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目的:探讨ROCK-Ⅰ蛋白活性的改变对卵巢癌细胞恶性表型的影响。方法:将ROCK-Ⅰ的显性激活突变体p160△3以脂质体介导,转染人卵巢癌细胞系SW626、SKOV3、A2780和CaOV-3细胞,采用RT-PCR与Westernblot印迹法检测转染前后p160△3mRNA和蛋白的表达,Boyden小室观察该突变体对4种卵巢癌细胞株侵袭及迁移能力的影响,MTT比色法测定转染前后细胞增殖及粘附能力的变化。结果:转染后,ROCK-Ⅰ的显性激活突变体p160△3在细胞内获得有效表达。转染了p160△3的SW626、SK-OV3、A2780和CaOV-3细胞的侵袭能力较各自的对照组分别提高(31.1±6·4)%、(33.0±2.0)%、(24.5±8)%及(39.4±2.8)%;同时四株细胞的随机运动能力较各自的对照组分别提高(31.9±1.0)%、(31.5±1.9)%、(23.0±0.5)%及(38.7±1·2)%,定向运动能力较各自的对照组分别提高(33.9±1.1)%、(33.0±3.6)%;(25.0±3.9)%及(40·2±2.6)%。转染后两株细胞的体外粘附能力及增殖能力均未显示出明显变化。空质粒pCAG-myc转染组与对照组间差异无显著统计学意义(P>0.05)。结论:ROCK-Ⅰ蛋白活性的改变与人卵巢癌细胞的体外侵袭与迁移密切相关,ROCK-Ⅰ蛋白活性的提高明显促进卵巢癌肿瘤细胞的侵袭与迁移。
Objective: To investigate the effect of ROCK-Ⅰ protein activity on the malignant phenotype of ovarian cancer cells. Methods: The dominant-negative mutant p160 △ 3 of ROCK-Ⅰ was transfected into human ovarian cancer cell lines SW626, SKOV3, A2780 and CaOV-3 by lipofectamine 2000. RT-PCR and Western blotting were used to detect the expression of The expression of p160 △ 3 mRNA and protein were detected before and after staining. The effect of the mutant on the invasion and migration of the four ovarian cancer cell lines was observed in Boyden chamber. The proliferation and adhesion of cells were detected by MTT assay. Results: After transfection, p160 △ 3, a dominant activating mutant of ROCK-Ⅰ, was efficiently expressed in cells. The invasion ability of SW626, SK-OV3, A2780 and CaOV-3 cells transfected with p160 △ 3 were (31.1 ± 6.4)%, (33.0 ± 2.0)% and (24.5 ± 8) (31.9 ± 1.0)%, (31.5 ± 1.9)%, (23.0 ± 0.5)%, (38.7 ± 1)% and (39.4 ± 2.8)% respectively in the control group compared with the control group 2)%, and the motor ability of directional movement increased by (33.9 ± 1.1)%, (33.0 ± 3.6)%, (25.0 ± 3.9)% and (40.2 ± 2.6)% respectively compared with the corresponding control group. After transfection, the adhesion and proliferation ability of the two cells did not show obvious changes in vitro. There was no significant difference between empty plasmid pCAG-myc transfection group and control group (P> 0.05). CONCLUSION: The change of ROCK-Ⅰ activity is closely related to the invasion and migration of human ovarian cancer cells in vitro. The increase of ROCK-Ⅰ activity obviously promotes the invasion and migration of ovarian cancer cells.