【摘要】：目的:探討沉默EN2對(duì)人腎小管上皮細(xì)胞(HK-2細(xì)胞株)生物學(xué)行為的影響。方法:利用慢病毒載體介導(dǎo)沉默人腎小管上皮細(xì)胞HK-2細(xì)胞株EN2基因的表達(dá),轉(zhuǎn)染并篩選穩(wěn)定低表達(dá)EN2基因的人腎小管上皮細(xì)胞HK-2細(xì)胞株,以Lv-shEN2為實(shí)驗(yàn)組、Lv-shcon為空載組、control為空白對(duì)照組。RT-qPCR和Western blot分別檢測(cè)EN2 mRNA和蛋白的表達(dá)水平,MTT檢測(cè)HK-2細(xì)胞增殖能力,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期和凋亡,Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力,細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力。結(jié)果:設(shè)計(jì)與EN2互補(bǔ)的DNA序列,合成shRNA-EN2并克隆到慢病毒載體,同時(shí)設(shè)計(jì)非特異性shRNA生成的載體作為陰性對(duì)照。轉(zhuǎn)染HK-2細(xì)胞后,篩選獲得穩(wěn)定低表達(dá)EN2基因的HK-2細(xì)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(dá)分別是Lv-shEN2組2(-ΔΔCt)=0.58±0.02的1.9倍和1.7倍。Western Blot檢測(cè)各組灰度值Lv-shEN2組2576.65,Lv-shcon組1619.26,control組1478.85。提示轉(zhuǎn)染成功。MTT法分析結(jié)果采用單因素方差分析顯示三組細(xì)胞OD值差別(F=4.78,P=0.01)有統(tǒng)計(jì)學(xué)意義。與兩對(duì)照組細(xì)胞比較,Lv-shEN2組細(xì)胞OD值升高,提示抑制EN2蛋白可促進(jìn)人腎小管上皮細(xì)胞HK-2細(xì)胞的增殖活性。流式細(xì)胞儀檢測(cè)顯示Lv-shEN2組相對(duì)于兩對(duì)照組,細(xì)胞凋亡率下降。Lv-shEN2實(shí)驗(yàn)組、Lv-shcon空載組、control空白對(duì)照組HK-2細(xì)胞凋亡比例分別為9.79%,24.98%和24.9%。與兩對(duì)照組細(xì)胞比較,Lv-shEN2組細(xì)胞早期凋亡率和晚期凋亡率均下降,提示抑制EN2基因表達(dá)可減少HK-2細(xì)胞凋亡。細(xì)胞穿膜實(shí)驗(yàn)顯示Lv-shEN2實(shí)驗(yàn)組、Lv-shcon空載組、control空白對(duì)照組HK-2細(xì)胞穿膜細(xì)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(shí)驗(yàn)組穿膜細(xì)胞數(shù)與兩對(duì)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(xì)胞HK-2的侵襲能力。細(xì)胞劃痕試驗(yàn)各組0h,6h,12h和24h劃痕寬度采用單因素方差分析,結(jié)果顯示各組細(xì)胞遷移能力差別無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:下調(diào)HK-2細(xì)胞株EN2基因的表達(dá)促進(jìn)HK-2細(xì)胞增殖、抑制細(xì)胞凋亡、提高細(xì)胞侵襲能力,對(duì)細(xì)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(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R692

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