Cyclopamine对肝癌细胞增殖和凋亡的影响及其机制的研究

Effect of cyclopamine on proliferation and apoptosis of hepatocarcinoma cells and its mechanism

目的:探讨sonic hedgehog(Shh)信号通路的特异性抑制剂环杷明(cyclopamine)对血清饥饿的人肝癌Hep3B细胞增殖、早期凋亡和白蛋白(albumin,ALB)、甲胎球蛋白(α-fetoprotein,AFP)表达的影响。方法:采用WST-8法、激光共聚焦扫描显微术、FCM法、RT-PCR和电化学发光免疫法,检测血清饥饿状态下cyclopamine引起的Hep3B细胞增殖抑制、线粒体膜电位改变,以及对细胞分泌ALB和AFP的影响。结果:血清饥饿状态下cyclopamine可明显抑制Hep3B细胞的增殖。半定量测量结果显示,Hep3B细胞平均红绿荧光吸光度比值有所降低;FCM法检测结果显示,6h后线粒体膜电位较0h时明显降低,差异有统计学意义(P<0.05)。AFP mRNA及蛋白分泌水平均明显降低,但对ALB mRNA及蛋白分泌影响不明显。结论:Cyclopamine对Hep3B细胞增殖以及AFP mRNA和蛋白的分泌有明显抑制作用,同时促进线粒体膜电位的降低,提示cyclopamine具有抑制肿瘤细胞生长和促进早期凋亡发生的作用。

Objective ： To investigate the influence of cyclopamine, a specific inhibitor of sonic hedgehog （Shh） , on proliferation, early apoptosis and expression of albumin （ALB） and alpha fetoprotein （AFP） of malignant hepatocarcinoma cell line Hep3B after serum starvation. Methods： The effect of cyclopamine on proliferation of Hep3B cells induced by serum starvation was assayed by WST-8 method. Laser scanning confocal microscopy and flow cytometry （FCM） were used to examine the effects of cyclopamine on apoptosis of Hep3B cells. The effect of cyclopamine on the cell mitochondrial membrane potential was determined by JC-1 staining. Reverse transcription-polymerase chain reaction （RT-PCR） was applied to detect the mRNA expressions of ALB and AFP in Hep3B ceils. Furthermore, the production of ALB and AFP in cultured Hep3B cell supernatant were measured by electrochemiluminescence （ECL） immunoassay. Results：Cyclopamine significantly inhibites cell proliferation under the condition of serum depletion. The semiquantitative analysis showed that the average absorbance of red and green fluorescence was decreased. FCM method indicated that the mitochondrial membrane potential of Hep3B cells was significantly lower at 6 h after exposure to cyclopamine compared with that at 0 h, and the difference was significant （ P 〈 0.05 ）. The mRNA level and protein secretion of AFP protein were significantly decreased. But cyclopamine has no significant effects on the mRNA level and protein secretion of ALB. Conelusion：Cyclopamine has significantly inhibitory effects on the proliferation, AFP mRNA expression, and AFP protein secretion of malignant hepatoma carcinoma cells and simuhaneously induces mitochondrial membrane potential collapse, which suggestes that cyclopamine inhibites tumor growth and induces early apoptosis of malignant hepatocarcinoma ceils.