长链非编码RNA母系表达基因3在小儿先天性巨结肠中的作用及意义

发布时间：2018-03-16 06:48

本文选题：先天性巨结肠　切入点：MEG3　出处：《遵义医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:检测长链非编码RNA母系表达基因3(maternally expressed gene 3 MEG3)在小儿先天性巨结肠(Hirschsprung’s disease HD)病变肠管中的表达情况,并通过观察过表达MEG3对人神经母细胞瘤细胞株(SK-N-BE(2))增殖、克隆、侵袭及凋亡的影响,探讨MEG3在HD中的作用及作用机制。方法:用RT-q PCR方法检测17例HD患儿病变结肠组织和17例非HD也无肠道畸形婴儿正常结肠组织中MEG3的表达量,用PASS检测其效能,并用ROC曲线下面积(AUC)及95%可行区间表示MEG3在HD中的诊断效能,评价MEG3在HD中的诊断价值。应用细胞培养及转染技术,在SK-N-BE(2)细胞中转染MEG3过表达质粒,设立MEG3高表达组、空载体组及对照组,并应用CCK-8、Edu细胞增殖实验、平板克隆技术、TRANSWELL技术、流式细胞实验分别检测过表达MEG3对SK-N-BE(2)细胞的增殖能力、克隆能力、侵袭能力及凋亡情况的影响。结果:RT-qPCR结果显示:HD患儿病变结肠组织中MEG3表达量较正常结肠组织中MEG3表达量明显升高(P0.01),效能检测显示效能达标,实现100%的检测功率,ROC曲线显示:曲线下面积(AUC)为84.9%,(95%可行区间71.6%-98.3%),最佳阳性截断值(cut off value)为1.46,灵敏度为82.4%,特异度为82.4%。MEG3过表达质粒转染SK-N-BE(2)细胞,转染效率为35.4%,达到转染要求,MEG3过表达组细胞的增殖能力、克隆能力以及侵袭能力与空载体组及对照组比较明显降低(P0.01),流式细胞检测显示:MEG3高表达组与空载体组及对照组比较细胞凋亡明显增加(P0.01)。结论:HD患儿病变结肠组织中MEG3表达量明显升高,MEG3可能通过调控SK-N-BE(2)细胞的增殖、克隆、侵袭与凋亡参与HD的发病,在HD的早期诊断与治疗中具有一定临床价值。
[Abstract]:Objective: to detect the expression of long chain noncoding RNA gene 3maternally expressed gene 3 MEG3 in the intestinal duct of Hirschsprunggs disease (HD) in children with congenital megacolon, and to observe the proliferation and cloning of human neuroblastoma cell line SK-N-BEN 2 by overexpression of MEG3. To explore the role and mechanism of MEG3 in HD. Methods: the expression of MEG3 in colon tissues of 17 children with HD and 17 normal colonic tissues with non HD and no intestinal malformation was detected by RT-q PCR method. PASS was used to detect its efficacy, and the area under the ROC curve and the feasible range of 95% were used to express the diagnostic efficacy of MEG3 in HD, and to evaluate the diagnostic value of MEG3 in HD. By means of cell culture and transfection technique, MEG3 overexpression plasmid was transfected into SK-N-BE2) cells. The high expression group of MEG3, empty vector group and control group were established. The proliferation and cloning ability of SK-N-BE2) cells were detected by using CCK-8 Edu cell proliferation assay, plate cloning technique, TRANSWELL technique and flow cytometry, respectively. Results the expression of MEG3 in the diseased colon tissue of the children with head HD was significantly higher than that in the normal colon tissue (P 0.01), and the efficiency of the detection was up to the standard. The detection power curve of 100% showed that the area under the curve was 84.9% and the feasible range was 71.6-98.30.The optimal truncation value cut off value was 1.46, the sensitivity was 82.4%, the specificity was 82.4% and the specificity was 82.4%. The transfection efficiency was 35.4g / kg, and the proliferation ability of the cells in the overexpression group of MEG3 was reached. Compared with empty vector group and control group, clone ability and invasion ability decreased significantly (P 0.01). Flow cytometry showed that cell apoptosis in the high expression group of 10% MEG3 was significantly higher than that in empty vector group and control group. Conclusion the pathological changes of colon tissue in children with 20% HD were significantly higher than those in control group. The expression of MEG3 in SK-N-BE2) cells increased significantly, and MEG3 may regulate the proliferation of SK-N-BE2) cells. Cloning, invasion and apoptosis play an important role in the early diagnosis and treatment of HD.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.7

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