【摘要】：目的:探討沉默EN2對人腎小管上皮細胞(HK-2細胞株)生物學行為的影響。方法:利用慢病毒載體介導沉默人腎小管上皮細胞HK-2細胞株EN2基因的表達,轉染并篩選穩(wěn)定低表達EN2基因的人腎小管上皮細胞HK-2細胞株,以Lv-shEN2為實驗組、Lv-shcon為空載組、control為空白對照組。RT-qPCR和Western blot分別檢測EN2 mRNA和蛋白的表達水平,MTT檢測HK-2細胞增殖能力,流式細胞術檢測細胞周期和凋亡,Transwell實驗檢測細胞侵襲能力,細胞劃痕實驗檢測細胞遷移能力。結果:設計與EN2互補的DNA序列,合成shRNA-EN2并克隆到慢病毒載體,同時設計非特異性shRNA生成的載體作為陰性對照。轉染HK-2細胞后,篩選獲得穩(wěn)定低表達EN2基因的HK-2細胞株。RT-qPCR分析顯示EN2mRNA在Lv-shcon組2(-ΔΔCt)=1.07±0.07,control組2(-ΔΔCt)=1±0.14中的表達分別是Lv-shEN2組2(-ΔΔCt)=0.58±0.02的1.9倍和1.7倍。Western Blot檢測各組灰度值Lv-shEN2組2576.65,Lv-shcon組1619.26,control組1478.85。提示轉染成功。MTT法分析結果采用單因素方差分析顯示三組細胞OD值差別(F=4.78,P=0.01)有統(tǒng)計學意義。與兩對照組細胞比較,Lv-shEN2組細胞OD值升高,提示抑制EN2蛋白可促進人腎小管上皮細胞HK-2細胞的增殖活性。流式細胞儀檢測顯示Lv-shEN2組相對于兩對照組,細胞凋亡率下降。Lv-shEN2實驗組、Lv-shcon空載組、control空白對照組HK-2細胞凋亡比例分別為9.79%,24.98%和24.9%。與兩對照組細胞比較,Lv-shEN2組細胞早期凋亡率和晚期凋亡率均下降,提示抑制EN2基因表達可減少HK-2細胞凋亡。細胞穿膜實驗顯示Lv-shEN2實驗組、Lv-shcon空載組、control空白對照組HK-2細胞穿膜細胞數(shù)分別為18.5±5.62,0.5±1.12和0.5±0.76,單因素方差分析顯示Lv-shEN2實驗組穿膜細胞數(shù)與兩對照組之間不同(F=19.35,P=0.00),說明下調(diào)EN2蛋白可提高人腎小管上皮細胞HK-2的侵襲能力。細胞劃痕試驗各組0h,6h,12h和24h劃痕寬度采用單因素方差分析,結果顯示各組細胞遷移能力差別無統(tǒng)計學意義(P0.05)。結論:下調(diào)HK-2細胞株EN2基因的表達促進HK-2細胞增殖、抑制細胞凋亡、提高細胞侵襲能力,對細胞遷移能力無明顯影響。
[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 鹵0.07 control group (- 螖 Ct) = 1 鹵0.14 was 1.9 times as high as that in Lv-shEN2 group 2 (- 螖 Ct) = 0.58 鹵0.02 and 1.7 times as high as that in Lv-shEN2 group 2 (螖 Ct) = 0.58 鹵0.02. RT-qPCR analysis showed that the expression of EN2mRNA in Lv-shcon group 2 (- 螖 Ct) = 1.07 鹵0.07 was 1.9 times as much as that in Lv-shEN2 group 2 (螖 Ct) = 0.58 鹵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 鹵5.62 鹵1.12 and 0.5 鹵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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R692

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