TGFβ1siRNA对293细胞合成TGFβ1的干扰作用

发布时间：2018-04-19 09:40

本文选题：转化生长因子β1 + RNA干扰　；参考：《暨南大学》2007年硕士论文

【摘要】： 目的：探讨TGFβ1siRNA对TGFβ1基因表达的干扰作用，为进一步深入研究TGFβ1与高氧肺损伤发生发展机理提供一种新的工具；为研究早产儿CLD的基因治疗方法提供依据。方法：针对大鼠TGFβ1基因mRNA序列，，设计、合成携带3条TGFβ1siRNA和TGFβ1基因的绿色荧光蛋白融合表达质粒载体，并设阴性质粒组和空质粒组为对照，通过脂质体包裹分别转染293细胞。转染后24h、48h和72h收集细胞，在荧光显微镜下观察干扰效果，采用荧光定量PCR检测TGFβ1基因表达情况，并计算干扰效率。结果：(1)成功设计并构建出携带大鼠TGFβ1siRNA和TGFβ1的绿色荧光蛋白融合表达质粒载体。(2)脂质体包裹质粒转染293细胞后在荧光显微镜下观察，可见转染后24h、48h和72h TGFβ1siRNA质粒组细胞绿色荧光强度均明显弱于阴性质粒组细胞，空质粒载体组未产生绿色荧光。(3)荧光定量PCR检测转染后293细胞TGFβ1mRNA表达量，其中转染后48h阴性质粒组表达量最高(P＜0.05)，转染后24hTGFβ1siRNA质粒组表达量最低(P＜0.05)，空质粒组未见TGFβ1mRNA表达；转染后24h、48h和72h TGFβ1siRNA质粒组TGFβ1mRNA表达量均显著低于阴性质粒组(P＜0.01)，其基因干扰效率则依次递减，分别为97.2％、97.1％和67.7％。结论：研究证明自行设计的TGFβ1siRNA转染293细胞后24h、48h和72hTGFβ1siRNA均能够高效干扰TGFβ1基因的表达，其基因干扰效率随转染后时间的增加而递减。
[Abstract]:Objective: to investigate the interfering effect of TGF 尾 1siRNA on the expression of TGF 尾 1 gene, to provide a new tool for further study on the mechanism of TGF 尾 1 and hyperoxia lung injury, and to provide the basis for studying the gene therapy of CLD in premature infants.Methods: according to the mRNA sequence of rat TGF 尾 1 gene, three green fluorescent protein expression plasmids carrying TGF 尾 1siRNA and TGF 尾 1 genes were designed and synthesized. The negative plasmid group and the empty plasmid group were used as control group and transfected into 293 cells by liposome.The cells were collected at 24 h and 72 h after transfection. The interference effect was observed under fluorescence microscope. The expression of TGF 尾 1 gene was detected by fluorescence quantitative PCR, and the interference efficiency was calculated.Results the fusion expression plasmid of green fluorescent protein carrying rat TGF 尾 1siRNA and TGF 尾 1 was successfully designed and constructed. The liposome encapsulated plasmid was transfected into 293 cells and observed under fluorescence microscope.The green fluorescence intensity of the TGF 尾 1siRNA plasmid group was significantly lower than that of the negative plasmid group at 24 h or 72 h after transfection. No green fluorescence was produced in the empty plasmid vector group. The expression of TGF 尾 1mRNA in 293 cells was detected by fluorescence quantitative PCR.The expression of TGF 尾 1mRNA was the highest in the negative plasmid group 48 hours after transfection and the lowest in the 24hTGF 尾 1siRNA plasmid group after transfection (P < 0.05). No TGF 尾 1mRNA expression was found in the empty plasmid group.The expression of TGF 尾 1mRNA in 24 h and 72 h TGF 尾 1siRNA plasmid group was significantly lower than that in negative plasmid group (P < 0.01), and the gene interference efficiency was significantly decreased to 97.2% and 67.7%, respectively.Conclusion: the results showed that both the self-designed TGF 尾 1siRNA and 72hTGF 尾 1siRNA could interfere with the expression of TGF 尾 1 gene in 293 cells 24 h after transfection, and the gene interference efficiency decreased with the increase of transfection time.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R722;R346

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