胃肠病学
胃腸病學
위장병학
CHINESE JOURNAL OF GASTROENTEROLOGY
2014年
9期
540-543
,共4页
结肠肿瘤%鸟嘌呤核苷酸交换因子类%Rho蛋白%载体构建%转染
結腸腫瘤%鳥嘌呤覈苷痠交換因子類%Rho蛋白%載體構建%轉染
결장종류%조표령핵감산교환인자류%Rho단백%재체구건%전염
Colonic Neoplasms%Guanine Nucleotide Exchange Factors%Rho Protein%Vector Construction%Transfection
背景：研究表明GEF-H1能激活Rho蛋白，与肿瘤发生、发展、浸润、迁移等密切相关。目的：构建人pEGFP-GEF-H1载体并稳定转染结肠癌HCT116细胞后检测GEF-H1表达。方法：在线合成引物软件设计人GEF-H1基因引物，构建重组质粒pEGFP-GEF-H1，并转染结肠癌HCT116细胞，以转染空质粒和未转染的细胞分别作为空质粒组和空白对照组。荧光显微镜下观察绿色荧光蛋白表达，RT-PCR和蛋白质印迹法分别检测GEF-H1 mRNA和蛋白表达水平。结果：成功构建了人pEGFP-GEF-H1载体，测序结果表明序列正确。转染重组质粒pEGFP-GEF-H1后，荧光显微镜下可见较强的绿色荧光，GEF-H1 mRNA和蛋白表达水平均较空质粒组和空白对照组明显上调。结论：体外成功构建了人pEGFP-GEF-H1载体，且转染结肠癌HCT116细胞后高表达GEF-H1，为进一步探讨结肠癌与GEF-H1的关系提供了必要的实验材料。
揹景：研究錶明GEF-H1能激活Rho蛋白，與腫瘤髮生、髮展、浸潤、遷移等密切相關。目的：構建人pEGFP-GEF-H1載體併穩定轉染結腸癌HCT116細胞後檢測GEF-H1錶達。方法：在線閤成引物軟件設計人GEF-H1基因引物，構建重組質粒pEGFP-GEF-H1，併轉染結腸癌HCT116細胞，以轉染空質粒和未轉染的細胞分彆作為空質粒組和空白對照組。熒光顯微鏡下觀察綠色熒光蛋白錶達，RT-PCR和蛋白質印跡法分彆檢測GEF-H1 mRNA和蛋白錶達水平。結果：成功構建瞭人pEGFP-GEF-H1載體，測序結果錶明序列正確。轉染重組質粒pEGFP-GEF-H1後，熒光顯微鏡下可見較彊的綠色熒光，GEF-H1 mRNA和蛋白錶達水平均較空質粒組和空白對照組明顯上調。結論：體外成功構建瞭人pEGFP-GEF-H1載體，且轉染結腸癌HCT116細胞後高錶達GEF-H1，為進一步探討結腸癌與GEF-H1的關繫提供瞭必要的實驗材料。
배경：연구표명GEF-H1능격활Rho단백，여종류발생、발전、침윤、천이등밀절상관。목적：구건인pEGFP-GEF-H1재체병은정전염결장암HCT116세포후검측GEF-H1표체。방법：재선합성인물연건설계인GEF-H1기인인물，구건중조질립pEGFP-GEF-H1，병전염결장암HCT116세포，이전염공질립화미전염적세포분별작위공질립조화공백대조조。형광현미경하관찰록색형광단백표체，RT-PCR화단백질인적법분별검측GEF-H1 mRNA화단백표체수평。결과：성공구건료인pEGFP-GEF-H1재체，측서결과표명서렬정학。전염중조질립pEGFP-GEF-H1후，형광현미경하가견교강적록색형광，GEF-H1 mRNA화단백표체수평균교공질립조화공백대조조명현상조。결론：체외성공구건료인pEGFP-GEF-H1재체，차전염결장암HCT116세포후고표체GEF-H1，위진일보탐토결장암여GEF-H1적관계제공료필요적실험재료。
Background:Studies have shown that GEF-H1 can activate Rho protein, and is closely associated with tumorigenesis,tumor progression,infiltration,migration. Aims:To construct human pEGFP-GEF-H1 vector and stably transfect into colon cancer HCT116 cells,and determine the expression of GEF-H1. Methods:The gene sequence was designed according to the Online primers synthesis software for constructing recombinant plasmid pEGFP-GEF-H1. HCT116 cells were transfected with plasmid pEGFP-GEF-H1,empty plasmid,respectively,and HCT116 cells without transfection were served as blank control group. Expression of green fluorescence protein was observed under fluorescence microscope. mRNA and protein expressions of GEF-H1 were determined by RT-PCR and Western blotting,respectively. Results:Human pEGFP-GEF-H1 vector was successfully constructed and identified by sequence analysis. After transfection with pEGFP-GEF-H1,green fluorescence could be observed in HCT116 cells,and the mRNA and protein expressions of GEF-H1 were higher than those in empty plasmid group and blank control group. Conclusions:Recombinant plasmid pEGFP-GEF-H1 is successfully constructed in vitro,and transfection into HCT116 cells leads to high expression of GEF-H1, thereby can provide experimental materials needed for further study on correlation between colon cancer and GEF-H1.
