PTTG1在子宫内膜癌侵袭及转移中的作用

发布时间：2018-11-17 07:00

【摘要】：目的:探讨PTTG1(垂体肿瘤转化基因1)对人子宫内膜癌ISK细胞增殖、迁移及侵袭能力的影响。方法:1.细胞培养:常规对人子宫内膜癌ISK细胞进行细胞复苏、细胞换液、细胞传代、细胞冻存、细胞计数等处理。2.构建3条靶向PTTG1基因的shRNA干扰质粒(PTTG1-homo-509;PTTG1-homo-922;PTTG1-homo-596)。3.在lipo2000作用下瞬时转染靶向PTTG1基因的shRNA干扰质粒至ISK细胞中。4.36h后避光条件下观察瞬时转染结果(荧光显微镜下进行),即观察有绿色荧光表达的细胞所占的比例。5.通过Western blot方法检验PTTG1蛋白在各组中表达差异。6.通过MTT实验检测降表达PTTG1后ISK细胞生长增殖活性的变化。7.通过Transwell小室实验检测下调PTTG1后ISK迁移及侵袭力的变化差异。结果:1.瞬转ISK细胞36h后,荧光显微镜下观测到有绿色荧光表达的细胞数量占75%以上,表明瞬转实验成功。2.Western blot实验检测到干扰组-509、干扰组-922、干扰组-596中的PTTG1与内参β-actin灰度值比值均明显小于正常组及阴性对照组,且差异明显有统计学意义(P0.05)。这就说明3个干扰组中的PTTG1蛋白表达均受到了明显抑制,即构建的3条靶向PTTG1基因的shRNA干扰质粒均有效。3.在MTT实验中,干扰组(转染了有效PTTG1 shRNA质粒的细胞)中在各时间段的OD值均小于正常组及阴性对照组,说明干扰组中细胞增长较两个对照组明显减慢,差异且存在统计学意义(P0.05)。4.在Transwell小室实验中,干扰组(转染了有效PTTG1 shRNA质粒的细胞)中的穿膜细胞数较正常及阴性对照组明显减少,说明该组细胞的迁移及侵袭能力较以上两组细胞明显减弱,且差异明显有统计学意义(P0.05)。结论:1.PTTG1 sh RNA干扰质粒可使人子宫内膜癌ISK细胞中的PTTG1蛋白表达量明显降低并可抑制ISK细胞的增殖、迁移及侵袭力。2.PTTG1在子宫内膜癌的发生发展过程中起到了重要作用。
[Abstract]:Aim: to investigate the effects of PTTG1 (pituitary tumor transforming gene 1) on the proliferation, migration and invasion of human endometrial carcinoma ISK cells. Methods: 1. Cell culture: human endometrial carcinoma ISK cells were routinely treated with cell resuscitation, cell fluid exchange, cell passage, cell cryopreservation, cell count and so on. 2. Three shRNA interference plasmids (PTTG1-homo-509;PTTG1-homo-922;PTTG1-homo-596) targeting PTTG1 gene were constructed. ShRNA interference plasmids targeting PTTG1 gene were transiently transfected into ISK cells by lipo2000. After 4.36 h, the results of transient transfection (carried out under fluorescence microscope) were observed, that is, the proportion of cells with green fluorescent expression was observed. The expression of PTTG1 protein in each group was detected by Western blot method. 6. 6%. MTT assay was used to detect the growth and proliferation activity of ISK cells after down-expression of PTTG1. 7. 7%. The changes of ISK migration and invasiveness after down-regulation of PTTG1 were detected by Transwell chamber experiment. The result is 1: 1. After transient transfer of ISK cells for 36 h, the number of cells with green fluorescent expression was more than 75% under fluorescence microscope, which indicated that the transient experiment was successful. The ratio of PTTG1 to 尾-actin in interference group-596 was significantly lower than that in normal group and negative control group, and the difference was statistically significant (P0.05). This indicated that the expression of PTTG1 protein was significantly inhibited in the three interference groups, that is, the three shRNA interference plasmids targeting PTTG1 gene were all effective. In the MTT experiment, the OD values in the interference group (the cells transfected with the effective PTTG1 shRNA plasmid) were lower than those in the normal group and the negative control group, indicating that the cell growth in the interference group was significantly slower than that in the two control groups. The difference was statistically significant (P0.05). 4. In the Transwell chamber experiment, the number of perforated cells in the interference group (cells transfected with effective PTTG1 shRNA plasmids) was significantly lower than that in the normal and negative control groups, indicating that the migration and invasion ability of the cells in the interference group was significantly lower than that in the above two groups. The difference was statistically significant (P0.05). Conclusion: 1.PTTG1 sh RNA interference plasmid can significantly decrease the expression of PTTG1 protein in human endometrial carcinoma ISK cells and inhibit the proliferation of ISK cells. Migration and invasiveness. 2.PTTG1 plays an important role in the development of endometrial carcinoma.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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