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目的通过建立与肝组织生理和病理硬度相当的体外培养模型,探讨基底硬度和TGF-β1协同作用对肝细胞表型变化的影响。方法采用免疫荧光显微观测技术及Western Blotting等试验方法,研究肝细胞在不同硬度梯度的聚丙烯酰胺基底膜上的细胞形态调整、运动变化特征、骨架构象以及整合素、E-钙粘素、白蛋白和alpha-平滑肌动蛋白表达的差别,并通过图像分析软件对上述结果进行定量分析。结果 3.6 kPa基底膜上单个分散细胞运动变形活跃,但群体细胞极化变小,肌动蛋白沿皮质下呈环状排列,E-钙粘素和白蛋白表达高,整合素和alpha-平滑肌动蛋白的表达水平较低,加药组与对照组变化趋势一致;30 kPa基底膜上细胞运动变形欠活跃,加药组与对照组相比,E-钙粘素和白蛋白表达均下调,alpha-平滑肌动蛋白表达上调;30 kPa与3.6 kPa对照组相比、30 kPa与3.6 kPa加药组相比,E-钙粘素及白蛋白表达均下调(P<0.05),alpha-平滑肌肌动蛋白的表达上调(P<0.05)。10 kPa基底膜上对照组和加药组与30 kPa和3.6 kPa对照组和加药组相比,均无显著性差异。结论基底硬度增加可诱导肝细胞表型转化,并促进TGF-β1对肝细胞代谢行为的影响。
OBJECTIVE: To investigate the effects of substrate hardness and synergistic effect of TGF-β1 on the changes of hepatocyte phenotype by establishing an in vitro culture model that is comparable to the physiological and pathological hardness of liver tissue. Methods Immunofluorescence microscopy and Western Blotting were used to study the changes of cell morphology, motility, skeletal architecture and integrin, E-cadherin, Albumin and alpha-smooth actin expression differences, and through image analysis software for quantitative analysis of the above results. RESULTS: The single dispersed cells on 3.6 kPa basement membrane were active and deformable, but the population of cells became less polarized. Actin was arranged in a ring along the cortex with high expression of E-cadherin and albumin, integrin and alpha-smooth muscle The expression of E-cadherin and albumin in the drug-loaded group was lower than that in the control group - smooth actin expression was up-regulated; E-cadherin and albumin were down-regulated (P <0.05), alpha-smooth muscle motility was lower in 30 kPa compared with 3.6 kPa control group The protein expression was up-regulated (P <0.05). There was no significant difference between the control group and the dosing group on the 10 kPa basement membrane and the control group and the dosing group at 30 kPa and 3.6 kPa. Conclusion The increase of basal stiffness induces hepatocyte phenotype transformation and promotes the effect of TGF-β1 on the metabolism of hepatocytes.