脐动脉血管平滑肌中BKca通道与子痫前期的相关性研究
发布时间：2018-08-27 18:51
【摘要】：目的:子痫前期(Preeclampsia,PE)是妊娠期特有的多系统疾病,严重危及孕产妇和围产儿的生命健康。全身小血管痉挛为该病基本病理变化[1],其中BKca通道(Large conductance calcium activated potassium channels,BKca)是调节血管舒缩功能中最关键离子通道。本实验采用免疫组化、实时荧光定量PCR(QPCR)和Western blotting技术检测脐动脉血管平滑肌中BKca通道阳性率、m RNA水平和蛋白表达水平,了解脐动脉血管平滑肌中BKca通道的表达和功能的改变,探讨脐动脉血管平滑肌中BKca通道与子痫前期的关系。再此基础上,进一步探讨脐动脉血管平滑肌中BKca通道的改变与胎儿生长受限(FGR)的关系。为探索子痫前期的发病机制,并从中寻找有效防治子痫前期及重度子痫前期合并胎儿生长受限的靶点提供实验数据和理论依据。方法:(1)HE和疫组化:收集经剖宫产或经阴道分娩的脐动脉正常妊娠组(NP组)和子痫前期组(PE组)各30例作为研究对象,快速分离脐动脉,部分脐动脉用10%甲醛固定(其余脐动脉用不同的冻存管分装,用于实时荧光定量PCR和免疫印迹实验),石蜡包埋,然后分别按照HE染色和免疫组化步骤逐一进行,照相并比较BKca通道在NP组和PE组脐动脉血管平滑肌中表达有何不同。(2)实时荧光定量PCR(QPCR):提取脐动脉血管平滑肌总RNA,测出RNA浓度,将适量RNA逆转录成c DNA,通过QPCR反应得到相应的△Ct值,并换算成ΔΔCt值,ΔΔCt值=病例组ΔCt-正常组ΔCt,计算出2-△△Ct值进行t检验分析,比较NP组和PE组所检测基因BKcaαm RNA、BKcaβ_1 m RNA水平的差异。(3)Western blotting:液氮研磨脐动脉组织至粉末状,加1ml蛋白裂解液提取样本蛋白;样本蛋白电泳,转至PVDF膜上,抗体孵育后,成像系统显像保存,用Quantity-One软件测量灰度值分析,应用SPSS17.0软进行t检验分析,比较NP组和PE组脐动脉血管平滑肌中BKcaα亚基、BKcaβ_1亚基蛋白相对表达量的差别。再此基础上进一步比较正常妊娠组、子痫前期组和慢性高血压合并子痫前期组脐动脉血管平滑肌中BKcaα亚基、BKcaβ_1亚基蛋白相对表达量的差异;比较重度子痫前期合并胎儿生长受限组及无胎儿生长受限组脐动脉血管平滑肌中BKcaα亚基、BKcaβ_1亚基蛋白相对表达量的差异。结果以均数加减标准差(xˉ±s)表示,P0.05,P0.01均具有统计学意义。结果:(1)(1)HE染色:NP组和PE组各30例均为符合实验要求的脐动脉,HE染色后镜下观察,可见脐动脉血管平滑肌细胞呈长梭形,肌层稍厚,胞浆红染,胞核位于中央,单核,蓝染。脐动脉在子痫前期疾病状态下可能会出现血管平滑肌排列紊乱,但未见形态结构改变和动脉粥样硬化。(2)免疫组化:采用定性、半定量法分析。BKcaα亚基和β_1亚基在NP组与PE组脐动脉血管平滑肌中均有表达。BKcaα亚基和β_1亚基主要表达于脐动脉血管平滑肌胞膜,部分表达于胞浆中。BKcaα亚基的阳性率在NP组和PE组脐动脉血管平滑肌中无明显差异;PE组脐动脉血管平滑肌中BKcaβ_1亚基表达强度较NP组明显增强,有统计学意义(P0.05)。(2)QPCR实验结果:数据采用相对定量分析,即2-△△Ct法。BKcaα亚基m RNA水平在两组中无统计学差异(P=0.8080.05)。与NP组比较,PE组脐动脉血管平滑肌中BKcaβ_1亚基m RNA水平明显上调,差异有统计学意义(P=0.0150.05)。(3)Western blotting实验结果:(1)BKcaα亚基蛋白表达水平在NP组和PE组脐动脉血管平滑肌中无统计学差异(P=1.0000.05);与NP组比较,PE组脐动脉血管平滑肌中BKcaβ_1亚基蛋白表达水平上调,差异有统计学意义(P0.01)。(2)正常妊娠组、子痫前期组和慢性高血压合并子痫前期组:与正常妊娠组比较,BKcaα亚基蛋白表达水平在三组中均无统计学差异(P0.05)。与正常妊娠组比较,BKcaβ_1亚基蛋白表达水平在子痫前期组脐动脉血管平滑肌中表达明显上调;在慢性高血压合并子痫前期组脐动脉血管平滑肌中表达明显下调;与子痫前期组比较,BKcaβ_1亚基蛋白表达水平在慢性高血压合并子痫前期组脐动脉血管平滑肌中表达水平下调更明显,差异均有统计学意义(P10.05,P20.05,P30.01)。(3)比较重度子痫前期合并胎儿生长受限组和无胎儿生长受限组:BKcaα亚基蛋白相对表达量在两组中表达无明显差异(P0.05);BKcaβ_1亚基蛋白相对表达量在重度子痫前期合并胎儿生长受限脐动脉血管平滑肌中表达水平有所下调,差异具有统计学意义(*P0.05)。结论:(1)脐动脉在子痫前期疾病状态下,会出现血管平滑肌细胞排列紊乱,但未见形态结构改变和动脉粥样硬化。(2)PE组与NP组比较,脐动脉血管平滑肌中BKcaα亚基的表达无明显差异(P0.05),BKcaβ_1亚基表达水平明显升高(P0.05)。提示BKcaβ_1亚基的改变可能与PE的发生发展相关,BKcaα亚基可能没参与PE的发生发展。(3)PE的发生发展引起了BKcaβ_1亚基的改变,β_1亚基的改变可能参与、促进了PE的发生发展。(4)BKcaβ_1亚基蛋白相对表达量在重度子痫前期合并胎儿生长受限脐动脉血管平滑肌中表达水平有所下调,提示胎儿生长受限可能与BKcaβ_1亚基的改变有关。
[Abstract]:Objective: Preeclampsia (PE) is a multisystem disorder peculiar to pregnancy, which seriously endangers the life and health of pregnant women and perinatal infants. Systemic vasospasm is the basic pathological change of PE [1]. BKca channel (BKca) is the most important ion channel in regulating vasodilator function. In this study, immunohistochemistry, real-time fluorescence quantitative PCR (QPCR) and Western blotting were used to detect the positive rate of BKca channel, the level of M RNA and protein expression in the smooth muscle of umbilical artery. The expression and function of BKca channel in the smooth muscle of umbilical artery were investigated. Furthermore, the relationship between BKca channel changes in umbilical artery smooth muscle and fetal growth restriction (FGR) was further explored, which provided experimental data and theoretical basis for exploring the pathogenesis of preeclampsia and finding effective targets for preventing and treating preeclampsia and severe preeclampsia with fetal growth restriction. And epidemic histochemistry: 30 cases of normal pregnant umbilical artery group (NP group) and 30 cases of preeclampsia group (PE group) were collected. The umbilical artery was quickly separated and fixed with 10% formaldehyde (the remaining umbilical artery was packed with different cryopreserved tubes for real-time fluorescence quantitative PCR and immunoblotting), paraffin embedded. The expression of BKca channel in smooth muscle of umbilical artery in NP group and PE group was compared by HE staining and immunohistochemical staining. (2) Real-time fluorescence quantitative PCR (QPCR): Total RNA of smooth muscle of umbilical artery was extracted, RNA concentration was measured, appropriate RNA was reverse transcribed into C DNA, and corresponding Delta Ct was obtained by QPCR reaction. 鍊，
本文编号：2208128
[Abstract]:Objective: Preeclampsia (PE) is a multisystem disorder peculiar to pregnancy, which seriously endangers the life and health of pregnant women and perinatal infants. Systemic vasospasm is the basic pathological change of PE [1]. BKca channel (BKca) is the most important ion channel in regulating vasodilator function. In this study, immunohistochemistry, real-time fluorescence quantitative PCR (QPCR) and Western blotting were used to detect the positive rate of BKca channel, the level of M RNA and protein expression in the smooth muscle of umbilical artery. The expression and function of BKca channel in the smooth muscle of umbilical artery were investigated. Furthermore, the relationship between BKca channel changes in umbilical artery smooth muscle and fetal growth restriction (FGR) was further explored, which provided experimental data and theoretical basis for exploring the pathogenesis of preeclampsia and finding effective targets for preventing and treating preeclampsia and severe preeclampsia with fetal growth restriction. And epidemic histochemistry: 30 cases of normal pregnant umbilical artery group (NP group) and 30 cases of preeclampsia group (PE group) were collected. The umbilical artery was quickly separated and fixed with 10% formaldehyde (the remaining umbilical artery was packed with different cryopreserved tubes for real-time fluorescence quantitative PCR and immunoblotting), paraffin embedded. The expression of BKca channel in smooth muscle of umbilical artery in NP group and PE group was compared by HE staining and immunohistochemical staining. (2) Real-time fluorescence quantitative PCR (QPCR): Total RNA of smooth muscle of umbilical artery was extracted, RNA concentration was measured, appropriate RNA was reverse transcribed into C DNA, and corresponding Delta Ct was obtained by QPCR reaction. 鍊，
本文编号：2208128
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