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目的 研究新型水疱口炎病毒VSVΔM51联合新型小分子抑制剂—核糖体S6激酶1(RSK1)抑制剂BI-D1870和Polo样激酶1(PLK1)抑制剂BI2536对胶质瘤细胞的体外杀伤效果.方法 (1)将体外培养的GL261、CT2A、HS68细胞分为对照组、雷帕霉素组、BI-D1870组、BI-2536组、VSVΔM51组、雷帕霉素+VSVΔM51组、BI-D1870+VSVΔM51组、BI2536+VSVΔM51组,分别用100 nmol/L雷帕霉素,10μmol/L BI-D1870,100 nmol/L BI-2536预处理2 h后感染0.1感染复数(MOI)VSV△M51病毒,72 h后采用Alarma Blue法测定细胞的存活率;分别用上述药物预处理1 h后感染10 MOI VSV△M51病毒,24 h后活化Caspase-3染色检测GL261细胞的凋亡;Western blotting检测细胞凋亡蛋白聚ADP-核糖聚合酶(PARP)的表达;Annexin V-FITC/碘化丙啶(PI)双染法检测细胞凋亡.(2)将GL261、CT2A细胞细胞分为VSVΔM51组 、 雷帕霉素+VSVΔM51组 、BI-D1870+VSVΔM51组 、BI2536+VSVΔM51组,分别用上述药物预处理1 h后感染0.1 MOI VSV△M51病毒,48 h后荧光显微镜下观察细胞中绿色荧光蛋白(GFP)的表达;IVIS200活体成像系统检测4组细胞病毒荧光素酶的变化;(3)15只CT2A细胞颅内种植胶质瘤模型小鼠,按随机数字表法分为VSV△M51组、BID-1870+VSV△M51组和BI2536+VSVΔM51组,每组5只.后2组小鼠分别腹腔注射BI-1870(100 mg/kg)、静脉注射BI-2536(20 mg/kg),24 h后3组小鼠均静脉注射病毒VSV△M51,24 h、72 h后IVIS200活体成像系统检测病毒荧光素酶;15只GL261细胞颅内种植胶质瘤模型小鼠的分组和处理同上,48 h后荧光显微镜下观察病毒GFP的表达;病毒噬斑法检测小鼠的病毒滴度.结果(1)与对照组、 雷帕霉素组、VSVΔM51组、BI-D1870组、BI-2536组比较,雷帕霉素+VSVΔM51组、BI-D1870+VSVΔM51组、BI2536+VSVΔM51组细胞存活率显著降低,差异有统计学意义(P<0.05).活化Caspase-3染色检测显示对照组无凋亡,雷帕霉素组、BI-D1870组、BI-2536组、VSVΔM51组可见少量凋亡小体,但雷帕霉素+VSVΔM51组、BI-D1870+VSVΔM51组、BI2536+VSVΔM51组凋亡小体明显增多.Western blotting检测显示对照组、雷帕霉素组、BI-D1870组、BI-2536组、VSVΔM51组GL261、CT2A细胞活化PARP蛋白的表达少于雷帕霉素+VSVΔM51组、BI-D1870+VSVΔM51组、BI2536+VSVΔM51组.Annexin V-FITC/PI染色结果与活化Caspase-3染色结果一致.(2)与VSVΔM51组、雷帕霉素+VSVΔM51组比较,BI-D1870+VSVΔM51组、BI2536+VSVΔM51组细胞GFP的表达增强、病毒荧光素酶更亮,差异均有统计学意义(P<0.05).(3)72 h后VSV△M51组、BID-1870+VSV△M51组、BI2536+VSV△M51组CT2A细胞颅内种植胶质瘤模型小鼠荧光素酶亮度依次增加,差异有统计学意义(P<0.05).48 h后VSV△M51组、BID-1870+VSVΔM51组、BI2536+VSV△M51组GL261细胞颅内种植胶质瘤模型小鼠的病毒滴度依次增加,差异均有统计学意义(P<0.05).结论 2种小分子抑制剂都在一定程度上促进了VSV△M51病毒的复制,进而增强了对胶质瘤细胞的杀伤作用,并且其增效效果明显优于雷帕霉素.“,”Objective To study the in vitro killing effect of novel small molecule inhibitors, ribosomal S6 kinase1 (RSK1) inhibitor (BI-D1870) and polo-like kinase 1 (PLK1) inhibitor (BI2536), combined with recombinant attenuated vesicular stomatitis virus VSVΔM51 on various glioma cells. Methods (1) In vitro cultured GL261, CT2A and HS68 cells were divided into control group, rapamycin group, BI-D1870 group, BI-2536 group, VSVΔM51 group, rapamycin +VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 group; pretreatments with 100 nmol/L rapamycin, 10 μmol/L BI-D1870, and 100 nmol/L BI-2536 for 2 h were given to the cells from the above groups, respectively, and then, they were infected with VSVΔM51 virus at 0.1 mutiplicity of infection (MOI); at 72 h after treatments, the cell survival rate was determined by Alarma Blue method; VSV△M51 virus was infected at 10 MOI one h after pretreatment with the above drugs, apoptosis of GL261 cells was detected by cleaved caspase-3 staining 24 h after that; the expression of apoptotic protein polyadp-ribosomal polymerase (PARP) was detected by Western blotting; Annexin V-FITC/propidium iodide double staining was used to detect the cell apoptosis. (2) GL261 and CT2A cells were divided into VSVΔM51 group, rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+ VSVΔM51 group; VSV△M51 virus was infected at 0.1 MOI one h after pretreatment with the above drugs,; 48 h after treatments, fluorescence microscope was used to detect the expression of green fluorescent protein (GFP); IVIS200 in vivo imaging system was used to detect the changes of cell virus luciferase in the 4 groups. (3) Fifteen CT2A intracranial implanted glioma model mice were divided into VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group according to random number table method (n=5); mice in the latter two groups were intraperitoneally injected with BI-1870 (100 mg/kg) or intravenously injected with BI-2536 (20 mg/kg); 24 h after that, mice in the three groups were intravenously injected with virus VSVΔM51; virus luciferase was detected by IVIS200 in vivo imaging system 24 and 72 h after treatments; the grouping and treatments of GL261 intracranial glioma model mice were the same as above, the expression of virus GFP was observed under fluorescence microscope 48 h after treatments, and virus titers of these mice were detected by virus plaque assay. Results (1) As compared with the control group, rapamycin group, BI-D1870 group, BI-2536 group, and VSVΔM51 group, the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 group had significantly lower cell survival rate (P<0. 05); cleaved Caspase-3 staining showed no cell apoptosis in the control group, a small amount of apoptotic corpuscles in the rapamycin group, BI-D1870 group, BI-2536 group, and VSVΔM51 group, but obvious increased amount of apoptotic corpuscles in the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+ VSVΔM51 group; Western blotting indicated that GL261 and CT2A cells from the control group, rapamycin group, BI-D1870 group, BI-2536 group, and VSVΔM51 group had lower cleaved PARP expression level than those from the rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group, and BI2536+VSVΔM51 group. The results of Annexin V-FITC/propidium iodide double staining were consistent with those of cleaved Caspase-3 staining. (2) As compared with VSVΔM51 group and rapamycin+VSVΔM51 group, BI-D1870+VSVΔM51 group and BI2536+VSVΔM51 group had significantly increased GFP expression and statistically higher intensity of virus luciferase (P<0.05). (3) CT2A cells in the VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group had increased intensity of virus luciferase successively, with significant differences (P<0.05); GL261 cells in the VSVΔM51 group, BID-1870+VSVΔM51 group and BI2536+VSVΔM51 group had increased virus titers successively, with significant differences (P<0.05). Conclusion Both small molecule inhibitors promote the replication of VSVΔM51 virus and enhance the killing effect on glioma cells, and its synergistic effect is obviously better than rapamycin.