转化生长因子β1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

目的 观察不同活化状态肝星状细胞(HSC)对外源性转化生长因子-β_1(TGF-β_1)旁分泌刺激的生物学效应作用。方法 原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500 pmol/L TGF-β_1温育细胞24h,~3H—TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原蛋白表达沉积,~3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量。100pmol/L TGF-β_1温育细胞15～90min,northern blot法检测细胞Ⅰ型前胶原mRNA的表达水平。结果 TGF-β_1浓度依赖性抑制培养1d HSC的细胞增殖,10～500 pmol/L TGF-β_1浓度组细胞内~3H—TdR掺入率分别为对照组的52.8％～16.8％,与对照组比较,q值为5.44～10.37,P<0.01。但TGF-β_1对培养4d与7d的细胞增殖无影响。随细胞活化,HSC基础性α-SMA、Ⅰ型胶原蛋白与mRNA水平明显增加,而TGF-β_1刺激各培养时间HSC以上蛋白与基因的表达。培养1、4、7d HSC基础水平与TGF-β_1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1 200±708)dpm/孔;(2 966±1 701)dpm/孔与(6 160±1 123)dpm/孔;(2 580±767)dpm/孔与(4 583±1 467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05。以培养4d HSC

Effects of transforming growth factor β1 on the proliferation and type I collagen expression at different differential rat hepatic stellate cells

OBJECTIVES: To investigate the biological responses of cultured hepatic stellate cells (HSC) at different differentiated stages on exotic transforming growth factor (TGF-beta1). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.