hepaCAM基因对人膀胱癌T24细胞黏附性影响及其机制

目的 探讨转染外源性野生型hepaCAM基因对人类膀胱移行细胞癌T24细胞黏附性的影响及其机制.方法 用脂质体介导的基因转染技术,将pEGFP-N2-hepaCAM质粒转染人人膀胱癌T24细胞,用潮霉素G418(400 ms/L)筛选出阳性克隆.Western blot检测hepaCAM蛋白表达;T24细胞与血管内皮细胞黏附实验了解hepaCAM基因对T24细胞黏附力的影响;激光共聚焦显微镜观察T24细胞肌动蛋白细胞骨架的形态学变化;流式细胞仪检测T24细胞肌动蛋白的含量变化.结果 获得稳定表达hepaCAM基因的T24细胞克隆;Western blot证实hepaCAM蛋白的表达;T24细胞与血管内皮细胞黏附实验表明实验组细胞的黏附能力(0.182±0.012)%明显高于空白组(0.110±0.002)%及阴性对照组(0.105±0.004)%(P<0.05);实验组纤维状肌动蛋白荧光增强,亮度增加,按细胞极性方向排列紧密规则,空白组及阴性对照组纤维状肌动蛋白荧光弥散,亮度降低,极性消失,排列疏松不规则;流式细胞分析仪检测证明实验组纤维状肌动蛋白含量(60.71±8.25)%明显高于空白组(7.00±1.01)%及阴性对照组(8.50±1.26)%(P<0.05).结论 hepaCAM基因可显著增强肿瘤细胞的黏附力,其原因可能是通过重组人类膀胱移行细胞癌T24细胞肌动蛋白骨架而影响肿瘤细胞的侵袭与转移.

Effect of hepaCAM gene on cell adhesion of human bladder cancer cell line T24 and the mechanism

Objective To investigate the effects of exogenous wild hepaCAM gene transfection on the adhesiveness of I2.4 cells and the mechanism. Methods Plasmid pEGFP-N2-hepaCAM was transfected into bladder cancer cell line (T24) by LipofectamineTM 2000,and cells expressing hepaCAM stably was screened out by G418 (400 rag/L). Western bolt was used to detect the expression of target of hepaCAM in stable cells. The tumor cell adhere to the vascular endothelial cell to use to research the adhesiveness of hepaCAM gene on T24 cello Using laser confocal microscopy to detect the cytoskeleton in T24 cells. The actin content in T24 cell measured with flow cytometry. Results Acquired T24 cell clone with stable expression of hepaCAMo In vitro experiments as tumor cell adhere to the vascular endothelial ceil indicated the cell adhesion was apparently enhanced compared with the non-transfection (0.110±0.002)% and mock-vehicle groups (0.105±0.004) % (P<0.05). In hepaCAM group, the fluorescence of filamentous actin(F-actin) was strengthen. The structure of filamentous actin was condensed and regularity. In sham and control group,the result was opposite. The content of F-actin in hepaCAM group was apparently enhanced compared with the non-transfection (7.00±1.01 )% and mock-vehicle groups (8.50±1.26)% (P < 0.05). Conclusion The hepaCAM geue can enhance the adhesiveness of T24 cell by induce F-ac-tin cytoskeleton,and may impact the TCCB invasion and metastasis.