复方苦参注射液对人食管鳞癌EC9706细胞凋亡及生长抑制的影响

Apoptosis and growth arrest of human esophageal squamous cell carcinoma cell EC9706 induced by Fufangkushen injection

目的探讨复方苦参对人食管癌细胞株EC9706的生长抑制和诱导凋亡作用。方法体外实验分为复方苦参25．00μl／ml组、6．25μl／ml组及对照组。采用四甲基偶氮唑蓝（MTT）法检测细胞增殖活性，SP法检测增殖细胞核抗原（PCNA）表达。流式细胞仪测定细胞周期及细胞凋亡，Western印迹检测细胞半胱氨酸蛋白酶（caspase）-3、Bcl-2及Fas蛋白的表达。平板克隆形成实验测定细胞克隆形成率。裸鼠移植瘤实验检测复方苦参的体内抑瘤作用，分为200μl／d治疗组、25μl／d治疗组及生理盐水对照组。sP法检测移植瘤PCNA及Bcl02的表达，原位末端标记（TUNEL）法检测细胞凋亡。结果体外实验中25．00μl／ml组细胞48、72、96h增殖活性均低于对照组（均P〈0．01）；PCNA表达水平及体外克隆形成率均低于对照组（均P〈0．05）；细胞凋亡率、激活型caspase-3表达及Fas表达均高于对照组[（25．2±7．3）％比（3．4±1．5）％、（21．3±4．4）％比（1．8±0．6）％、（30．2±8．3）％比（5．4±1．6）％，均P〈0．01]，Bcl-2表达低于对照组（P〈0．01）。动物实验200μl／d治疗组瘤体质量低于生理盐水对照组[（987±386）mg比（1935±838）mg，P〈0．01]，凋亡指数高于生理盐水对照组[（33．8±8．7）％比（5．3±1．4）％，P〈0．01]。结论复方苦参能够抑制EC9706细胞增殖，诱导细胞凋亡，阻止移植瘤生长。诱导凋亡机制可能与EC9706细胞周期阻滞、Bcl-2表达下调、Fas表达上调及caspase-3激活有关。

Objective To investigate the inhibitory effect and apoptosis induction on human esophageal carcinoma EC9706 cell by Fufangkushen. Methods The experiment of Fufangkushen was designed into three groups including 25.00 μl/ml group, 6. 25 μl/ml group and control group in vitro. The method of MTT was used to evaluate the growth inhibition effects. Proliferating cell nuclear antigen （PCNA） was detected by immunohistochemistry （IHC） in vitro. The morphological changes of cells were observed under inverted microscope. FACS was used to analyze the distribution of cell cYcle and apoptosis. The expressions of Bcl-2, Fas and caspase-3 in EC9706 cells were detected by Western blotting. The clone formation in plate was used to test the capacity of cell clone formation. Nude mice experiments were conducted to investigate the tumor inhibition of Fufangkushen in vivo. The mice were divided into 3 groups of 200 μl/d treatment, 25 μl/d treatment and saline control. PCNA and Bcl-2 were detected by IHC. And the apoptotic index was detected by terminal transferase dUTP nick end labeling （TUNEL） on xenograft of nude mice. Results The proliferative capacities of 25.00 μl/ml group were lower than that of the control group at 48, 72, 96 h respectively（ all P 〈 0. 01 ）. IHC showed the PCNA expressions, cell clone formation rate were both lower than that of control group （in 25.00 μl/ml treatment group both P 〈 0. 05）. Many apoptotic cells could be observed. And the apoptotic rate was higher in 25.00 μl/ml group than that in the control group （ （25. 2 ± 7. 3 ） % vs （ 3.4 ± 1.5 ） %, P 〈 0. 01 ）. After a treatment of Fufangkushen, the activation of caspase-3 and theFas were higher（（21.3 ±4.4）% vs （1.8 ±0.6）%, （30.2 ±8.3）% vs （5.4 ± 1.6）%, both P 〈 0. 01 ）, the Bcl-2 were lower （P 〈 0.01 ） were observed in vitro. Comparing with the saline control group, the tumor weight in 200 μl/d treatment group were lower （（ 987 ± 386 ） vs （1935 ± 838） mg, P 〈0.01） and the apoptotie index higher （（33.8±8.7）% vs （5.3±1.4）%, P〈0. 01）. Conclusion Fufangkushen can inhibit the proliferation of EC9706 cells and induce the cellular apoptosis. The mechanism of apoptosis is probably associated with the arrest of cell cycle, the up-regulation of Fas, the down-regulation of Bcl-2 and the activation of caspase-3 in ESCC EC9706 cells.