蛋白酪氨酸磷酸酶SHP-2在乳腺癌细胞移动及粘附中的作用

探讨蛋白酪氨酸磷酸酶SHP 2在乳腺癌细胞MCF 7的移动及粘附中的作用 .利用基因重组技术分别将野生型SHP 2与突变型SHP 2与绿色荧光蛋白GFP的基因片段构成重组质粒 (SHP 2 GFP、SHP 2C >S GFP) .脂质体转染法分别转入MCF 7中 ,表达成功后筛选并建立SHP 2 GFP和SHP 2C >S GFP细胞株 .荧光显微镜观察细胞移动情况 ,免疫印迹法检测粘附分子E 钙粘蛋白和金属蛋白酶MMP 1及MMP 9的表达 .实验后建立SHP 2 GFP及SHP 2C >S GFP细胞株 ,同时观察到SHP 2C >S GFP细胞的形态发生明显改变 :从梭形状态变成圆形状态 .荧光显微镜发现 ,MCF 7细胞和SHP 2 GFP、SHP 2C >S GFP转染的细胞在 3h、6h、9h的移动情况分别是MCF 7为 10 %、2 3%、5 4% ,SHP 2 GFP为 15 %、4 9%、98% ,SHP 2C >S GFP为 4 %、11%、30 % .免疫印迹结果表明 ,SHP 2C >S GFP细胞的E 钙粘蛋白表达比SHP 2 GFP细胞明显升高 (P <0 0 5 ) .MMP 1及MMP 9的表达量在SHP 2 GFP细胞中有所增强 (P <0 0 5 ) .实验表明 ,SHP 2可能通过调节粘附分子和基质金属磷酸酶而在细胞移动、粘附中发挥重要作用

Roles of Protein Tyrosine Phosphatase SHP-2 in Cell Migration and Adhesion in Breast Cancer Line MCF-7

The roles of protein tyrosine phosphatase SHP 2 in breast cancer(MCF 7) cell adhesion and migration were investigated. For expression of wide type SHP 2 GFP and mutant type SHP 2C>S GFP protein, a recombinant vector was constructed by inserting a GFP cDNA fragment into SHP 2 and SHP 2C>S at a Xba I site. Then Perfect TM Lipids containing 1 μg of the recombinant DNA plasmid were transfected into MCF 7 cells and the resistant cell lines were selected with hygromycin. Cell migrations were observed under a fluorescence microscope .The exprssions of E cadherin and MMP 1 and MMP 9 were detected by immunoblot. SHP 2 GFP and SHP 2C>S GFP cell lines were constructed. The morphology of the mutant transfected cell lines varied from shuttle to round. The migrations of three cell lines at 3 hours, 6 hours,9 hours were as follows: GFP transfected cell line: 10%,23%,54%; SHP 2 GFP transfected cell lines:15%,49%,98% and SHP 2C>S GFP transfeted cell lines: 4%,11%,30%.The expression of E cadherin was higher in SHP 2C>S GFP transfected cell lines than that in SHP 2 GFP transfected cell lines, while the expression of MMP 1 and MMP 9 was higher in SHP 2 GFP transfected cell lines. It is considered that the protein tyrosine phosphatase SHP 2 plays a role in cell migration and adhesion mediated by regulating the expression of cadherin and MMPS.