摘要
目的研究人内皮型一氧化氮合酶运输介导物（endothelial nitric oxide synthase traffic inducer, NOSTRIN）基因在体外培养的人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）中的过度表达对该细胞生物学特性的影响。方法脂质体介导转染体外培养的HU-VEC，G418筛选阳性克隆。免疫组织化学法检测第八因子相关抗原（FⅧRAg）的表达；细胞计数法绘制细胞生长曲线以观察细胞的增殖能力；Western印迹检测细胞中NOSTRIN和eNOS蛋白质的表达；分光光度法和硝酸还原酶法分别测定细胞培养上清中eNOS的活性和NO代谢产物亚硝酸基／亚硝基（NO2^-／NO3^-）水平；光镜、电镜观察细胞的形态和超微结构。结果转染前后细胞均为血管内皮来源细胞；转染NOSTRIN基因后细胞的增殖能力明显降低；Western印迹显示细胞转染NOSTRIN基因后其表达量明显增高，而eNOS蛋白质的表达量在各组细胞之间无差异（P〉0．05）；转染NOSTRIN的细胞培养上清中eNOS活性[（11．727±3．09）U／ml]与转染空载体细胞[（22．69±3．36）U／ml]和对照组细胞[（21．85±3．47）U／ml]比较降低显著（P〈0．01），同时转染NOSTRIN细胞培养上清中NO2^-／NO3^-[（25．56±2．49）μmol／L]，与转染空载体细胞[（48．37±2．61）μmol／L]和对照组[（49．06±2．78）μmol／L]比较显著降低（P〈0．01）；光镜和电镜观察看到转染NOSTRIN后对HUVEC有损伤作用（HUVEC拉长、微绒毛变短、细胞膜损伤，胞浆内有脂滴空泡，核膜不完整）。结论NOSTRIN的高表达影响了HUVEC的生物学特性，对HUVEC有明显的损伤作用。
Objective To study the overexpression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） in cultured human umbilical vein endothelial cells （HUVEC） and its effect on the bioactivity of these cells. Methods Recombinant pcDNA3. 1-NOSTRIN and empty vector were transfected into cultured human umbilical vein endothelial cells mediated by lipofectamine. HUVEC without transfection served as control and positive clones were selected by G418. Factor Ⅷ related antigen was examined by immunohistochemical analysis. Growth curve of HUVEC were made in both transfected and non-transfected cells. Western blot analysis was applied to detect NOSTRIN and eNOS expression. The eNOS activity was assayed by spectrophotometry and NO2^-/NO3^- and the stable metabolic end products of NO was measured with nitrate reductase. Morphologic changes and ultrastructure were observed in both transfected and non-transfected cells. Results Immunohistochemical analysis confirmed the vascular endothelial origin of the cells both before and after transfection and demonstrated that NOSTRIN was expressed in cytomembrane and cytoplasm in all groups. The proliferation was significantly decreased in transfected NOSRIN cells. Western analysis showed that all cell lines expressed 58kDa NOSTRIN, but the level was much higher in transfected cells with NOSTRIN gene （P〈0.01） and eNOS protein was expressed in all groups, but no differences was found among the three groups （P〉0.05）. The activity of eNOS was significantly decreased in pcDNA3.1-NOSTRIN transfected cells [ （11. 727±3.69） U/roll than those of transfected cells with empty vector [（22.69±3.36） U/ml] and control group [（21.85±3.47） U/ml] （P〈0.01）. The amountof NO2^-/NO3^- on NOSTRIN transfected cells [（25.56±2.49） μmol/L] was significantly lower than those of transfected cells with empty vector [ （48.37 ± 2.61） μmol/L] and control group [ （49.06 ± 2.78） μmol/L]（P〈0. 01）. The HUVEC revealed elongation, deformity of karyotheca, cytomembrane injury, massive lipids in cytoplasm and the shortening of the microvilli after transfected under light and electron microscope. Conclusions The overexpression of NOSTRIN has significant effect on the bioactivity of HUVEC and can damage the HUVEC indicating its important role in the pathogenesis of preeclampsia.
出处
《中华围产医学杂志》
CAS
2007年第1期14-18,73,共6页
Chinese Journal of Perinatal Medicine
