【摘要】 运用定量显微形态分析技术、免疫荧光实验技术和MTT分析法,分别对裱衬纤维连接蛋白(fibronectin,FN)和应用肌球蛋白轻链激酶(myosin light chain kinase,MLCK)抑制剂ML-7作用下,肝细胞L02与肝癌细胞HepG2细胞骨架装配和Rho蛋白表达变化,以及细胞迁移能力进行检测和定量表征,了解肝癌细胞侵袭转移的胞内分子基础。结果显示:1)L02Rho蛋白表达水平明显低于HepG2,裱衬FN低浓度(1～5μg/mL)使L02Rho蛋白表达进一步下调,HepG2Rho蛋白表达水平却明显增高,而裱衬FN高浓度(10～40μg/mL)L02Rho蛋白升高超过对照组,HepG2Rho蛋白表达下降,且远低于对照组水平;2)随着裱衬FN浓度的增加,HepG2增殖抑制明显;3)裱衬低浓度FN使HepG2迁移运动能力增强,而高浓度FN使细胞净位移和迁移轨迹离散度减少;4)ML-7低浓度(6μmol/L)使L02Rho蛋白表达下调,而对HepG2Rho蛋白表达无明显影响,L02细胞骨架解聚较HepG2明显;ML-7高浓度(10μmol/L)HepG2Rho蛋白表达减少,且迁移速率降低;L02Rho蛋白表达无进一步下调。说明:1)细胞粘附状态对调节HepG2和L02Rho蛋白表达呈相反趋势;2)HepG2对骨架收缩抑制的响应较L02迟缓;3)HepG2Rho蛋白表达水平与其迁移能力呈正相关。更多还原

【Abstract】 Using quantitative microscopic morphological analysis,immunofluescence,and cell proliferation evaluation with MTT,the authors explored the effects of FN coating and ML-7 treatment,which inhibit the activity of MLCK,on the Rho protein expression and cytoskeleton assembly in hepatic cells(L02) and hepatoma cells(HepG2).Cell motility was aslo quantified under the experimental conditions.The study is expected to deepen understanding on the molecular basis for hepatoma cell migration.The results are,(1) L02 cells’ Rho protein expression is lower than HepG2 cells.The level of Rho protein expression in L02 cells was down-regulated when FN was coated in a concentration range of 1 ～5 μg/mL,which contrasted the case for HepG2 cells where the Rho protein expression increased significantly.The level of L02 Rho protein expression was higher than control with higher FN concentration range of 10～40 μg/mL.Again,this was in contrast with the case for HepG2 cells,where Rho protein expression became lower than the control values.(2) The inhibition of HepG2 cells proliferation increased significantly with the increasing coating concentration of FN.(3) For HepG2 cells,displacement and migration trajectory increased significantly with low FN coating concentration of 1 ～ 5 μg/mL,however these parameters reduced at higher concentrations of 10 ～40 μg/mL.(4) With the addition of 6 μmol/L ML-7,Rho protein expression decreased in L02 cells,yet this concentration had no significant effects in HepG2 cells.When treated with a higher concentration 10 μmol/L of ML-7,cell migration rate and Rho protein expression were reduced in HepG2 cells,However,the expression of Rho protein was not down-regulated further in L02 cells.It is shown that,(1) There is an opposite tendency for regulating the Rho protein expression by the cell adhesion status in HepG2 vs L02 cells;(2) The response to inhibition of the cytoskeleton contraction in HepG2 cells is more slowly than in L02 cells.(3) Rho protein expression levels and cell motility are positively correlated for HepG2 cells.更多还原