蛋白激酶C对大肠癌HT-29细胞骨架影响的形态学研究

Morphological study on effects of protein kinase C on large intestine carcinoma HT-29 cells cytoskeleton

目的:从形态学角度探讨蛋白激酶C(protein kinase C,PKC)对大肠癌细胞骨架的调节作用。方法:采用考马斯亮蓝法和间接免疫荧光法显示PKC激活剂PMA和抑制剂Staurosporine(SP)对大肠癌HT-29细胞骨架微丝纤维、微管蛋白(tubulin)、黏着斑纽蛋白(vinculin)表达的影响,研究PKC对HT-29细胞骨架调节作用。结果:HT-29细胞骨架微丝、微管纤维和黏着斑数量丰富,但纤维短,排列紊乱,无分极现象;而PMA处理可使细胞骨架出现分极现象,骨架微丝、微管纤维和黏着斑数量减少,向两极放射排列,纤维稀疏,并可见向外伸展的板层(lamellae)结构和板状伪足(lamellipodia);而SP可抑制PMA的作用,使细胞骨架微丝、微管纤维和黏着斑数量增多、增粗,排列规则,边界完整,形成完整的骨架网络结构。结论:PMA通过激活PKC表达,促进骨架蛋白微丝、微管的解聚和黏着斑的解体,有利于细胞骨架的重组,促进细胞改变形态利于扩散、运动;SP可拮抗PMA作用,通过抑制PKC表达,导致骨架蛋白不能解聚或解体,不利于细胞骨架的重组,影响细胞的移动。推测PKC对细胞侵袭、转移的调节可能部分涉及了其调节细胞骨架的机制。

Objective：To evaluate the effects of protein kinase C （PKC） on large intestine carcinoma cells（ HT- 29 ） cytoskeleton by morphological study. Methods ： Cytoskeleton components, such as microfilaments, tubulin, vinculin changes of large intestine carcinoma HT - 29 cells by using PKC activator PMA and inhibitor SP were displayed by coomassie staining and indirect immunofluorescence staining, in order to study the effects of PKC on HT -29 cells cytoskeleton. Results ： Abundant cytoskeleton components of HT - 29 cell, such as microfilaments, microtubules and the focal contact,were observed, but skeleton fibres were short, arrangement was in disorder and not polarized. PMA could induce the cytoskeleton components of HT - 29 cell to be polarized and to be decreased, and the invasive structures of lamellae and lamellipodia were also observed. However, SP could inhibit the effects induced by PMA, and induce the cytoskeleton components of HT - 29 cell to be increased markedly and to be arranged trimly, and to form the intact cytoskeleton framework. Conclusion：Activation of PKC by treatment with PMA causes depolymerization and reorganization of microfilaments,microtubules and focal adhesions. SP can inhibit the effects of PMA, and not further causes depolymerization and reorganization of microfilaments, microtubules and focal adhesions. We propose that HT - 29 cells invasion and metastasis adjuested by PKC may involve the mechanism of effecting cytoskeleton with PKC.