【作者】 李文波；

【导师】 周建大；

【作者基本信息】 中南大学 ， 烧伤整形外科， 2011， 硕士

【摘要】 目的：在前期的研究基础上,探讨MicroR-33对黑色素瘤细胞株增殖和凋亡的影响。方法：构建靶向MicroR-33的高表达mimics及inhibitor干扰单链,运用脂质体转染技术将其导入黑色素瘤高转移细胞B16F10中,使MicroR-33基因高表达或表达抑制,用实时荧光定量PCR(Real-timePCR)验证MicroR-33基因的差异表达。用MTT法检测各组B16F10细胞的细胞增殖情况,流式细胞术检测细胞凋亡率和细胞周期,最后统计分析MicroR-33对B16F10黑色素瘤细胞增殖和凋亡的影响。结果：1.MicroR-33 mimics及inhibitor转染后,转染细胞内可见cy3荧光物质,提示转染成功。2.Real-time PCR检测结果,MicroR-33 mimics组中MicroR-33基因相对表达量(1.7733E3±2.4583E2)明显高于空白组和mimics对照组(0.9340±0.2913)(t=12.487,P=0.006<0.05)；MicroR-33 inhibitor组中MicroR-33基因相对表达量(0.6973±0.1958)低于空白组和inhibitor对照组(1.0200±0.0848),但未达到显著性差异；而mimics对照组,inhibitor对照组和空白组比较也无显著差异性(P>0.05)。3.转染48h后各组B16F10黑色素瘤细胞镜下显示MicroR-33 mimics组细胞密度较其他几组明显稀疏。MTT绘制细胞生长曲线发现,与空白组相比,MicroR-33 mimics组细胞生长呈下降趋势,且在转染后48h(1.1875±0.0502)(t=-14.5,P=0.044<0.05)及72h(1.7500±0.0933)(t=-13.59,P=0.047<0.05),此趋势具有显著性差异。而MicroR-33 inhibitor组,mimics对照组和inhibitor对照组细胞生长趋势与空白组比较均无显著差异性(P>0.05)。4.流式细胞术检测细胞凋亡率结果,MicroR-33 mimics组B16F10黑色素瘤细胞凋亡率(1.8050±0.2050)%明显高于空白组(1.13±0.1414)%(t=15.000,P=0.042<0.05)；而mimics对照组(1.3650±0.0212), MicroR-33 inhibitor组(1.4450±0.0212)%,inhibitor对照组(1.3700±0.2546)%细胞凋亡率与空白组比较均无显著差异性(P>0.05)。5.细胞周期结果显示MicroR-33 mimics组黑色素瘤细胞G1期细胞比例(62.7±1.7321)与空白组(53.5±1.9630)相比显著增加(t=4.98,P=0.016<0.05),S期细胞比例(23.4±2.5044)与空白组(32.6±2.5566)相比显著减少(t=-4.998,P=0.004<0.05),而G2期细胞比例无显著变化。MicroR-33 inhibitor组,mimics对照组,inhibitor对照组与空白组比较各周期细胞比例无明显变化(P>0.05)。结论：MicroR-33在高表达时能抑制B16F10黑色素瘤细胞的增殖,促进细胞凋亡更多还原

【Abstract】 Objective:Based on preliminary study, to approach the effect of MicroR-33 to melanoma cells line proliferation and apoptosis.Methods:Construct targeted MicroR-33 over-expression mimics and inhibitor, then use gene transfer technology to transfer them into B16F10 melanoma cells.Let the expression of MicroR-33 up or down. The differences of MicroR-33 gene expression were validated by Real-time quantitative PCR; the cell growth curve were draw by MTT; the apoptosis rate and cell cycle were measured by flow cytometry. Finally, using statistical method to analyse the effect of MicroR-33 on B16F10 cell proliferation and apoptosis..Results:1. After transfection, cy3 fluorescent material can be observed in the transfected cells.2. Real-time PCR verified that the relative MicroR-33 gene expression of MicroR-33 mimics group (1.7733E3±2.4583E2)was significantly higher than the non-transfected group and the mimics control group (0.9340±0.2913) (t=12.487, P=0.006<0.05); The relative MicroR-33 gene expression in MicroR-33 inhibitor group (0.6973±0.1958)was partly lower than the non-transfected group and the inhibitor control group(1.0200±0.0848) but not reach the significant difference (P>0.05). There was also no significant difference between the non-transfected group, the mimics control group and the inhibitor control group (P>0.05).3. From visual field of microscope we found the cells density of MicroR-33 mimics group significant lower than other group after transfection 48h.Cell growth curve showed that compare with non-transfected group, the growth trendency of MicroR-33 mimics group declined.And after transfection 48h (1.1875±0.0502)(t=-14.5, P=0.044< 0.05) and72h (1.7500±0.0933) (t=-13.59, P=0.047<0.05), the trend showed significant difference.The growth trendency of the MicroR-33 inhibitor group, mimics control group and inhibitor control group was similar to the non-transfected group.4. Flow cytometry demonstrated the apoptotic ratio of MicroR-33 mimics group(1.8050±0.2050)%remarkably increased, higher than non-transfected group (1.13±0.1414)%(t=l5.000, P=0.042<0.05). The apoptotic ratio of mimics control group (1.3650±0.0212)%, MicroR-33 inhibitor group (1.4450±0.0212)%and inhibitor control group (1.3700±0.2546)%, was no significant difference, respectively, compared with the non-transfected group (P>0.05).5.Cell cycle result showed the proportion of Gl period in MicroR-33 mimics group (62.7±1.7321) compared with non-transfected group (53.5±1.9630) remarkably increased(t=4.98,P=0.016＜0.05), the proportion of S period (23.4±2.5044) compared with non-transfected group (32.6±2.5566) remarkably decreased (t=-4.998,P=0.004<0.05); the proportion of G2 period didn’t change a lot compared with non-transfected group.Cell cycle of MicroR-33 inhibitor group,mimics control group and inhibitor control group didn’t change a lot compared with non-transfected group (P>0.05)Conclusion:MicroR-33 over expression can restrain the proliferation of B16F10 cells, promote B16F10 cells apoptosis.更多还原