Liu Cheng-hai, Xan Hong-ping
Shanghai University of Traditional Chinese Medicine, Institute of Liver Disease, Shanghai 200032, China.
Zhonghua Gan Zang Bing Za Zhi. 2003 Dec;11(12):731-4.
To investigate the biological responses of cultured hepatic stellate cells (HSC) at different differentiated stages on exotic transforming growth factor (TGF-beta1).
HSC was isolated from rat and primarily cultured in uncoated disc for 1 d, 4 d and 7 d, when the cells were at quiescent, intermediate activated and full activated stages respectively. The cells were incubated with 10 pmol/L to 500 pmol/L TGF-beta1 for 24 h, cell proliferation was measured with [3H] TdR incorporation, alpha-smooth muscle actin (alpha-SMA) and type I collagen protein were assayed with Western blot, and total protein secretion in the culture supernatant was analyzed by [3H] proline pulse and collagenase digestion. HSC was treated with 100 pmol/L TGF- beta1 for 15 min to 90 min, and type I pro-collagen mRNA level was assayed by Northern blot.
TGF-beta1 remarkably inhibited d1 HSC proliferation, the percentage of [3H] TdR incorporation at 10 pmol/L to 500 pmol/L TGF-beta1 was 52.8% to 16.8% of the control, q value was 5.44 to 10.37 and P<0.01 vs control. But TGF-beta1 had no influence on d4 and d7 HSC. As the cells cultivation prolonged and activated, the basal levels of alpha-SMA, type I collagen and gene expression increased gradually. TGF-beta1 increased the above protein and gene expression. The basal and TGF-beta1 stimulated total protein secretion levels at d1-d7 HSC were 804+/-274 vs 1200+/-708; 2966+/-1701 vs 6160+/-1123, t=3.84, P<0.01; 2580+/-767 vs 4583+/-1467, t=2.96, P<0.05. While d4 HSC showed the strongest response of total protein secretion and alpha-SMA expression.
TGF-beta1 remarkably inhibited quiescent HSC proliferation, and promoted HSC collagen production at both quiescent and activated HSC. Intermediate HSC had the strongest response to TGF-beta1, while activated HSC lost the response to TGF-beta1 inhibitory growth, and TGF-beta1 exerted divergent actions on HSC as the cells activated.
研究不同分化阶段的培养肝星状细胞（HSC）对外源性转化生长因子（TGF-β1）的生物学反应。
从大鼠分离HSC并在未包被的培养皿中进行原代培养1天、4天和7天，此时细胞分别处于静止期、中间激活期和完全激活期。将细胞与10 pmol/L至500 pmol/L的TGF-β1孵育24小时，用[3H]TdR掺入法检测细胞增殖，用蛋白质印迹法检测α-平滑肌肌动蛋白（α-SMA）和I型胶原蛋白，并用[3H]脯氨酸脉冲和胶原酶消化法分析培养上清液中的总蛋白分泌。用100 pmol/L的TGF-β1处理HSC 15分钟至90分钟，用Northern印迹法检测I型前胶原mRNA水平。
TGF-β1显著抑制第1天HSC的增殖，10 pmol/L至500 pmol/L的TGF-β1作用下[3H]TdR掺入百分比为对照组的52.8%至16.8%，q值为5.44至10.37，与对照组相比P<0.01。但TGF-β1对第4天和第7天的HSC无影响。随着细胞培养时间延长和激活，α-SMA、I型胶原蛋白的基础水平及基因表达逐渐增加。TGF-β1增加上述蛋白和基因表达。第1天至第7天HSC的基础及TGF-β1刺激后的总蛋白分泌水平分别为804±274对1200±708；2966±1701对6160±1123，t=3.84，P<0.01；2580±767对4583±1467，t=2.96，P<0.05。而第4天的HSC在总蛋白分泌和α-SMA表达方面表现出最强反应。
TGF-β1显著抑制静止期HSC增殖，并促进静止期和激活期HSC的胶原生成。中间激活期的HSC对TGF-β1反应最强，而激活期的HSC失去对TGF-β1抑制生长的反应，且随着细胞激活，TGF-β1对HSC发挥不同作用。
