外源性转化生长因子β3对HSC—T6细胞内源性转化生长因子β3表达的影响

Effects of exogenous TGF-β3 on the expression of endogenous TGF-β3 in hepatic stellate cell-T6 （HSC-T6）

目的通过对大鼠肝星状细胞株（HSC-T6）中内源性转化生长因子β3（TGF-β3）mRNA和蛋白水平的检测，探讨外源性TGF-β3对HSC-T6分泌内源性TGF-β3的影响。方法体外培养HSC-T6，未加入外源陛TGF-β3培养的细胞为对照组，加入外源性TGF-β3（10ng／m1）培养的细胞为TGF—p3组。（1）分别在1、2、4、12h和24h收集细胞，用实时荧光定量PCR法检测内源性TGF-β3mRNA表达；（2）分别在0、1、6h和12h收集细胞，用Western b1ot法检测细胞内TGF-β3蛋白的表达；（3）分别在0、1、2、4、8、14h和24h收集细胞上清液，用酶联免疫吸附法（ELISA）检测细胞外总TGF-β3蛋白的表达；加入外源性TGF-β3培养HSC-T6 2h，然后换用无血清的DMEM培养液继续培养细胞至2．25、2．5、3、4、6、10h和14h收集细胞上清液，用ELISA法检测细胞外内源性TGF-β3蛋白的表达。对数据进行单因素方差分析。结果（1）TGF-β3组细胞内TGF-β3mRNA表达水平明显增高，于2h达峰值（2．796±0．518），为对照组（1．022±O．038）的2．74倍（P〈0．05）；随后缓慢降低，维持在1．45倍水平达10h，24h时表达水平仍为对照组的1．18倍（P〈0．05）。（2）不同时间点细胞内TGF-β3表达水平均无明显变化（P〉0．05）。（3）TGF-β3组细胞外总TGF-βD3含量明显增加，于4h时达高峰[（18．931±2．904）ng／ml]，约为诱导量[（10．026±0．022）ng／m1]的1．89倍，随后开始下降；内源性TGF-β3于诱导后3h达高峰[（0．835±0．027）ng／m1]，为对照组[（0．026±0．022）ng／m1]的32．12倍，之后开始逐渐降低，直至维持一个弱增长的状态（P〈0．05）。结论外源性TGF-β3可促进HSC-T6细胞分泌大量内源性TGF-β3。

Objective To investigate the effects of exogenous TGF-β3 on the expression of endogenous TGF-β3 in hepatic stellate cell （HSC）. Methods HSCs were cultured and divided into two groups： TGF-β3 group and blank control group, the cells of TGF-β3 group were exposed to TGF-β （10 ng/ml）, whereas the blank control group was not treated. The cells were incubated in the presence of exogenous TGF-β and then （1） were harvested at Oh, lh, 2h, 4h, 12h, 24h, and real time PCR was performed to detect the mRNA expression of endogenous TGF-β3. （2） The cells were collected at Oh, lh, 6h, 12h, and western-blot was used to detect the protein synthesis of endogenous TGF-β3 in HSC; （3） The cell culture supematant was harvested at Oh, lh, 2h, 4h, 8h, 14h, 24h, and ELISA was performed to measure the total protein of extracellular TGF-β3; HSCs were treated with TGF-β3 （10ng/ml） for 2h. The cells were then incubated in serum-free medium and the cell culture supernatant was harvested at 2.25h, 2.5h, 3h, 4h, 6h, 10h and 14h. ELISA was used to detect the extracellular secret ion of endogenous TGF-β3 by HSCs. Results （1） Exogenous TGF-β3 treatment induced a marked increase in TGF-β3 mRNA expression. By 2h of exogenous TGF-β3 treatment, maximal TGF-β3 mRNA expression levels （2.796 ±0.518） of 2.74 fold above control values （1.022 ±0.038） was reached （P 〈 0.05）. Thereafter, TGF-β3 mRNA expression level declined, and the expression level was maintained at level of 1.45-fold for at least 10h and was 1.18-fold above control values by 24h TGF-β3 treatment （P 〈 0.05）; （2） No significant difference about the intracellular protein expression level of endogenous TGF-β3 was found between two groups. （P 〉 0.05）; （3） The total expression level of TGF-β reached a peak [（18.931± 2.904） ng/ml] at 4h after TGF-β3 treatment （1.89-fold higher than basic TGF-β3 （10ng/ml）. After that, it slowly declined. The expression peak [（0.835 ±0.027） ng/ml] induction of extracellular secreted TGF-β3 was at 3h （32.12-fold higher than control [（0.026 ± 0.022） ng/nil], （P 〈 0.05）. Thereafter, TGF-β3 slowly decreased after the peak time, and their expressions were still.statistically significant as compared to the control （P 〈 0.05）. Conclusion Exogenous TGF-β3 could increase the expression of endogenous TGF-βl3 mRNA and extracellular secreted TGF-β3 protein obviously.