【摘要】：目的:子癇前期(Preeclampsia,PE)是妊娠期特有的多系統(tǒng)疾病,嚴重危及孕產(chǎn)婦和圍產(chǎn)兒的生命健康。全身小血管痙攣為該病基本病理變化[1],其中BKca通道(Large conductance calcium activated potassium channels,BKca)是調(diào)節(jié)血管舒縮功能中最關鍵離子通道。本實驗采用免疫組化、實時熒光定量PCR(QPCR)和Western blotting技術檢測臍動脈血管平滑肌中BKca通道陽性率、m RNA水平和蛋白表達水平,了解臍動脈血管平滑肌中BKca通道的表達和功能的改變,探討臍動脈血管平滑肌中BKca通道與子癇前期的關系。再此基礎上,進一步探討臍動脈血管平滑肌中BKca通道的改變與胎兒生長受限(FGR)的關系。為探索子癇前期的發(fā)病機制,并從中尋找有效防治子癇前期及重度子癇前期合并胎兒生長受限的靶點提供實驗數(shù)據(jù)和理論依據(jù)。方法:(1)HE和疫組化:收集經(jīng)剖宮產(chǎn)或經(jīng)陰道分娩的臍動脈正常妊娠組(NP組)和子癇前期組(PE組)各30例作為研究對象,快速分離臍動脈,部分臍動脈用10%甲醛固定(其余臍動脈用不同的凍存管分裝,用于實時熒光定量PCR和免疫印跡實驗),石蠟包埋,然后分別按照HE染色和免疫組化步驟逐一進行,照相并比較BKca通道在NP組和PE組臍動脈血管平滑肌中表達有何不同。(2)實時熒光定量PCR(QPCR):提取臍動脈血管平滑肌總RNA,測出RNA濃度,將適量RNA逆轉錄成c DNA,通過QPCR反應得到相應的△Ct值,并換算成ΔΔCt值,ΔΔCt值=病例組ΔCt-正常組ΔCt,計算出2-△△Ct值進行t檢驗分析,比較NP組和PE組所檢測基因BKcaαm RNA、BKcaβ_1 m RNA水平的差異。(3)Western blotting:液氮研磨臍動脈組織至粉末狀,加1ml蛋白裂解液提取樣本蛋白;樣本蛋白電泳,轉至PVDF膜上,抗體孵育后,成像系統(tǒng)顯像保存,用Quantity-One軟件測量灰度值分析,應用SPSS17.0軟進行t檢驗分析,比較NP組和PE組臍動脈血管平滑肌中BKcaα亞基、BKcaβ_1亞基蛋白相對表達量的差別。再此基礎上進一步比較正常妊娠組、子癇前期組和慢性高血壓合并子癇前期組臍動脈血管平滑肌中BKcaα亞基、BKcaβ_1亞基蛋白相對表達量的差異;比較重度子癇前期合并胎兒生長受限組及無胎兒生長受限組臍動脈血管平滑肌中BKcaα亞基、BKcaβ_1亞基蛋白相對表達量的差異。結果以均數(shù)加減標準差(xˉ±s)表示,P0.05,P0.01均具有統(tǒng)計學意義。結果:(1)(1)HE染色:NP組和PE組各30例均為符合實驗要求的臍動脈,HE染色后鏡下觀察,可見臍動脈血管平滑肌細胞呈長梭形,肌層稍厚,胞漿紅染,胞核位于中央,單核,藍染。臍動脈在子癇前期疾病狀態(tài)下可能會出現(xiàn)血管平滑肌排列紊亂,但未見形態(tài)結構改變和動脈粥樣硬化。(2)免疫組化:采用定性、半定量法分析。BKcaα亞基和β_1亞基在NP組與PE組臍動脈血管平滑肌中均有表達。BKcaα亞基和β_1亞基主要表達于臍動脈血管平滑肌胞膜,部分表達于胞漿中。BKcaα亞基的陽性率在NP組和PE組臍動脈血管平滑肌中無明顯差異;PE組臍動脈血管平滑肌中BKcaβ_1亞基表達強度較NP組明顯增強,有統(tǒng)計學意義(P0.05)。(2)QPCR實驗結果:數(shù)據(jù)采用相對定量分析,即2-△△Ct法。BKcaα亞基m RNA水平在兩組中無統(tǒng)計學差異(P=0.8080.05)。與NP組比較,PE組臍動脈血管平滑肌中BKcaβ_1亞基m RNA水平明顯上調(diào),差異有統(tǒng)計學意義(P=0.0150.05)。(3)Western blotting實驗結果:(1)BKcaα亞基蛋白表達水平在NP組和PE組臍動脈血管平滑肌中無統(tǒng)計學差異(P=1.0000.05);與NP組比較,PE組臍動脈血管平滑肌中BKcaβ_1亞基蛋白表達水平上調(diào),差異有統(tǒng)計學意義(P0.01)。(2)正常妊娠組、子癇前期組和慢性高血壓合并子癇前期組:與正常妊娠組比較,BKcaα亞基蛋白表達水平在三組中均無統(tǒng)計學差異(P0.05)。與正常妊娠組比較,BKcaβ_1亞基蛋白表達水平在子癇前期組臍動脈血管平滑肌中表達明顯上調(diào);在慢性高血壓合并子癇前期組臍動脈血管平滑肌中表達明顯下調(diào);與子癇前期組比較,BKcaβ_1亞基蛋白表達水平在慢性高血壓合并子癇前期組臍動脈血管平滑肌中表達水平下調(diào)更明顯,差異均有統(tǒng)計學意義(P10.05,P20.05,P30.01)。(3)比較重度子癇前期合并胎兒生長受限組和無胎兒生長受限組:BKcaα亞基蛋白相對表達量在兩組中表達無明顯差異(P0.05);BKcaβ_1亞基蛋白相對表達量在重度子癇前期合并胎兒生長受限臍動脈血管平滑肌中表達水平有所下調(diào),差異具有統(tǒng)計學意義(*P0.05)。結論:(1)臍動脈在子癇前期疾病狀態(tài)下,會出現(xiàn)血管平滑肌細胞排列紊亂,但未見形態(tài)結構改變和動脈粥樣硬化。(2)PE組與NP組比較,臍動脈血管平滑肌中BKcaα亞基的表達無明顯差異(P0.05),BKcaβ_1亞基表達水平明顯升高(P0.05)。提示BKcaβ_1亞基的改變可能與PE的發(fā)生發(fā)展相關,BKcaα亞基可能沒參與PE的發(fā)生發(fā)展。(3)PE的發(fā)生發(fā)展引起了BKcaβ_1亞基的改變,β_1亞基的改變可能參與、促進了PE的發(fā)生發(fā)展。(4)BKcaβ_1亞基蛋白相對表達量在重度子癇前期合并胎兒生長受限臍動脈血管平滑肌中表達水平有所下調(diào),提示胎兒生長受限可能與BKcaβ_1亞基的改變有關。
[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，

本文編號：2208128