EN2基因沉默对人肾小管上皮细胞生物学行为的影响
发布时间：2018-10-17 11:23
【摘要】：目的:探讨沉默EN2对人肾小管上皮细胞(HK-2细胞株)生物学行为的影响。方法:利用慢病毒载体介导沉默人肾小管上皮细胞HK-2细胞株EN2基因的表达,转染并筛选稳定低表达EN2基因的人肾小管上皮细胞HK-2细胞株,以Lv-shEN2为实验组、Lv-shcon为空载组、control为空白对照组。RT-qPCR和Western blot分别检测EN2 mRNA和蛋白的表达水平,MTT检测HK-2细胞增殖能力,流式细胞术检测细胞周期和凋亡,Transwell实验检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力。结果:设计与EN2互补的DNA序列,合成shRNA-EN2并克隆到慢病毒载体,同时设计非特异性shRNA生成的载体作为阴性对照。转染HK-2细胞后,筛选获得稳定低表达EN2基因的HK-2细胞株。RT-qPCR分析显示EN2mRNA在Lv-shcon组2(-ΔΔCt)=1.07±0.07,control组2(-ΔΔCt)=1±0.14中的表达分别是Lv-shEN2组2(-ΔΔCt)=0.58±0.02的1.9倍和1.7倍。Western Blot检测各组灰度值Lv-shEN2组2576.65,Lv-shcon组1619.26,control组1478.85。提示转染成功。MTT法分析结果采用单因素方差分析显示三组细胞OD值差别(F=4.78,P=0.01)有统计学意义。与两对照组细胞比较,Lv-shEN2组细胞OD值升高,提示抑制EN2蛋白可促进人肾小管上皮细胞HK-2细胞的增殖活性。流式细胞仪检测显示Lv-shEN2组相对于两对照组,细胞凋亡率下降。Lv-shEN2实验组、Lv-shcon空载组、control空白对照组HK-2细胞凋亡比例分别为9.79%,24.98%和24.9%。与两对照组细胞比较,Lv-shEN2组细胞早期凋亡率和晚期凋亡率均下降,提示抑制EN2基因表达可减少HK-2细胞凋亡。细胞穿膜实验显示Lv-shEN2实验组、Lv-shcon空载组、control空白对照组HK-2细胞穿膜细胞数分别为18.5±5.62,0.5±1.12和0.5±0.76,单因素方差分析显示Lv-shEN2实验组穿膜细胞数与两对照组之间不同(F=19.35,P=0.00),说明下调EN2蛋白可提高人肾小管上皮细胞HK-2的侵袭能力。细胞划痕试验各组0h,6h,12h和24h划痕宽度采用单因素方差分析,结果显示各组细胞迁移能力差别无统计学意义(P0.05)。结论:下调HK-2细胞株EN2基因的表达促进HK-2细胞增殖、抑制细胞凋亡、提高细胞侵袭能力,对细胞迁移能力无明显影响。
[Abstract]:Aim: to investigate the effect of silencing EN2 on the biological behavior of human renal tubular epithelial cells (HK-2 cells). Methods: lentivirus vector was used to mediate the silencing of EN2 gene expression in human renal tubular epithelial cell HK-2 cell line, and to transfect and screen HK-2 cell line with stable low expression of EN2 gene. Lv-shEN2 was used as experimental group, Lv-shcon as no-load group and control as blank control group. RT-qPCR and Western blot were used to detect the expression of EN2 mRNA and protein, MTT was used to detect the proliferation of HK-2 cells, flow cytometry was used to detect cell cycle and apoptosis, and Transwell assay was used to detect the ability of cell invasion. Cell migration ability was measured by cell scratch assay. Results: DNA sequence complementary to EN2 was designed, shRNA-EN2 was synthesized and cloned into lentivirus vector, and non-specific shRNA vector was designed as negative control. The expression of EN2mRNA in Lv-shcon group 2 (- 螖 Ct) = 1.07 卤0.07 control group (- 螖 Ct) = 1 卤0.14 was 1.9 times as high as that in Lv-shEN2 group 2 (- 螖 Ct) = 0.58 卤0.02 and 1.7 times as high as that in Lv-shEN2 group 2 (螖 Ct) = 0.58 卤0.02. RT-qPCR analysis showed that the expression of EN2mRNA in Lv-shcon group 2 (- 螖 Ct) = 1.07 卤0.07 was 1.9 times as much as that in Lv-shEN2 group 2 (螖 Ct) = 0.58 卤0.02 and 1.7 times. Western Blot in Lv-shEN2 group (2576.65 Lv-shcon group). The results of MTT analysis showed that the OD values of the three groups were significantly different (FF4.78% P0. 01). Compared with the control group, the OD value of Lv-shEN2 group increased, suggesting that inhibition of EN2 protein could promote the proliferation of HK-2 cells in human renal tubular epithelial cells. Flow cytometry analysis showed that the apoptosis rate of Lv-shEN2 group was lower than that of two control groups. The percentage of HK-2 cell apoptosis in Lv-shEN2 experimental group, Lv-shcon no-load group and control blank control group was 9.79% and 24.9%, respectively. Compared with the control group, the early and late apoptosis rates of Lv-shEN2 cells decreased, suggesting that inhibiting the expression of EN2 gene can reduce the apoptosis of HK-2 cells. Cell perforation test showed that the number of HK-2 cells in Lv-shEN2 experimental group, Lv-shcon no-load group and control blank control group were 18.5 卤5.62 卤1.12 and 0.5 卤0.76, respectively. Univariate analysis of variance showed that the number of perforating cells in Lv-shEN2 experimental group was different from that in two control groups (F _ (19.35) P ~ (0.00), indicating that EN2 eggs were down-regulated. White can enhance the invasiveness of HK-2 in human renal tubular epithelial cells. Single factor analysis of variance (ANOVA) showed that there was no significant difference in cell migration ability between each group (P0.05). Conclusion: down-regulating the expression of EN2 gene in HK-2 cell line can promote the proliferation of HK-2 cells, inhibit cell apoptosis, improve the ability of cell invasion, and have no significant effect on the ability of cell migration.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R692
本文编号：2276501
[Abstract]:Aim: to investigate the effect of silencing EN2 on the biological behavior of human renal tubular epithelial cells (HK-2 cells). Methods: lentivirus vector was used to mediate the silencing of EN2 gene expression in human renal tubular epithelial cell HK-2 cell line, and to transfect and screen HK-2 cell line with stable low expression of EN2 gene. Lv-shEN2 was used as experimental group, Lv-shcon as no-load group and control as blank control group. RT-qPCR and Western blot were used to detect the expression of EN2 mRNA and protein, MTT was used to detect the proliferation of HK-2 cells, flow cytometry was used to detect cell cycle and apoptosis, and Transwell assay was used to detect the ability of cell invasion. Cell migration ability was measured by cell scratch assay. Results: DNA sequence complementary to EN2 was designed, shRNA-EN2 was synthesized and cloned into lentivirus vector, and non-specific shRNA vector was designed as negative control. The expression of EN2mRNA in Lv-shcon group 2 (- 螖 Ct) = 1.07 卤0.07 control group (- 螖 Ct) = 1 卤0.14 was 1.9 times as high as that in Lv-shEN2 group 2 (- 螖 Ct) = 0.58 卤0.02 and 1.7 times as high as that in Lv-shEN2 group 2 (螖 Ct) = 0.58 卤0.02. RT-qPCR analysis showed that the expression of EN2mRNA in Lv-shcon group 2 (- 螖 Ct) = 1.07 卤0.07 was 1.9 times as much as that in Lv-shEN2 group 2 (螖 Ct) = 0.58 卤0.02 and 1.7 times. Western Blot in Lv-shEN2 group (2576.65 Lv-shcon group). The results of MTT analysis showed that the OD values of the three groups were significantly different (FF4.78% P0. 01). Compared with the control group, the OD value of Lv-shEN2 group increased, suggesting that inhibition of EN2 protein could promote the proliferation of HK-2 cells in human renal tubular epithelial cells. Flow cytometry analysis showed that the apoptosis rate of Lv-shEN2 group was lower than that of two control groups. The percentage of HK-2 cell apoptosis in Lv-shEN2 experimental group, Lv-shcon no-load group and control blank control group was 9.79% and 24.9%, respectively. Compared with the control group, the early and late apoptosis rates of Lv-shEN2 cells decreased, suggesting that inhibiting the expression of EN2 gene can reduce the apoptosis of HK-2 cells. Cell perforation test showed that the number of HK-2 cells in Lv-shEN2 experimental group, Lv-shcon no-load group and control blank control group were 18.5 卤5.62 卤1.12 and 0.5 卤0.76, respectively. Univariate analysis of variance showed that the number of perforating cells in Lv-shEN2 experimental group was different from that in two control groups (F _ (19.35) P ~ (0.00), indicating that EN2 eggs were down-regulated. White can enhance the invasiveness of HK-2 in human renal tubular epithelial cells. Single factor analysis of variance (ANOVA) showed that there was no significant difference in cell migration ability between each group (P0.05). Conclusion: down-regulating the expression of EN2 gene in HK-2 cell line can promote the proliferation of HK-2 cells, inhibit cell apoptosis, improve the ability of cell invasion, and have no significant effect on the ability of cell migration.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R692
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