小白菊内酯对人肝癌细胞HepG-2细胞Caspase-3和NF-κB蛋白表达的影响

Experimental study on the effect of parthenolide on human hepatocellular carcinoma HepG-2 cells

目的:研究小白菊内酯对人肝癌细胞Hep G-2增殖抑制和诱导凋亡作用的机制。为小白菊内酯在临床上早日应用于肝癌患者提供理论及实验依据。方法:用体外培养的方法培养人肝癌Hep G-2细胞;用四唑盐(MTT)比色法检测小白菊内酯对人肝癌Hep G-2细胞的抗增殖及细胞毒作用;用倒置显微镜、HE染色光学显微镜;用免疫组织化学染色法检测小白菊内酯作用人肝癌Hep G-2前后Caspase-3、NF-κB蛋白阳性表达变化的情况。通过以上实验研究小白菊内酯在体外对人肝癌Hep G-2在细胞增殖、细胞凋亡等方面的影响及其作用机制。结果:(1)MTT法结果显示:小白菊内酯对人肝癌Hep G-2细胞的作用影响具有浓度和时间依赖性;(2)细胞形态学观察:倒置显微镜观察到对照组细胞贴壁生长,伸展性良好,细胞呈梭形和多角形,细胞之间连接紧密。小白菊内酯作用人肝癌Hep G-2 24h以后,增殖速度减慢,细胞贴壁能力下降,细胞之间连接疏松。药物作用48h以后,增殖速度大大减慢,大部分细胞变圆漂浮在培养瓶中。药物作用72h以后,几乎无活细胞,并且可见死细胞碎片;HE染色光学显微镜下观察:对照组细胞呈梭形和多角形,细胞之间连接紧密,细胞胞浆丰富,细胞核完整;药物作用24h以后,细胞伸展减弱,有一部分细胞呈现圆形,细胞之间连接疏松,核浓缩,染色质出现边聚;药物作用48h后视野中可见凋亡细胞和死亡细胞,细胞浆浓缩、染色质浓缩成新月形和"出泡"现象的变化;(3)免疫组化结果显示:实验组Caspase-3表达增加,NF-κB表达降低,与对照组相比具有显著性差异(P<0.05)。结论:小白菊内酯对人肝癌Hep G-2细胞有明显的抑制增殖作用,呈现出浓度和时间的依赖性;小白菊内酯对人肝癌Hep G-2细胞株有诱导凋亡作用,其诱导人肝癌Hep G-2细胞凋亡机制可能与NF-k B、Caspase-3等信号转导有关。

Objective： To study the mechanism of action of parthenolide on human hepatocellular carcinoma Hep G- 2 cell proliferation inhibition and apoptosis induction effect; and to provide theoretical and experimental basis for parthenolide in clinical application as soon as possible in patients with hepatocellular carcinoma. Methods：Human hepatocellular carcinoma Hep G- 2 cells were cultured the in vitro. cultured human hepatocellular carcinoma Hep G- 2cells; four tetrazolium（ MTT） was used to detectthan the antiproliferative and cytotoxic effect of detection of parthenolide assay in human hepatocellular carcinoma cell line Hep G- 2. By inverted microscope,HE staining light microscope was used to observeation of parthenolide effect of human hepatocellular carcinoma Hep G- 2cells and cell morphology change with the changes before and after drug detection. Immunohistochemistry staining of parthenolide was used to detect human hepatocellular carcinoma Hep G- 2 and expression of Caspase- 3,NF- κB protein positive changes in the situation. Through the above experiment of parthenolide in vitro on human hepatocellular carcinoma Hep G- 2 effects on cell proliferation,apoptosis and its mechanism of action were studied. Results：（ 1） MTT method results showed that： the effect of parthenolide was dependent on the concentration and time in affecting human hepato cellular carcinoma Hep G- 2 cells. in a concentration and time dependent;（ 2） the morphology of cells was observed by inverted microscope observation. The ： to control growth of adherent cells in control group was observed,stretching for good,Cells were spindle and polygonal cells wereare connected tightly. Parthenolide effect of human hepatocellular carcinoma Hep G- 2 24 h,proliferation,cell adherence ability drops,loose connections between cells,some cells became round floating in the culture flask. After Ddrug effecteds after 48 h,the proliferation speed slowed down considerably,Most of the cells became round floating in the culture flask. Drug effects after 72 h,almost no live cells,dead cells and debris visible. We and observed under light microscope with HE staining： group cells were spindle and polygonal control,cells wereare connected tightly,rich in cytoplasm,nucleus integrity; drug effect after 24 h,cell spreading weakened,some cells showed round,osteoporosis,connections between cells pyknosis,chromatin appeared margination; apoptotic cells and cell death after 48 h in the view of drug effect,cytoplasm condensed,dyeing into crescent and the ＂ bubble＂ phenomenon of condensation;（ 3） The results of immunohistochemistry showed that： the experimental group increased the expression of Caspase- 3,but reduced the expression of NF- κB, which compared with the control group with significant difference（ P〈 0. 05）. Conclusions： 1Parthenolide has obvious effect on inhibiting the proliferation of human hepatocellular carcinoma Hep G- 2 cells,showing a concentration and time dependence; 2Parthenolide to induces the apoptosis of human hepatocellular carcinoma cell line Hep G- 2,The possible mechanism of human hepatocellular carcinoma Hep G- 2 cells inducesd apoptosis and NF- k B,Caspase- 3 and other signal transduction.