本文選題：心鈉素 + 胚胎干細(xì)胞　； 參考：《山東師范大學(xué)》2007年碩士論文

【摘要】： 目的: L型鈣通道是電壓門控離子通道,在動(dòng)作電位的平臺(tái)期參與心肌細(xì)胞外鈣內(nèi)流,并在心肌收縮偶聯(lián)過程中起重要作用。心鈉素是心臟分泌的一種多肽,其可下調(diào)L型鈣電流,但作用機(jī)制還不清楚。本文旨在探索心鈉素對小鼠胚胎干細(xì)胞分化的心肌細(xì)胞L型鈣電流的調(diào)控機(jī)制。 方法:應(yīng)用胚胎干細(xì)胞懸滴培養(yǎng)的方法誘導(dǎo)分化為心肌細(xì)胞,利用膠原酶Ⅱ消化法獲得不同時(shí)期的單個(gè)跳動(dòng)的心肌細(xì)胞;利用全細(xì)胞膜片鉗技術(shù),記錄心鈉素刺激下心肌細(xì)胞的L型鈣電流及相關(guān)信號(hào)通道的抑制劑或激活劑對L型鈣電流的調(diào)控。免疫熒光法測細(xì)胞中特異蛋白ANP和肌球蛋白MHC表達(dá);檢測不同時(shí)期小鼠胚胎心臟中ANP表達(dá)水平。 結(jié)果:利用懸滴法培養(yǎng)小鼠胚胎干細(xì)胞并分化為跳動(dòng)的EB,酶消化得到了單個(gè)跳動(dòng)的心肌細(xì)胞,免疫熒光檢測到心肌特異的а-MHC表達(dá),并且在早期ESMC中檢測到心肌細(xì)胞動(dòng)作電位和鈣電流。 ANP在EDS和LDS心肌細(xì)胞中對動(dòng)作電位有顯著負(fù)變時(shí)效應(yīng),并且能夠降低EDS和LDS心肌細(xì)胞的基礎(chǔ)的和Iso激活的L型鈣電流。ANP降低ILcal途徑,獨(dú)立于NO途徑、PTX敏感的G蛋白(Go,Gi)和可溶性GC。ANP對ILcal的抑制是通過其受體激活了顆粒性GC、cGMP依賴的PDEⅡ途徑,PDEⅡ水解cAMP導(dǎo)致其濃度降低,減少了其調(diào)控的鈣通道開放頻率。 RT-PCR結(jié)果顯示在胚胎發(fā)育過程中ANP的表達(dá)持續(xù)升高,并且在8天胚胎中已經(jīng)表達(dá),第12天ANP強(qiáng)烈表達(dá),隨后ANP表達(dá)基本沒變化,但是出生后分泌增多,成體后表達(dá)量降低。免疫熒光檢測ANP的定位,在胚胎期在細(xì)胞質(zhì)中分散表達(dá),早期細(xì)胞ANP的表達(dá)出現(xiàn)極性。
[Abstract]:Aim: L-type calcium channel is a voltage-gated ion channel which participates in extracellular calcium influx during the plateau phase of action potential and plays an important role in the process of cardiac contractile coupling. Atrial natriuretic peptide (ANP) is a polypeptide secreted by the heart, which can down-regulate L-type calcium current, but the mechanism is unclear. The aim of this study was to explore the regulation mechanism of atrial natriuretic peptide (ANP) on L-type calcium current in cardiomyocytes differentiated from mouse embryonic stem cells. Methods: embryonic stem cells were induced to differentiate into cardiomyocytes by suspension culture of embryonic stem cells, single beating cardiomyocytes were obtained by collagenase 鈪，

本文編號(hào)：1908990