妊娠期高血压病患者胎儿脐静脉内皮细胞模型的建立及血管紧张素Ⅱ表达的检测

目的：探索建立妊娠期高血压病患者胎儿脐静脉内皮细胞模型的优化方法并对该细胞模型中血管紧张素Ⅱ表达量进行分析。方法：取妊高症孕妇脐带（约8cm）,用PBS冲洗和灌注胰蛋白酶（浓度为1%）消化法分离脐静脉内皮细胞,用McCoy＇s 5A培养基进行原代和继代培养;用吖啶橙（AO）细胞荧光染色进行细胞的形态学鉴定;用免疫组织化学分析所分离细胞的来源;用Western blotting检测血管紧张素Ⅱ（AngⅡ）的表达情况。结果：利用1%的胰蛋白酶灌注消化可以获得大量的原代细胞,并且在不添加任何生长因子的情况下,使用McCoy＇s 5A培养基进行原代和继代培养可以实现连续传代6代以上,且细胞状态良好,生长稳定。AO染色鉴定显示细胞边缘平整,细胞核小且形态完整,符合正常细胞的形态。免疫组化鉴定结果表明在所分离的细胞中人VⅡI因子及血管内皮生长因子（VEGF）相关抗原呈强阳性。Western blotting检测发现在所分离的细胞中AngⅡ表达量远远高于普通细胞（90.20±5.84）%,P=0.0130]。结论：1%浓度的胰蛋白酶灌注消化法与McCoy＇s 5A培养基培养法是体外建立妊娠期高血压病患者胎儿脐静脉内皮细胞模型的高效经济的方法,该模型高表达血管紧张素Ⅱ,推测AngⅡ可能是诱发妊高症的原因之一。

Study on Establishment of Human Umbilical Vein Endothelial Cells of Hypertensive Disorder Complicating Pregnancy and Angiotensin II Expression

Objective： To study on establishment of human umbilical vein endothelial cells model of hypertensive disorder corn plicating pregnancy（ HDCP）, as well as to assay the angiotensin Ⅱ expression. Methods： Human umbilical vein endothelial cells were isolated by filling umbilical weins with digestive 1% Trypsin after PBS washed, then the cells were cultured in McCoy＇s 5Ac cell media; The morphology and source of isolated cells were assayed by acridine orange staining and immunohistochemistry technique; the differential expressions of Ang II in hUVECs between HDCP and WT were examined by western blotting. Results： Large numbers of primary cells can be get with 1% Trypsin digestive, which can be cultured into 6th passage in McCoy＇s SAc cell media without any growht factors. The AO staining indicated that the ceils had slippery verges and inerratic nucleolus. The results of immunohistoehemistry indicated that hVIII and hVEGF were expressed highly in the isolated ceils. The expression level of Ang II in HDCP hUVECs （90.20 ±5.84）%, P = 0. 0130）was much high than WT＇s. Conclusion： Perfusion with 1% Trypsin and cultured with McCoy＇s 5Ac cell media is an effective method for estalishing human umbilical vein endothelial cells of hypertensive disorder complicating pregnancy, and in which the expression level of Ang Ⅱ is high.