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目的通过比较融合生长肝细胞(L02)与肝癌细胞(HCCLM3)对不同硬度基底(0.5、4 kPa和玻璃)的响应,探查两类细胞融合生长差异的成因。方法运用实时显微摄影技术、免疫荧光染色、流式细胞技术及Western Blot-ting等试验方法分别检测融合生长L02和HCCLM3细胞在不同硬度基底上的形态特征、骨架构象、E-cad和Integrinβ1表达分布,以及Src激酶活性水平的变化。结果 (1)融合生长L02细胞呈圆形或立方形,HCCLM3细胞呈多角形,铺展和极化更为明显;随基底硬度增加,L02细胞圆度随时间变化幅度较小,HCCLM3细胞变化幅度较大。(2)融合生长的L02细胞皮质下呈现环形骨架,E-cad定位于细胞-细胞接触处,HCCLM3中皮质骨架环不完整,胞内骨架沿基底呈放射状分布,E-cad呈弥散分布于细胞质中。(3)随基底硬度增加,L02和HCCLM3细胞中E-cad表达显著降低(P<0.01),而p-Src和Integrinβ1的表达量则显著增加(P<0.01),HCCLM3较L02细胞变化明显。结论皮质下环形骨架装配与E-cad在细胞-细胞定位呈正相关,基底硬度对肝癌细胞钙黏素与整合素黏附系统平衡调节的影响较对肝细胞的影响明显。
Objective To investigate the genesis differences between two types of cell fusion by comparing the response of L02 and HCCLM3 to different hardness substrates (0.5, 4 kPa and glass). Methods The morphological characteristics, skeletal architecture, expression of E-cad and Integrinβ1 in L02 and HCCLM3 cells were detected by real-time microscopy, immunofluorescence, flow cytometry and Western Blot-ting respectively. Distribution, and changes in Src kinase activity. Results (1) L02 cells fused and formed round or cuboid shape, HCCLM3 cells were polygonal, spreading and polarization more obvious; with the increase of substrate hardness, L02 cells roundness changes with time, HCCLM3 cells more than the magnitude of change Big. (2) The fused L02 cells showed a ring-shaped skeleton under the cortex, E-cad located at the cell-cell contact, HCCLM3 cortical framework ring is not complete, the intracellular skeleton is radially distributed along the base, E-cad was diffusely distributed in the cytoplasm in. (3) With the increase of substrate hardness, the expression of E-cadherin in L02 and HCCLM3 cells was significantly decreased (P <0.01), while the expression of p-Src and Integrinβ1 was significantly increased (P <0.01), while HCCLM3 was more obvious than L02 cells. Conclusions The assembly of cortical annular framework and E-cad has a positive correlation with cell-cell localization. The effect of base hardness on the balance regulation of HCC cadherin and integrin adhesion system is more significant than that of hepatocyte.