过表达mi RNA-21对肾小管上皮细胞TGF-β/Smad信号通路Smad 7蛋白的影响

The Effect of mi RNA-21 on TGF-β/Smad Signaling Pathway Smad 7 Protein in Renal Tubular Epithelial Cells

目的:探究mi RNA-21过表达对TGF-β/Smad信号通路Smad7蛋白的影响。方法:采用mi RNA-21过表达慢病毒载体和慢病毒空载体分别感染高糖和低糖培养的人肾近端小管上皮细胞HK-2细胞,根据培养基类型和是否感染病毒将细胞分组为高糖mi RNA-21过表达慢病毒组、低糖mi RNA-21过表达慢病毒组、高糖空载组、低糖空载组、高糖细胞组和低糖细胞组,用荧光倒置显微镜观察感染后表达绿色荧光蛋白(GFP)细胞数,通过流式细胞术检测转染率;采用实时荧光定量PCR检测感染后细胞mi RNA-21的表达量,采用Western blot检测Smad7蛋白的表达。结果:感染mi RNA-21过表达慢病毒后,荧光显微镜下HK-2细胞有明显绿色荧光(约为60%),感染率结果显示转染率>60%,实时荧光定量PCR结果显示感染mi RNA-21过表达慢病毒后,高糖转染组细胞mi RNA-21表达量高于高糖细胞组和高糖空载组(P<0.05);Weston blot结果显示高糖转染组Smad 7蛋白的表达低于高糖转染空载病毒组、低糖细胞组、高糖细胞组(P<0.05)。结论:mi RNA-21可以通过抑制Smad7蛋白的表达来调控TGF-β/Smad信号通路。

Objective： To explore the effect of mi RNA-21 overexpression on TGF-β/Smad signaling pathway Smad7 protein. Methods： The mi RNA-21 overexpression lentiviral vector was used to infect HK-2 cells cultured with high glucose and low sugar respectively. According to the type of culture medium and whether or not the virus was infected,the cells were divided into high glucose mi RNA-21 overexpression lentivirus group,low glucose mi RNA-21 overexpression lentiviral group,high glucose and empty virus group,low sugar empty virus group,high glucose cell group and low glucose cell group. The expression of green fluorescent protein（ GFP） cells（ green fluorescence） after infection was observed by fluorescence inverted microscope,and the transfection rate was detected by flow cytometry. The expression of mi RNA-21 in infected cells was detected by real-time quantitative PCR,and the expression of Smad7 protein was detected by Western blot. Results： When infected with mi RNA-21 overexpressing lentivirus,HK-2 cells showed significant green fluorescence（ about 60%） under fluorescence microscope. The infection rate showed that transfection rate was 60% or more. The results of real-time quantitative PCR showed that the expression of mi RNA-21 in the high glucose transfection group was higher than that in the high glucose cell group as well as the high glucose and empty virus group after infection with mi RNA-21 overexpression lentivirus（ P〈0. 05）. The results of Weston blot showed that the expression of Smad 7 protein in high glucose transfection group was lower than that in high glucose empty virus group,low sugar cell group and high glucose cell group（ P〈0. 05）. Conclusion： mi RNA-21 can regulate the TGF-β/Smad signaling pathway by inhibiting the expression of Smad7 protein.