表皮松解性掌跖角化病KRT9R163W突变对细胞影响的研究

发布时间：2018-05-12 17:27

  本文选题：HaCaT细胞 + RNA干扰　； 参考：《新疆医科大学》2017年硕士论文

【摘要】：目的:利用siRNA靶向沉默人角质形成细胞(HaCaT细胞)KRT9R163W表达,通过荧光定量聚合酶链式反应(qPCR)方法检测HaCaT细胞角蛋白9(KRT9)基因mRNA水平表达,进一步共聚焦显微镜观察转染前后细胞骨架结构变化,探讨KRT9R163W突变的基因功能在表皮松解性掌跖角化病的发病机制中的作用。方法:1.构建KRT9R163W质粒转染入HaCaT细胞过表达。2.设计针对KRT9 R163W基因的特异性siRNA,利用脂质体转染HaCaT细胞,并设立阴性对照组、阳性对照组及空白对照组。3.通过荧光定量聚合酶链式反应(qPCR)方法检测HaCaT细胞KRT9基因mRNA水平表达。4.共聚焦显微镜观察siRNA转染前后细胞骨架结构变化。结果:1.KRT9 R163WsiRNA转染HaCaT细胞48后,细胞的KRT9基因mRNA表达水 平明显降低,KRT9R163W+siRNA-R163W组与 KRT9-R163W 组 KRT9 基因 mRNA相对表达量分别为0.242 ±0.051,1.063 ±0.159针对突变位点设计的siRNA能显著抑制KRT9R163W的相对表达量(P0.05)。2.KRT9R163W高表达时,细胞骨架结构变成错综复杂的网状结构,且部分细胞骨架结构消失。3.siRNA特异性沉默KRT9R163W基因后,细胞骨架结构恢复正常的束状排列。结论:内源性角蛋白9(KRT9)是维持细胞正常骨架结构的重要蛋白,KRT9R163W高表达可影响细胞的正常结构,进一步通过siRNA技术验证KRT9R163W为新疆维吾尔族表皮松解性掌跖角化病大家系致病的关键基因,为表皮松解性掌跖角化病发病机制研究及靶点治疗提供新的理论基础。
[Abstract]:Objective: to silence the expression of KRT9R163W in human keratinocytes by targeting siRNA, and to detect the expression of KRT9R163W in HaCaT cells by quantitative polymerase chain reaction (QPCR). Further confocal microscopy was used to observe the changes of cytoskeletal structure before and after transfection and to explore the role of KRT9R163W mutation gene function in the pathogenesis of epidermolysis palmoplantar keratosis. Method 1: 1. KRT9R163W plasmid was constructed and transfected into HaCaT cells. The specific siRNAs targeting KRT9 R163W gene were designed and transfected into HaCaT cells by liposome, and negative control group, positive control group and blank control group were established. The expression of KRT9 gene mRNA in HaCaT cells was detected by fluorescence quantitative polymerase chain reaction (QPCR). The changes of cytoskeleton structure before and after siRNA transfection were observed by confocal microscope. Results 1. KRT9 R163WsiRNA was transfected into HaCaT cells 48. The relative expression of KRT9 gene mRNA in KRT9R163W siRNA-R163W group and KRT9-R163W group was 0.242 卤0.051 卤1.063 卤0.159, respectively, which could significantly inhibit the relative expression of KRT9R163W. 2. KRT9R163W was highly expressed in KRT9R163W and KRT9R163W, respectively. The cytoskeleton structure became a complex reticular structure, and some cytoskeletal structures disappeared. 3. After silencing the KRT9R163W gene, the cytoskeleton structure returned to normal bundles. Conclusion: the high expression of KRT9R163W is an important protein to maintain the normal cytoskeleton structure of cells. Furthermore, KRT9R163W was proved to be the key gene of epidermolysis palmoplantar keratosis in Uygur Uygur nationality by siRNA technique, which provided a new theoretical basis for the study of pathogenesis and target therapy of epidermolytic palmoplantar keratosis.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.5
，

本文编号：1879486