siRNA沉默TPX2对人宫颈癌HeLa细胞放射敏感性的影响

发布时间：2018-03-11 12:16

本文选题：TPX2　切入点：siRNA　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的对于分期较晚不考虑外科治疗的宫颈癌患者,主要的治疗手段之一是放疗。肿瘤细胞对射线杀伤是否敏感直接决定了疗效和疾病预后。DNA修复蛋白与肿瘤的放射敏感性关系密切。TPX2主要在胞核中起作用,依赖细胞周期并受其调节,与有丝分裂中纺锤体的准确合成及结构稳定有直接关系。TPX2基因异常表达直接产生的结果是中心体出现反常扩增及异倍体形成,同时与肿瘤转移和复发明显相关。已有研究证实了射线依赖的?-H2AX形成量与DNA双链断裂点的数量之间有相关关系。最新的研究发现胞内TPX2蛋白发生不同程度的消耗时,射线诱导的?-H2AX的数量会呈现短暂增加的现象。本实验主要利用RNAi技术,设计合成靶基因TPX2的小干扰RNA,观察导入si RNA后HeLa细胞中TPX2 mRNA和蛋白表达的改变,以及HeLa细胞放射敏感性的变化,为宫颈癌的放射增敏提供了研究方向。方法选择人宫颈癌HeLa细胞,根据小干扰RNA设计原则设计靶基因TPX2的si RNA,共设计3对分别为si RNA-1、si RNA-2和si RNA-3,同时设计阴性对照si RNA。利用ribo FECTTMCP转染试剂,将si RNA转染入HeLa细胞,对转染后各组细胞内TPX2 mRNA和蛋白质的表达情况进行观察。主要采用RT-PCR、Western Blotting法,于转染后48 h进行,提取总RNA和总蛋白后检测TPX2 mRNA和蛋白表达情况,并筛选出最佳转染组及最佳转染时间。实验分组:目的基因组、阴性对照组、空白对照组。利用ribo FECTTMCP将si RNA转染入宫颈癌HeLa细胞中进行4 Gy-X线照射,利用CCK8法和流式细胞术观察TPX2表达下调的HeLa细胞对射线的敏感性的改变。结果转染48 h后提取总RNA,利用RT-PCR观察转染后各组细胞TPX2 mRNA的相对表达量发现,转染针对靶基因TPX2的si RNA的各组细胞均表现出TPX2基因表达抑制效应,以si RNA-2效果最明显,其TPX2的相对含量为(0.133±0.008)与空白对照组(1.000)及阴性对照组(0.981±0.814)相比有统计学意义(P0.05)。转染后继续培养48 h,用Western Blot法检测HeLa细胞TPX2蛋白的表达。结果发现si RNA-2组TPX2蛋白表达明显降低,蛋白相对含量为(0.518±0.685),空白对照组为(1.064±0.794),阴性对照组为(0.981±0.814),si RNA-2组与阴性对照组及空白对照组比较,有统计学意义(P0.05)。HeLa细胞放射敏感性的检测:转染后给予4 Gy X线照射,(1)利用CCK8检测各组细胞OD值,并计算细胞增殖活力,沉默TPX2组为(0.147±0.003),阴性对照组为(0.224±0.009),空白对照组为(0.353±0.012),统计学分析得出F=9.243,P=0.0015,差异有统计学意义(P0.05);(2)用流式细胞术检测各组细胞凋亡率发现各组细胞均出现凋亡增加,其中沉默TPX2组细胞凋亡率为(12.68±0.03%),阴性对照组为(9.37±0.11%),空白对照组为(5.81±0.21%),进行单因素方差分析,F=10.242,P0.0001,差异有统计学意义(P0.05)。结论针对靶基因TPX2的si RNA成功转染HeLa细胞,并能发挥基因表达的特异性抑制作用,目的基因的mRNA和蛋白的表达都受到抑制,同时可以提高HeLa细胞的放射敏感性。
[Abstract]:Objective to treat cervical cancer patients whose stage is late and surgical treatment is not considered. One of the main therapeutic methods is radiotherapy. The sensitivity of tumor cells to radiation directly determines the effect and prognosis of disease. DNA repair protein is closely related to the radiosensitivity of tumor. TPX2 mainly plays a role in the nucleus. Depending on and regulated by cell cycle, the abnormal expression of TPX2 gene is directly related to the accurate synthesis and structural stability of spindle in mitosis. The abnormal amplification of centrosome and the formation of aneuploidy are the results of abnormal expression of TPX2 gene. There is also a clear correlation between tumor metastasis and recurrence. There is a correlation between the amount of H2AX formation and the number of double strand breaks in DNA. The number of H2AX increased briefly. In this experiment, we designed and synthesized the small interference RNAs of target gene TPX2 by RNAi technique, observed the changes of TPX2 mRNA and protein expression in HeLa cells after transfection of si RNA, and the radiosensitivity of HeLa cells. Methods Human cervical cancer HeLa cells were selected. Si RNAs of target gene TPX2 were designed according to the principle of small interfering RNA design. Three pairs of siRNA-1 siRNA-3 were designed respectively. Si RNA was transfected into HeLa cells by ribo FECTTMCP transfection reagent. After transfection, the expression of TPX2 mRNA and protein in the cells was observed. The expression of TPX2 mRNA and protein was detected by RT-PCR Western Blotting method at 48 hours after transfection. The total RNA and total protein were extracted, and the expression of TPX2 mRNA and protein were detected after transfection. The best transfection group and optimal transfection time were selected. The experimental groups were as follows: objective genome, negative control group and blank control group. Si RNA was transfected into HeLa cells of cervical cancer by ribo FECTTMCP for 4 Gy-X irradiation. The changes of radiosensitivity of HeLa cells with down-regulated TPX2 expression were observed by CCK8 assay and flow cytometry. Results Total RNAs were extracted 48 h after transfection, and the relative expression of TPX2 mRNA was observed by RT-PCR. Si RNA transfected with target gene TPX2 showed inhibitory effect on the expression of TPX2 gene, especially si RNA-2. The relative content of TPX2 was 0.133 卤0.008), which was significantly higher than that of control group (0.133 卤0.008) and negative control group (0.981 卤0.814). The expression of TPX2 protein in HeLa cells was detected by Western Blot assay at 48 h after transfection. The results showed that the expression of TPX2 protein in si RNA-2 group was significantly lower than that in control group. The relative content of protein was 0.518 卤0.685g, 1.064 卤0.794 in the blank control group, 0.981 卤0.814 RNA-2 in the negative control group, and compared with the negative control group and the blank control group. Detection of radiosensitivity of P0.05U. HeLa cells: after transfection, 4 Gy X ray irradiation was given. OD value of each group was measured by CCK8, and cell proliferation activity was calculated. The rate of apoptosis was 0.147 卤0.003 in the silencing TPX2 group, 0.224 卤0.009 in the negative control group and 0.353 卤0.012 in the blank control group. The apoptosis rate of silencing TPX2 group was 12.68 卤0.03, that of negative control group was 9.37 卤0.11, and that of blank control group was 5.81 卤0.21. The single factor ANOVA analysis showed that the cell apoptosis rate was significantly different (P 0.05). Conclusion the si RNA for target gene TPX2 was successfully transfected into HeLa cells. Both mRNA and protein expression of the target gene were inhibited, and the radiosensitivity of HeLa cells was enhanced.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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