【作者】 王勇；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 放射医学， 2013， 硕士

【摘要】 实验目的利用慢病毒沉默CD133基因探讨其对人肝癌CD133+-HepG2干细胞放射敏感性的影响。第一部分：CD133+-HepG2细胞的分选及“干性”鉴定实验方法：利用免疫磁珠分选技术从HepG2肝癌细胞系中分选出CD133+及CD133-细胞亚群,并用流式细胞术检测分选前后CD133的表达率；对分选得到的CD133+-HepG2肝癌细胞进行体外成球能力及NOD/SCID小鼠皮下成瘤能力检测,并取皮下移植瘤进行HE染色观察肿瘤组织结构；克隆形成试验对比CD133+细胞与CD133-细胞克隆形成能力。实验结果：从HepG2肝癌细胞系中利用免疫磁珠分选技术成功分选出CD133’与CD133-细胞,并用流式细胞术检测HepG2细胞的CD133基础表达率为(1.36±0.20)%,通过分选后CD133表达率上升到(87.62±1.92)%。随后无血清培养结果显示CD133+HepG2细胞能在含EGF、FGF、LIF的无血清培养液中能成球生长,而CD133--HepG2细胞则无法在相同的培养液中生存。小鼠皮下成瘤实验结果显示1×103个CD133+细胞即可在NOD/SCID小鼠皮下形成移植瘤,而相同条件下CD133细胞均也不能成瘤。HE染色结果显示,由CD133+细胞形成的皮下移植瘤在显微镜下组织细胞呈排列致密,细胞核清晰可见。平板克隆形成试验显示,CD133+细胞比CD133细胞具有更强的克隆形成能力,其克隆形成率为：35.03±2.35%,而CD133-细胞克隆形成率仅为：6.4±0.72%。两者具有显著差异,P<0.01。实验结论：上述实验结果表明通过免疫磁珠分选得到的CD133+-HepG2细胞比CD133--HepG2细胞具有更强的体外成球和体内成瘤能力及克隆形成能力,是具有肿瘤干细胞特性的肝癌细胞亚群。第二部分携带CD133干扰序列的慢病毒感染CD133+-HepG2实验方法：利用慢病毒介导的RNA干扰技术,以CD133基因为沉默靶点,按预实验得出的MOI值20对CD133+-HepG2肝癌细胞进行感染,以转染shCD133组为实验组、转染shNC组为阴性对照组,未处理的CD133+-HepG2细胞为空白对照组；3天后在荧光显微镜下观察慢病毒的感染效率；克隆形成实验检测三组细胞克隆形成能力；RT-PCR和Western Blot检测三组细胞CD133mRNA及蛋白的表达；实验结果：接种慢病毒后3天,在倒置荧光显微镜观察到绿色荧光蛋白(GFP)的表达,第5天达到高峰,其感染阳性效率达80%以上；克隆形成实验结果显示沉默CD133后的CD133+肝癌干细胞增殖能力明显减弱(P<0.01)；RT-PCR和Western Blot结果显示慢病毒介导的CD133shRNA能有效下调CD133的mRNA和蛋白表达(P<0.01);实验结论：上述实验结果表明慢病毒介导的shRNA能显著下调CD133+肝癌干细胞CD133的表达,并能显著抑制其增殖能力。第三部分克隆形成实验检测各组细胞放射敏感性并探讨其可能机制。实验方法：采用克隆形成实验检测各组细胞克隆形成率(Plating efficiency PE), PE=克隆数目/接种细胞数×10096,以及存活分数(survival fraction SF), SF=受照射细胞的克隆形成率／对照组细胞克隆形成率×100%。绘制剂量存活曲线并计算放射生物学参数。利用流式细胞仪检测三组细胞细胞周期及凋亡情况。实验结果：阳性感染组的剂量存活曲线较其他两组整体下移。根据多靶单击模型计算出各组SF2、D0、Dq及N值结果表明,阳性感染组的PE、SF、SF2、D0、Dq及N值均较空白组和阴性感染组明显减小,其放射增敏比(SER)为：1.37±0.02。差异具有统计学差异,P<0.01。流式细胞术检测各组细胞周期及凋亡情况,结果发现阳性感染组其S期明显减少G2期增多,细胞凋亡也明显增加。差异有统计学意义P<0.05。实验结论：沉默CD133基因后,CD133+-HepG2细胞放射敏感性明显增强,细胞周期的改变与细胞凋亡率的增加是其可能的增敏机制。更多还原

【Abstract】 To detect the effects of CD133-downrultation on the radiosensitivity of CD133+HepG2cancer stem cells (CSCs) in hepatocelluar carcinoma.Part1Sorting of CD133+-HepG2cells and checking of their stemness properties.Methods:MACS was used to isolate CD133+and CD133-cells from HepG2cell line. Flow cytometry was used to detect the expression of CD133before and after cells isolation. Spheres-forming assay in vitro and the NOD/SCID mice transplantation tumor experiments in vivo were performed to validate the cancer stem-like properties of sorted CD133+cells. Colony forming assay was used to compare the colony-formation ability between CD133+and CD133-cells.Results:CD133+and CD133-cells were successfully sorted by MACS, and1.36±0.20%and87.62±1.92%CD133+cells were detected by flow cytometry before and after isolation, respectively. Spheres-forming assay showed that CD133+cells could form spheres and in serum-free culture media with grow factors, whereas the CD133-cells showed absent failed to stay alive in the serum-free culture media. NOD/SCID mice tumorigenicity assay showed that just1,000CD133+cells could form subcutaneous xenografts5weeks after inoculation, whereas10,000CD133-cells could not form tumors in the same condition. Hematoxylin and eosin (H&E) stating analysis showed a highly cellular mass and clear nucleis below the CD133+cells injection site. Colony forming assay showed that CD133+cells have greater ability to form clones than CD133-cells, which had a higher cloning efficiency (35.03±2.35)%compared to CD133-cells’ cloning efficiency (6.4±0.72)%(P<0.01).Conclusion:The results revealed that CD133+HepG2cells demonstrated higher tumor spheres formation ability, colony forming ability and tumorigenesis capacity than CD133-cells, which could be considered as CSC-like subsets of liver cancer cells.Part2Infection of CD133+HepG2by lentivirus with CD133sequenceMethods:Targeted silence towards CD133gene was performed and experimental groups were divided into blank control group, negative-transfection group and positive-tranfection group. The efficiency of infection was observed by inverted fluorescence microscope. RT-PCR and Western blot were used to detect the gene and protein expressions of CD133. Colony formation assay was applied to detect the proliferative ability of cells.Results:The expression of green fluorescence protein were detected under inverted fluorescence microscope3days after infection, which peaked on the fifth day. The infection rate was more than80%. The expression level of CD133in mRNA and protein level was significantly decreased in positive-transfection group(P<0.01). Colony formation assay showed that the proliferation ability of CD133+liver cancer stem cells was decreased after CD133-downregulation(P<0.01).Conclusion:The results showed that lentivirus-mediated shRNA could down-regulate the expression of CD133in the CD133+liver cancer stem cells, and inhibit their proliferative ability.Part3Detection of radiosensitivity by colony formation assay and exploration of mechanismsMethods:Colony formation assay was applied to detect colony formation efficiency and survival rate after irradiation with different doses; survival curve was drawn and radiobiology parameters were counted. Flow cytometry was used to test cell cycle and apoptosis rate.Results:The dose survival curve of positive-transfected group was down overall than the other two groups. The values of radiobiology parameters PE, SF, SF2, Do, Dq and N were decreased obviously compared to negative-transfection group and blank control group. The SER (sensitive enhancement ratio) was1.37±0.02(P<0.01). The results of flow cytometry and apoptosis assay showed that S phase cells decreased in posi tive--transfection group whereas G2phase cells and apoptosis rate increased significantly (P<0.05).Conclusion:After CD133-downregulation, the radiosensitivity was obviously increased. The change of cell cycle and the increasement of apoptosis rate may be the mechanisms to enhance radiosensitivity.更多还原