三维和二维培养对砷诱导膀胱上皮细胞间充质转化的影响

Effects of three-and two-dimensional culture on arsenic induced mesenchymal transformation of bladder epithelial cells

目的 比较三维(3D)和二维(2D)培养下慢性砷暴露的膀胱上皮细胞间充质转化(epithelial mesenchymal transition, EMT)的相关因子表达。方法 2D培养人膀胱上皮细胞(SV-HUC-1)暴露于0、0.5μmol/L亚砷酸钠(NaAsO_(2))40周,分别于2D和3D(采用Matrigel构建)培养5 d,采用反转录聚合酶链反应(RT-qPCR)和免疫印迹法(Western blot)检测EMT的E-cadherin、Vimentin mRNA和蛋白的水平。结果 膀胱上皮细胞株SV-HUC-1在3D培养中呈球形生长,3 d后逐渐聚团。从mRNA水平看,在2D与3D培养中As暴露组较对照组的E-cadherin水平显著下降(P<0.01),Vimentin水平显著升高(P<0.05);在两种培养方式下As暴露组间差异也有统计学意义(P<0.01)。2D培养中,与对照组比较,As暴露组的E-cadherin蛋白表达显著下降(P<0.001),Vimentin蛋白表达显著升高(P<0.01);3D培养条件下,染毒前后两种蛋白水平差异无统计学意义。结论 膀胱上皮细胞株SV-HUC-1在3D培养环境下细胞呈球形;长期低剂量As暴露的膀胱上皮细胞株E-cadherin和Vimentin的表达变化在2D培养条件下更显著。

Objective To compare the expression of bladder epithelial mesenchymal transition(EMT) induced by chronic arsenic exposure under three dimensions(3 D) and two dimensions(2 D).Methods SV-HUC-1 cells were treated with NaAsO(0.5 μmol/L) for 40 weeks, then, cultured in 2 D and 3 D(built with Matrigel) conditions for 5 days, respectively.RT-qPCR and Western blot were used to measure the levels of E-cadherin and Vimentin mRNA,and the proteins contents of EMT.Results The results showed that the bladder epithelial cell line SV-HUC-1 showed spherical growth in 3 D culture and gradually aggregated after 3 days.In terms of mRNA level, E-cadherin level in NaAsOexposed group was significantly decreased compared with control group both in 2 D and 3 D culture(P<0.01),while the mRNA level of Vimentin had increased significantly(P<0.05),additionally, there were significant differences between the exposed groups under two culture methods(P<0.01).In 2 D culture, the protein expression of E-cadherin in exposed group was significantly decreased compared with the control group(P<0.001),the Vimentin protein expression was significantly increased(P<0.01);while in 3 D culture, there was no significant difference in the proteins expression before and after exposure.Conclusion The epithelial cell line SV-HUC-1 of bladder was grown in spherical shape under 3 D cell culture, the expression of E-cadherin and Vimentin in bladder epithelial cell lines exposed to long-term low-dose sodium arsenite were more significant under 2 D culture condition.