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目的:观察MARK2基因对He La细胞极性与增殖的影响,探讨其可能的机制。方法:脂质体介导MARK2质粒转染He La细胞,G418抗性筛选获得稳定转染的细胞株。TRITC-Phalloidin染色观察细胞极性,平板克隆形成试验研究细胞贴壁生长能力,流式细胞术分析细胞周期各时相细胞的比例,Western blot检测总Rb蛋白及磷酸化Rb蛋白的水平。结果:成功构建稳定转染MARK2的He La细胞系,与空质粒对照组细胞相比,MARK2蛋白表达增强后He La细胞形态改变、上皮细胞极性恢复;MARK2表达增强后He La细胞形成平板克隆数量明显减少[(303.67±7.77)vs.(111.67±7.64),P=0.000],细胞周期G1期比例明显增加[(48.82±0.84)%vs.(72.01±2.13)%,P=0.000]、S期比例减少[(36.60±0.58)%vs.(16.03±3.85)%,P=0.010],Rb蛋白的磷酸化水平明显降低[(7.66±0.74)vs.(1.10±0.13),t=15.053,P=0.003]。结论:在He La细胞中,MARK2通过抑制Rb蛋白的磷酸化,使细胞周期停滞在G1期,抑制细胞增殖。MARK2能够重建He La细胞上皮极性,协同调控细胞极性与细胞增殖两条通路。
Objective: To observe the effect of MARK2 gene on the polarity and proliferation of HeLa cells and to explore its possible mechanism. Methods: HeLa cells transfected with MARK2 plasmid were transfected by liposome, and stable transfected cell lines were obtained by G418 resistance screening. The polarity of the cells was observed by TRITC-Phalloidin staining. The adherent growth ability of the cells was determined by plate colony formation assay. The ratio of phase cells was analyzed by flow cytometry. The levels of total Rb protein and phosphorylated Rb protein were detected by Western blot. RESULTS: HeLa cell line stably transfected with MARK2 was successfully constructed. Compared with the empty plasmid control group, the expression of MARK2 protein increased and the He La cell morphology changed and the polarity of epithelial cells recovered; HeLa cells formed after MARK2 expression was enhanced. The number was significantly decreased [(303.67±7.77) vs. (111.67±7.64), P=0.000], and the proportion of G1 phase in the cell cycle was significantly increased [(48.82±0.84)% vs. (72.01±2.13)%, P=0.000], The proportion of S phase decreased [(36.60±0.58)% vs. (16.03±3.85)%, P=0.010], and the phosphorylation level of Rb protein was significantly decreased [(7.66±0.74) vs. (1.10±0.13), t=15.053 , P=0.003]. CONCLUSION: In HeLa cells, MARK2 inhibits the phosphorylation of Rb protein, arrests the cell cycle in G1 phase, and inhibits cell proliferation. MARK2 can reconstruct the epithelial polarity of He La cells and coordinate the two pathways of cell polarity and cell proliferation.