CAJ | 학술논문

目的：研究磷脂酰肌醇3－激酶（PI3K）p55γ调节亚基N末端24个氨基酸（N24）过表达对胃癌细胞MGC803黏附性的影响，并初步探讨其作用的分子机制。方法：通过脂质体介导用pEGFP－N24和pEGFP－C1质粒分别进行基因转染，建立稳定表达融合蛋白GFP－N24和GFP的MGC803细胞系。倒置显微镜下观察转染细胞的形态学变化，免疫印迹实验鉴定转染基因的蛋白表达，细胞黏附实验检测转染细胞的黏附性，免疫印迹实验检测细胞黏附分子上皮细胞钙黏蛋白（ E－cadherin ）和钙紧张素（β－catenin ）的表达，酶谱实验检测基质金属蛋白酶9（MMP9）和尿激酶型纤溶酶原活化因子（ uPA）的表达。结果：建立了稳定表达融合蛋白GFP－N24的MGC803／GFP－N24细胞系和表达 GFP的 MGC803／pEGFP－C1细胞系，但 GFP－N24的表达量明显低于 GFP。基因转染使MGC803细胞的形态由铺路石样转变为成纤维细胞样，胞浆分泌颗粒明显增加。表达GFP－N24的MGC803细胞在纤黏连蛋白和胶原上的黏附性与对照组细胞相比明显下降（P＜0．05）。 GFP－N24的表达使MGC803细胞黏附分子E－cadherin的表达增高，而Wnt信号通路关键分子β－catenin的表达降低，但不影响肿瘤转移相关蛋白MMP9和uPA的表达与分泌。结论： PI3K p55γN 末端氨基酸过表达通过影响 E－cadherin 和β－catenin 的表达而抑制胃癌MGC803细胞的黏附性。
목적：연구린지선기순3－격매（PI3K）p55γ조절아기N말단24개안기산（N24）과표체대위암세포MGC803점부성적영향，병초보탐토기작용적분자궤제。방법：통과지질체개도용pEGFP－N24화pEGFP－C1질립분별진행기인전염，건립은정표체융합단백GFP－N24화GFP적MGC803세포계。도치현미경하관찰전염세포적형태학변화，면역인적실험감정전염기인적단백표체，세포점부실험검측전염세포적점부성，면역인적실험검측세포점부분자상피세포개점단백（ E－cadherin ）화개긴장소（β－catenin ）적표체，매보실험검측기질금속단백매9（MMP9）화뇨격매형섬용매원활화인자（ uPA）적표체。결과：건립료은정표체융합단백GFP－N24적MGC803／GFP－N24세포계화표체 GFP적 MGC803／pEGFP－C1세포계，단 GFP－N24적표체량명현저우 GFP。기인전염사MGC803세포적형태유포로석양전변위성섬유세포양，포장분비과립명현증가。표체GFP－N24적MGC803세포재섬점련단백화효원상적점부성여대조조세포상비명현하강（P＜0．05）。 GFP－N24적표체사MGC803세포점부분자E－cadherin적표체증고，이Wnt신호통로관건분자β－catenin적표체강저，단불영향종류전이상관단백MMP9화uPA적표체여분비。결론： PI3K p55γN 말단안기산과표체통과영향 E－cadherin 화β－catenin 적표체이억제위암MGC803세포적점부성。
AIM:To investigate the effect of the N-terminal 24-amino acid (N24) overexpression in p55γre-gulatory subunit of phosphatidylinositiol 3-kinase ( PI3K) on the cell adhesion of human gastric carcinoma cell MGC 803. METHODS:MGC803 cells, which stably expressed GFP-N24 fusion protein and GFP alone , were generated by transfec-tion with pEGFPN-24 plasmid and control plasmid pEGFP-C1, respectively.The morphological change of the cells was ob-served under inverted microscope .The expression of GFP-N24 fusion protein was detected by Western blot .The adhesion of the cells was determined by cell adhesion assay .The effects of GFP-N24 fusion protein on the expression of E-cadherin andβ-catenin were analyzed by Western blot .The expression and secretion of matrix metalloproteinase 9 (MMP9) and u-rokinase-type plasminogen activator ( uPA ) in the MGC803 cells were detected by gelatin zymography .RESULTS:MGC803/GFP-N24 cell line steadily expressed GFP-N24 fusion protein and MGC803/GFP cell line steadily expressing GFP were successfully established , but the expression of fusion protein GFP-N24 was lower than that of the control protein GFP.The morphological changes of the transfected cells from paving stone to fibroblast cell form after gene transfection , and the cytoplasm secretory granules were increased significantly .The cell adhesion to fibronectin and collagen decreased after GFP-N24 transfection .GFP-N24 fusion protein increased the expression of cell adhesion molecule E-cadherin and de-creased the wnt signal pathway molecule β-catenin in the MGC803 cells.However, it did not affect the expression and se-cretion of tumor metastasis-related proteins MMP9 and uPA.CONCLUSION:Overexpression of N24p55γinhibits cell ad-hesion by influencing the expression of E-cadherin and β-catenin in the MGC803 cells.