT-PCR test showed that the △Ct values in Groups A-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263, 2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (P0.05). But in Groups E and F, the promotive tendency became less significant(P0.05). Conclusion TGF-β1 can promote the production of CTGF in the mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.