论文部分内容阅读
目的:探讨ROCK-I蛋白表达或活性改变对人卵巢癌细胞运动表型的影响。方法:将ROCK-I的反义寡核苷酸(ASODN)或其显性激活突变体p160Δ3用脂质体介导,转染人卵巢癌细胞系CAOV-3细胞,用RT-PCR与W estern blot印迹法检测转染前后ROCK-I和p160Δ3 mRNA和蛋白的表达;rhodam ine-phalloid in染色显示ROCK-I ASODN和p160Δ3对细胞骨架的影响;Transwell小室和划痕实验观察ROCK-I表达降低及其活性提高后卵巢癌细胞系CAOV-3运动能力的改变。结果:转染ROCK-I ASODN后,CAOV-3细胞内ROCK-I蛋白的表达明显减少,最大抑制率可达49%;转染的p160Δ3在细胞内获得有效表达。转染ROCK-I ASODN后CAOV-3细胞伪足消失,肌动蛋白纤维减少且变得无序,而转染p160Δ3后细胞伪足增多变长,肌动蛋白纤维亦明显增多,且沿细胞长轴分布。伤口愈合实验显示,ROCK-I ASODN明显抑制了CAOV-3细胞的迁移,p160Δ3则显著增强了其迁移能力;转染10μmol/L或20μmol/L ROCK-I ASODN后细胞的随机运动能力分别为其对照组的(72.0±1.3)%和(55.9±2.5)%,定向运动能力分别为其对照组的(72.5±3.4)%和(54.5±1.9)%;转染p160Δ3后,与对照组相比细胞的随机运动能力提高(38.7±1.2)%,定向运动能力提高(40.2±2.6)%。结论:ROCK-I蛋白表达或活性改变与人卵巢癌细胞CAOV-3的运动能力密切相关,ROCK-I可能成为治疗卵巢癌肿瘤细胞转移的新靶点。
Objective: To investigate the effect of ROCK-I protein expression or activity on the kinetic phenotype of human ovarian cancer cells. METHODS: The antisense oligonucleotide of ROCK-I (ASODN) or its dominant-negative mutant p160Δ3 was transfected into human ovarian cancer cell line CAOV-3 by lipofectamine 2000. RT-PCR and Western blot The expression of ROCK-I and p160Δ3 mRNA and protein before and after transfection was detected by Western blotting. The effect of ROCK-I ASODN and p160Δ3 on cytoskeleton was observed by rhodam ine-phalloid in staining. The decrease of ROCK-I expression was observed by Transwell chamber and scratch assay The activity of ovarian cancer cell line CAOV-3 after exercise increased. RESULTS: After ROCK-I ASODN was transfected, the expression of ROCK-I protein was significantly decreased in CAOV-3 cells with the maximum inhibition rate of 49%. The transfected p160Δ3 was effectively expressed in cells. After transfection with ROCK-I ASODN, the pseudopods of CAOV-3 cells disappeared and the actin fibers decreased and became disordered. After transfection with p160Δ3, the pseudopodia increased and the actin fibers increased obviously. Shaft distribution. Wound healing experiments showed that ROCK-I ASODN significantly inhibited the migration of CAOV-3 cells, p160Δ3 significantly enhanced its migration ability; transfected with 10μmol / L or 20μmol / L ROCK-I ASODN cells were random movement capacity (72.0 ± 1.3)% and (55.9 ± 2.5)% in the control group were (72.5 ± 3.4)% and (54.5 ± 1.9)% in the control group, respectively. After transfection of p160Δ3, The random exercise capacity of the cells increased by 38.7 ± 1.2% and the ability of directional movement increased by 40.2 ± 2.6%. CONCLUSION: The change of ROCK-I protein expression or activity is closely related to the motor ability of CAOV-3 in human ovarian cancer cells. ROCK-I may be a new target for the treatment of ovarian cancer cell metastasis.