本文選題：HaCaT細胞 + RNA干擾��； 參考：《新疆醫(yī)科大學》2017年碩士論文

【摘要】：目的:利用siRNA靶向沉默人角質形成細胞(HaCaT細胞)KRT9R163W表達,通過熒光定量聚合酶鏈式反應(qPCR)方法檢測HaCaT細胞角蛋白9(KRT9)基因mRNA水平表達,進一步共聚焦顯微鏡觀察轉染前后細胞骨架結構變化,探討KRT9R163W突變的基因功能在表皮松解性掌跖角化病的發(fā)病機制中的作用。方法:1.構建KRT9R163W質粒轉染入HaCaT細胞過表達。2.設計針對KRT9 R163W基因的特異性siRNA,利用脂質體轉染HaCaT細胞,并設立陰性對照組、陽性對照組及空白對照組。3.通過熒光定量聚合酶鏈式反應(qPCR)方法檢測HaCaT細胞KRT9基因mRNA水平表達。4.共聚焦顯微鏡觀察siRNA轉染前后細胞骨架結構變化。結果:1.KRT9 R163WsiRNA轉染HaCaT細胞48后,細胞的KRT9基因mRNA表達水 平明顯降低,KRT9R163W+siRNA-R163W組與 KRT9-R163W 組 KRT9 基因 mRNA相對表達量分別為0.242 ±0.051,1.063 ±0.159針對突變位點設計的siRNA能顯著抑制KRT9R163W的相對表達量(P0.05)。2.KRT9R163W高表達時,細胞骨架結構變成錯綜復雜的網(wǎng)狀結構,且部分細胞骨架結構消失。3.siRNA特異性沉默KRT9R163W基因后,細胞骨架結構恢復正常的束狀排列。結論:內源性角蛋白9(KRT9)是維持細胞正常骨架結構的重要蛋白,KRT9R163W高表達可影響細胞的正常結構,進一步通過siRNA技術驗證KRT9R163W為新疆維吾爾族表皮松解性掌跖角化病大家系致病的關鍵基因,為表皮松解性掌跖角化病發(fā)病機制研究及靶點治療提供新的理論基礎。
[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 鹵0.051 鹵1.063 鹵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.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R758.5
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本文編號：1879486