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目的探讨鳖甲煎改良方含药血清对大鼠肝星状细胞(hepatic stellate cell,HSC)-T6增殖、周期及凋亡的影响。方法分别用高剂量[28.4 g/(kg·d)]、中剂量[14.2 g/(kg·d)]与低剂量[7.1 g/(kg·d)]的鳖甲煎改良方及与鳖甲煎改良方高剂量相同剂量的鳖甲煎丸灌胃大鼠以制备含药血清。将等体积不同剂量的鳖甲煎改良方含药血清分别处理HSC-T6细胞,以等体积鳖甲煎丸含药血清处理的细胞为阳性对照组,生理盐水处理的细胞为空白对照组,正常大鼠血清处理的细胞为正常对照组。CCK-8检测不同处理组HSC-T6细胞的增殖情况,并绘制其增殖曲线;流式细胞仪(flow cytometry,FCM)检测经不同处理的HSC-T6细胞的周期,Annexin V-PI染色法检测其凋亡;在mRNA及蛋白水平分别检测经不同处理的HSC-T6细胞的增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)基因及其编码蛋白的表达变化。结果 CCK-8检测结果显示,鳖甲煎改良方含药血清对HSC-T6细胞增殖具抑制作用,高剂量与中剂量组的增殖速度慢于正常对照组、阳性对照组和空白对照组(P<0.05,P<0.01),且该抑制作用呈剂量-效应关系。FCM检测证实,鳖甲煎改良方含药血清可将HSC-T6细胞阻滞于S期,能诱导HSC-T6细胞的凋亡,与正常对照组比较,其凋亡诱导率差异具有统计学意义(P<0.01)。mRNA与蛋白水平检测结果显示,与空白对照组和阳性对照组比较,高剂量组能在基因与蛋白水平显著下调HSC-T6细胞PCNA的表达。结论鳖甲煎改良方含药血清能明显抑制HSC-T6细胞的增殖,诱导HSC-T6细胞的凋亡,并能下调其PCNA基因及其编码蛋白的表达。
Objective To investigate the effects of Biejiajian Modified Recipe on the proliferation, cell cycle and apoptosis of hepatic stellate cell (HSC) -T6 in rats. Methods High dose [28.4 g / (kg · d)], middle dose [14.2 g / (kg · d)] and low dose [7.1 g / (kg · d) Jiajian modified high-dose high dose of the same dose of biejia Pill fed rats to prepare containing serum. The same volume of different doses of Biejiajian modified prescription serum containing HSC-T6 cells were treated with an equal volume of Biejiaorou containing serum-treated cells as a positive control group, saline-treated cells as a blank control group, normal Rat serum-treated cells were normal control group. The proliferation of HSC-T6 cells in different treatment groups was detected by CCK-8 and the proliferation curve was drawn. The cycle of HSC-T6 cells treated with different treatments was detected by flow cytometry (FCM) The apoptosis and apoptosis of HSC-T6 cells were detected by flow cytometry. The expression of PCNA gene and its encoded protein in HSC-T6 cells treated with different treatments were detected at mRNA and protein levels respectively. Results The results of CCK-8 test showed that BJJT-containing serum could inhibit the proliferation of HSC-T6 cells. The proliferation rate of HSC-T6 cells was lower than that of the normal control group, the positive control group and the blank control group (P <0.05, P <0.01), and the inhibition was dose-effect relationship. FCM test confirmed that BMRA modified drug-containing serum could arrest HSC-T6 cells in S phase and induce apoptosis of HSC-T6 cells, compared with the normal control group, the apoptosis induction rate was statistically significant (P <0.01). The mRNA and protein levels of HSC-T6 cells in high-dose group were significantly lower than those in blank control group and positive control group (P <0.05). Conclusion Modified Biejia decoction can significantly inhibit the proliferation of HSC-T6 cells, induce the apoptosis of HSC-T6 cells, and down-regulate the expression of PCNA gene and its encoded protein.