华蟾素联合丝裂霉素诱导人宫颈癌细胞株Hela细胞发生凋亡的实验研究

Study on Apoptosis of Human Cervical Carcinoma Hela Cell Induced by Bufonin and Mitomycin

目的探讨抗癌药物华蟾素与丝裂霉素的联合作用诱导Hela细胞发生凋亡的机制。方法体外培养人宫颈癌Hela细胞,以不同浓度的华蟾素与丝裂霉素刺激Hela细胞,倒置显微镜下观察细胞形态,MTT法检测细胞的增殖,凝胶电泳检测细胞DNA片段化,FITC-Annexin V检测细胞凋亡的发生,RT-PCR检测TNF-α的表达。结果经药物作用后的细胞在倒置显微镜下观察,6 h后开始出现明显的凋亡现象;电泳结果呈200 bp大小的典型梯状带;FITC-Annexin V检测到凋亡早期细胞膜呈强绿色荧光,12 h后胞核逐渐被碘化丙啶染成红色,24 h后大部分细胞被染成红色;RT-PCR产物大小为299 bp。结论华蟾素和丝裂霉素能使细胞生长受到明显的抑制,能诱导Hela细胞发生凋亡,能诱导DNA片段化和TNF-α的表达。

Objective To approach the apoptosis mechanism of the anti - cancer drugs of bufonin and mitomycin to human cervical carcinoma cell Hela. Methods Human cervical carcinoma Hela cells were cultured in vitro, and stimulated with different concentration of bufonin and mitomycin. The cell morphology was observed under the inverted microscope, the cell proliferation was detected by MTT. DNA gragmentation was detected by agarose gel electrophoresis. Cell apoptosis was inspected by FITC- Annexin V. In addition, the expression of TNF - α in Hela cell was observed by RT - PCR. Results After treating by the drugs for six hours, the cell apoptosis was apparently appeared, the result of the apoptotic DNA was presented to a 200bp fragment after the agarose gel electrophoresis. In the prophase of the apoptosis, the membrane was dyed into green fluorescence, after 12 hours, the nucleus was dyed by propidium iodide gradually, and after 24 hours, most of the cells were dyed into red color. The product of the RT- PCR was 299bp. Conclusions The combined use of bufonin and mitomycin can inhibit the cell growth, induce the apoptosis of the Hela cell, and induce fragments of DNA and expression of TNF -α.