血管紧张素Ⅱ诱导人胎肾系膜细胞凋亡及机制的初步研究

ANGIOTENSIN INDUCES APOPTOSIS OF MESANGIAL CELLS OF HUMAN FETAL KIDNEYS AND PRELIMINARY STUDIES ON ITS MECHANISM

观察了血管紧张素Ⅱ（ＡｎｇⅡ）的１型（ＡＴ１）和２型（ＡＴ２）受体及白细胞介素－１β转化酶（ＩＣＥ）在人胎肾系膜细胞表达情况及其与肾小球系膜细胞凋亡的关系。采用６个月龄人胎肾培养肾小球系膜细胞，经１０－８、１０－７、１０－６和１０－５ｍｏｌ／Ｌ浓度的ＡｎｇⅡ诱导后，用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术检测了胎肾系膜细胞的ＡＴ１、ＡＴ２受体和ＩＣＥ基因的ｍＲＮＡ表达情况。经ＡｎｇⅡ刺激后ＡＴ１受体ｍＲＮＡ表达有明显增强，并呈浓度依赖性，而ＡＴ２受体和ＩＣＥ基因的ｍＲＮＡ表达仅在１０－５ｍｏｌ／Ｌ的ＡｎｇⅡ刺激后才检测到。用１０－５ｍｏｌ／Ｌ浓度的ＡｎｇⅡ诱导系膜细胞凋亡，分别于２４、４８和７２ｈ收集细胞进行检测，应用碘化丙啶染色（ＰＩ）、原位末端标记染色（ＴＵＮＥＬ）、染色质ＤＮＡ电泳和流式细胞仪等定性和定量方法观察系膜细胞的凋亡改变。经１０－５ｍｏｌ／Ｌ浓度的ＡｎｇⅡ刺激７２ｈ后，肾小球系膜细胞出现了典型的凋亡征象，包括细胞核固缩和碎裂，染色质ＤＮＡ片段化，流式细胞仪检测到标志着细胞凋亡的Ａｏ峰；应用ＡＴ１受体抗体封闭后对这种凋亡过程无显著影响。结果表明，ＡｎｇⅡ诱导的系膜细胞凋亡可能是通过其ＡＴ２受?

The relationship between the expressions of Angiotensin ( Ang )type 1 (AT1) and type 2 (AT2) receptor genes and interleukin1 coverting enzyme (ICE) gene and apoptosis were explored in glomerular mesangial cells (GMCs) of human fetal kidneys. GMCs, cultured from the human fetal kidneys at the age of 6 months, were treated with Ang at the concentrations of 10-8,10-7,10-6 and l0-5mol/L for 24, 48 and 72 h, respectively. The mRNA expression of AT1 and AT2 receptors and ICE were examined with RTPCR. The apoptosis of GMCs was identified by means of both qualitative and quantitative methods including propidium iodide (PI) and TUNEL staining, detection of a typical ladder shape of genomic DNA with electrophoresis, and flow cytometry analysis. Results showed that Ang could upregulate the AT1 receptor mRNA expression in a dosedependent manner and induce the AT2 receptor and ICE mRNA expressions only at 10-5mol/L.Ang triggered GMCs apoptotic changes including small and fragmented nuclei, genomic DNA fragmentation and hypodiploid DNA peaks in flow cytometry analysis. The blocker of AT1 receptor made little effect on Ang induced apoptosis. It is concluded that Ang could induce apoptosis of human mesangial cells probably via its AT2 receptor, and ICE gene might be involved in the apoptotic process.