一氧化氮合成酶在人肾小球系膜细胞中表达的研究

The study on the expression of nitric oxide synthase in human glomerular mesangial cells.

目的探讨肾小球系膜细胞 (MC)诱生型一氧化氮合成酶 (iNOS)的表达。方法采用 4个月龄水囊引产的胎儿 ,取胎肾 ,用不锈钢网筛分离技术 ,培养原代系膜细胞 ,用胰酶传代 ,取Ⅲ代细胞用于实验。设实验组和对照组 ,观察脂多糖 (LPS)刺激后不同时间 (4、6、2 4、4 8小时 )及一氧化氮合成酶抑制剂 (L NAME)刺激后同一时间 (2 4小时 )对诱生型一氧化氮合成酶表达的影响。结果细胞为梭形、星形或树枝状 ,核居中央 ,呈圆形或椭圆形 ;免疫荧光结果为抗结蛋白抗体、抗Ⅳ型胶原、抗纤维连接蛋白 (FN)、抗层粘连蛋白 (LN)阳性 ;抗角蛋白、抗Ⅷ因子抗体阴性 ;在未刺激的MC中无iNOSmRNA的表达 ;LPS刺激后 4小时 ,即可检测到iNOS ;6小时后开始明显增加 ;2 4小时达到高峰 ;至 4 8小时iNOS仍较高 ;L NAME为iNOS特异性的抑制剂。结论肾小球MC确含有iNOS ,且在生理状态下呈稳定状态 ,病理状态下 (如LPS刺激后 )表达上调。

Objective To explore the expression of induced nitric oxide(iNOS) in human glomerular mesangial cells(MC). Methods Four months old fetus of induced labor by water cyst was adopted for fetal kidney.And then the stainless steel sieve technique was used to culture primary of mesangial cells which was passed with trypsin. And then III generation cells were taken for experiments. The cells were divided into experiment group and control group in order to observe the effect of LPS stimulation at different time points(4?6?24?48 h) and nitric oxide synthase inhibitor(L NAME) at the same time(24h) on iNOS. Results Human mesangial cells were spindle, astroid, and dendritic shaped, with a round or ellipsoidal nucleus in the center. Immunofluorescence results were positive for antidesmin, anti type IV collagen, anti fibronectin(FN), and anti laminin(LN), negative for anti keratin and anti VIII factor antibody. iNOSmRNA did not express in MC which was not stimulated, while iNOS could be detected 4 h after LPS stimulation, which was significantly increased 6h later, peaked 24h later and remained high 48h later. L NAME was a specific inhibitor of iNOS. Conclusion There is iNOS in MC which keeps stable under physiological state while will be upregulated under pathological state.