RNA干扰抑制水通道蛋白3对肠粘膜上皮屏障影响的实验研究

发布时间：2018-05-13 04:11

本文选题：水通道蛋白 + RNA干扰　；参考：《南京医科大学》2012年硕士论文

【摘要】：目的：探讨RNA干扰下调人AQP3基因表达对肠粘膜上皮屏障的影响，并探讨其可能的作用机制。方法：在Caco-2细胞系中检测AQP3基因的表达，构建沉默AQP3mRNA表达的shRNA慢病毒载体，，转染Caco-2细胞系，建立稳定转染细胞系，荧光定量PCR以及Western blot验证其在mRNA和蛋白水平的干扰效率。体外建立肠粘膜上皮屏障，分为实验分为三组：空白对照组（CON）、阴性对照组(NC)、AQP3干扰组(RNAi)，分别在第7，14，21，28天检测细胞跨膜电阻（TEER），荧光黄(Luciferyellow，LY)的通透性和易位的大肠杆菌（E.coli C25）的数量。Western blot验证紧密连接（TJ）相关蛋白的表达情况。并且采用免疫细胞化学法观察紧密连接相关蛋白，如Occludin蛋白，Claudin-1蛋白的分布和结构变化。结果：荧光定量PCR及Western blot结果显示shRNA慢病毒载体可有效沉默AQP3在Caco-2细胞系中的表达，与空载质粒组相比，RNA干扰组在第7，14，21天TEER明显降低，荧光黄的通透性增大并且易位的大肠杆菌（E.coli C25）的数量明显增多，各检测时间点RNAi组和空载质粒组比较均存在统计学差异（P㩳0.05)。Western blot结果显示AQP3干扰组紧密连接相关蛋白Occludin以及Claudin-1的表达明显降低。免疫细胞化学结果显示Caco-2细胞间Occludin以及Claudin-1主要表达在细胞膜和/或胞浆中，相邻Caco-2细胞间TJ结构遭到破坏。结论：靶向AQP3的shRNA技术通过对AQP3的有效沉默可明显降低肠粘膜屏障的完整性，为进一步探讨肠粘膜屏障损伤治疗提供新的思路。
[Abstract]:Aim: to investigate the effect of RNA interference on intestinal mucosal epithelial barrier and its possible mechanism. Methods: to detect the expression of AQP3 gene in Caco-2 cell line, construct the shRNA lentivirus vector which silenced the expression of AQP3mRNA, transfect Caco-2 cell line, establish stable transfection cell line, test its interference efficiency at mRNA and protein level by fluorescence quantitative PCR and Western blot. To establish intestinal mucosal epithelial barrier in vitro, The experiment was divided into three groups: blank control group (Conn), negative control group (control group) and negative control group (control group). The transmembrane resistance (TJ) and the translocation of E. coli C25 (E. coli C25) were detected on day 714, 21 and 28, respectively. Western blot was used to verify the close connection of TJ). Expression of related proteins. Immunocytochemistry was used to observe the distribution and structural changes of tightly linked proteins, such as Occludin protein Claudin-1. Results: the results of fluorescence quantitative PCR and Western blot showed that shRNA lentivirus vector could effectively silence the expression of AQP3 in Caco-2 cell line. Compared with the control group, the TEER of shRNA lentivirus interference group was significantly lower than that of the blank plasmid group on day 7, 14 and 21. The permeability of fluorescent yellow increased and the number of E. coli C25 translocated increased significantly. There was statistical difference between RNAi group and no-load plasmid group at each time point. The results of Western blot showed that the expression of Occludin and Claudin-1 in AQP3 interference group was significantly lower than that in control group. The results of immunocytochemistry showed that Occludin and Claudin-1 were mainly expressed in the cell membrane and / or cytoplasm of Caco-2, and the structure of TJ between adjacent Caco-2 cells was destroyed. Conclusion: shRNA targeting AQP3 can significantly reduce the integrity of intestinal mucosal barrier through the effective silencing of AQP3, which provides a new idea for further study on the treatment of intestinal mucosal barrier injury.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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