硫酸右旋糖苷抑制人胃癌细胞粘附以及整合素β_1基因表达的机制研究

A mechanism study of the inhibition of the adhesion of human gastric cancer MKN1 cells and suppression of the expression of integrin β_1 gene by dextran sulfate

目的通过观察人胃癌MKN1细胞的粘附过程和测定整合素β1cDNA的表达水平,研究硫酸右旋糖苷(dextransulfate,DS)对人胃癌MKN1细胞粘附的抑制作用的机制。方法分别在培养液中加入DS或PBS对MKN1细胞进行培养,用荧光免疫细胞染色法和共聚焦显微镜对固定细胞和活细胞在培养盘上的贴壁过程及形态变化进行观察,用RT-PCR测定整合素β1的cDNA表达。结果MKN1细胞通过变形,形成伪足粘附到培养盘,并与其他细胞接触形成单细胞层。免疫染色可见整合素β1在细胞膜上有强烈的表达,并且形成整合素集落。DS抑制整合素β1的表达并使已经形成的整合素集落区域减少;使MKN1细胞的伪足缩短,表现出维持圆形的倾向。在使用DS培养4h后仍有部分细胞处于游离状态,分别收集游离细胞和贴璧细胞,用RT-PCR测定整合素β1的cDNA表达。实验组贴壁细胞和游离细胞内整合素β1的cDNA的表达水平分别减少到对照组细胞的74%和38%。结论DS对人胃癌细胞MKN1粘附过程的抑制与整合素β1表达的减弱有关。

Aim To observe the adhesive process of the human gastric cancer cell line MKN1, and study the expression of integrin β1 ;to investigate the mechanism of the inhibition of the adhesion process of MKN1 cells by destran sulfate （DS）. Methods The MKN1 cells were cultured with DS or PBS, then stained with immunofluorescent cytochemistry and observed in fixed or living conditions with confocal laser scanning microscope. RT-PCR was used to analyze the cDNA expression of MKN1 cells. Results MKN1 cells adhered to culture dishes by the process Of forming filopodia, changed into a flat shape, and then adhered to other cells to form a cell-monolayer. Integrin β1 was intensively expressed in the cell membrane, where integrin β1 formed clusters. DS inhibted the expression of integrin β1 in cell membrane ,and decreased the area of integrin β1 clusters. DS-treated cells also tended to maintain a round shape by contracting the filopodia. In DS- containing culture dishes, some cells kept floating 4 hours after seeding. DS decreased the level of the cDNA expression of the adhered cells to 74% and of the floating cells to 38% of that of the cells in un-treated group, respectively. Conclusion The inhibition of the adhesion of MKN1 cells by DS was related to the suppression of the expression of integrin β1.