| Objective: Transforming growth factor-β(TGF-β), a pleiotropic cytokine involved in cell differentiation and proliferation, is ubiquitously expressed in cells and tissues. TGF-β1 plays a pivotal role in the process of liver fibrosis while TGF-β3 can inhibit the expression of TGF-β1, result in resistance to hepatic fibrosis. To increase the expression of endogenous TGF-β3, we stimulate hepatic stellate cell (HSC) with exogenous TGF-β3, and investigate the effects of exogenous TGF-β3 on the expression of endogenous TGF-β3 and proliferao-tion of HSC.Methods: HSCs were cultured and divided into two groups: TGF-β3 group and blank con-trol group. The cells of TGF-β3 group were exposed to exogenous TGF-β3, whereas the blank control group was not treated. (1)The cells were harvested at 1h,2h,4h,12hå'Œ24h, and real time PCR was performed to detect the mRNA expression of endogenous TGF-β3.(2)The cells were collected at 0h,1h,6h,12h, and Western-blots were used to detect the protein synthesis of endogenous TGF-β3 in HSC;(3)The cell culture supernatant was harvested at 0h,2h,4h,8h,14h,24h,and ELISA was performed to measure the total protein of of extracellular TGF-β3;HSCs were treated with or without TGF-β3 (10ng/ml) for 2h. The cells were then incubated in TGF-β3-free medium and the cell culture superna-tant were harvested at 2.25h,2.5h,3h,4h,6h,10h and 14h. The ELISA was used to detect the extracellular secretion of endogenous TGF-β3 by HSC. (4) The effects of exogenous TGF-β3 on proliferation of HSC: HSCs were seeded in 96-well plates and divided into group A and B. The cells of group A were grown in the presence of exogenous TGF-β3 of different concentrations for 24 hours; the cells of group B were treated with TGF-β3 (5ng/ml) for 24 and 48 hours, and evaluated for proliferation ability by MTT method. (5)Cell morphology: The difference of HSC shape between the blank control group and TGF-β3 group was observed under inverted microscope.Results: (1)Exogenous TGF-β3 treatment up-regulated the expression of TGF-β3 mRNA, maximal expression level of TGF-β3 mRNA to 3.11 fold above control values was achieved at 2h after exogenous TGF-β3 treatment,(P<0.05). Thereafter, TGF-β3 mRNA expression levels declined, so that expression levels were maintained at levels of 1.56-fold for at least 10h and were 1.30-fold above control values by 24h after TGF-β3 treatmen(tP<0.05)(.2)No significant difference about the intracellular protein expression level of endogenous TGF-β3 was found at the time points indicated(.P>0.05);(3)The total content of TGF-β3 stimulated by exogenous TGF-β3 reached a peak at 4h after TGF-β3 treatment (1.89-fold higher than basic TGF-β3). After that, it slowly declined. The expression peak induction of extracellular secreted TGF-β3 was at 3h (32.36-fold higher than control, P<0.05). Thereaf-ter, TGF-β3 slowly decreased after the peak time, and their expression were still statistical significant compared with control (P<0.05). (4)Lower concentration of exogenous TGF-β3(0.001-0.08ng/ml)had no effect on proliferation of HSC, and relatively high concentra-tions exogenous TGF-β3 (greater than 0.32ng/ml) had the promotional effects on prolifera-tion of the HSC in vitro. (5) The results of the inverted microscope showed that cell gross shape was not affected by exogenous TGF-β3. Conclusions: (1)Exogenous TGF-β3 up-regulates the expression of endogenous TGF-β3 mRNA and extracellular secreted TGF-β3 protein obviously.(2)TGF-β3 could promote the proliferation of HSC. (3) Exogenous TGF-β3 treatment makes no difference to HSC gross morphology. |
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