雷帕霉素对人急性淋巴细胞白血病细胞SUP-B15的增殖、凋亡与自噬的影响

目的探讨雷帕霉素(RAPA)对人急性淋巴细胞白血病SUP-B15细胞的增殖、自噬与凋亡的影响。方法常规方法复苏、传代培养SUP-B15细胞,设置空白对照组(正常培养)、雷帕霉素处理组。处理24、48、72 h后收集细胞,采用MTT比色法检测细胞的活性和增殖能力;采用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;吖啶橙单染及透射电镜观察细胞自噬现象。结果 MTT检测结果显示,RAPA对SUP-B15细胞增殖有较强抑制作用(IC50=18.174 nmol/L),且抑制作用随药物剂量增加和作用时间延长而增强;流式细胞仪检测细胞凋亡结果显示,不同浓度RA-PA作用可诱导SUP-B15细胞凋亡,作用48、72 h细胞凋亡较24 h时明显;RAPA作用48 h时的细胞凋亡随药物浓度增大而增多,72 h时高剂量药物(50、100 nmol/L)引起的细胞凋亡较低剂量药物(10、20 nmol/L)少;吖啶橙单染荧光显微镜观察显示,与空白对照组相比,雷帕霉素处理组细胞胞质中出现较多红色斑点的酸性膜泡(即自噬小体),且随药物浓度增大红色斑点增多;透射电镜观察结果显示,雷帕霉素处理组细胞胞质内可见较多自噬小体和自噬溶酶体,线粒体内嵴明显紊乱,细胞核不规则,染色质边缘化。结论雷帕霉素对SUP-B15细胞生长有较强的抑制作用;雷帕霉素可诱导SUP-B15细胞发生自噬和凋亡,且自噬的发生先于凋亡出现,一定强度的自噬活性可诱导细胞凋亡增加。

Study on the Effects of Rapamycin on Cell Proliferation, Apoptosis and Autophagy in Human Acute Lymphoblastic Cell Line SUP-B15

Objective To explore the effects of Rapamycin （RAPA） on cell proliferation,apoptosis and autophagy in hu- man acute lyrnphoblastic cell line SLIP - B15. Methods SUP - B15 cells were recovered and cultured with conventional methods and were studied in logarithmic phase. A control group （cells cultured without RAPA） and ten rapamycin treatment groups were set. Cells were collected after 24 h, 48 h or 72 h treatement. Cell viability and proliferation were assessed by MTT colorimetric assay. Cell appoptosis was detected by Annexin V- FITC/PI double staining flowcytometry. Cell autophagy was ob- served by fluorescence microscopy after acridine orange staining and by transmission electron microscopy. Results MTT assay showed that RAPA produced strong inhibition on the proliferation of SUP- B15 cells in a time- and dose- dependent manner （the ICs0 value is 18. 174 nmol/L）. Flowcytometry indicated that RAPA could induce apoptosis in SUP - B15 cells at various concentrations, and the effect was more obvious after the cells were treated for 48 h, 72 h than for 24 h. RAPA treatment for 48 h induced cell apoptosis in a dose - dependent manner, while RAPA treatment for 72h induced less cell apoptosis in higher doses （50, 100 nmol/L） than in lower doses （10, 20 nmol/L）. Fluorescence microscopy after acridine orange staining found that the SLIP-B15 cells incubated with different concentrations of RAPA for 72 h emitted more red fluorescein in acidic vesicular or- ganelles （autophagosome） than the control cells, and it was more obvious with the increase of RAPA concentration. Transmission electron microscopy revealed some autolysosome and vacuoles in SUP - B15 cells treated with 100 nmol/L rapamycin for 72 h, and mitochondria cristae chaos, mesoplast anomalism and chromatin marginalization were evident. Conclusions Rapamycin can significantly inhibit the proliferation of SUP - B15 cells in a time - and dose - dependent manner. It can induce autophagy and apoptosis in SUP - B15 cells. Autophagy occurred before apoptosis. Excessive autophagy activity can induce apoptosis.