重组人干扰素α-2b-卡介苗对膀胱癌MB49细胞的体外作用

Effect of recombinant hIFN-αt-2b-BCG on mouse bladder tumor MB49 cells in vitro

目的探讨重组人干扰素α-2b-卡介苗（hIFN—α-2b—BCG）对膀胱肿瘤细胞的直接效应及其作用机制。方法采用体外实验，将重组hIFN-α-2b—BCG直接作用于小鼠膀胱癌MB-49细胞，荧光染色后，在光镜和电镜下观察细胞的变化。采用四甲基偶氮唑蓝（MTT）和流式细胞仪（FCM），检测野生型BCG和重组hIFN—α-2b—BCG对MB49细胞的影响。结果重组hIFN-α-2b—BCG与MB49细胞共培养后，光镜下见肿瘤细胞变圆，增殖速度明显减缓，部分细胞脱壁，胞浆呈大量空泡和颗粒状。透射电子显微镜下可见肿瘤细胞表面微绒毛脱落，细胞质坏死，大量空泡，细胞表面有出泡现象。吖啶橙染色后，在荧光显微镜下可见肿瘤细胞聚集成团状的细胞球体，胞突消失，凋亡小体出现。Hoechst33258染色后，可见大量肿瘤细胞呈明亮的蓝色荧光。MTT测定结果显示，随共培养时间延长，重组hIFN—α-2b—BCG可不同程度抑制肿瘤细胞的生长，抑制率高于野生BCG。FCM检测结果显示，24h后野生型BCG诱导凋亡率为10．8％，重组hIFN—α-2b—BCG诱导凋亡率为19．7％；48h后野生型BCG诱导凋亡率为20．9％，重组hIFN—α-2b—BCG诱导凋亡率为46．6％。两者比较，差异有统计学意义（P〈0．01）。重组hIFN-α-2b—BCG上调MB49细胞MHC—I表达，具有时间依赖性，且上调比例明显高于野生型BCG（P〈0．01）。结论重组hIFN—α-2b—BCG成功诱导肿瘤细胞凋亡，有更高的调节免疫原性、抗肿瘤作用和细胞毒性。

Objective To investigate the antitumor effect of recombinant IFN-α-2b-BCG on mouse bladder cancer MB49 cells in vitro, and to explore its antitumor mechanisms. Methods MB49 cells were coultured with recombinant BCG or wild BCG, and than were examined by light and transmission electron microscopy. The cell growth was assessed by MTT assay, and apoptosis rate and MHC-I of the MB49 cells was detected by flow cytometry using AO and Hoechst33258 fluorescence immunostaining. Results The hIFN-α-2b-BCG-treated tumor cells showed slow growth, detachment of some cells, and various degree of degeneration. Light microscopy revealed organelle disorganization, chromatin aggregation, nuclear pyknosis, and cytolysis in some cells. Cellular membrane bulged and some bubbles were seen under fluorescence microscope using AO staining. Hoeehst33258 assay also depicted frequent apoptosis in the tumor cells. The MTT assay showed that rBCG more actively than the wild BCG inhibited the proliferation of MB49 cells. The apoptosis rate of the recombinant BCG group was 19.7% and 46.6% at the time point of 24 h and 48 h, respectively, significantly higher than 10.8% and 20.9% , respectively, in the wild BCG group. The results of flow cytometry indicated that both types of BCG enhanced the expression of MHC-I in the MB49 cells, but more effective in the recombinant BCG group. Conclusion The recombinant hIFN-α-2b-BCG has more strong immuno-modulatory properties, anti-tumor effect on MB49 cells and induces apparent cytotoxicity in the bladder cancer cells in vitro.