本文選題：Galectin-1 + 鈣離子通道��； 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:由慢性壓力后負(fù)荷增大而引起的病理性心肌肥大是一種較常見的心肌疾病,細(xì)胞內(nèi)鈣離子穩(wěn)態(tài)對(duì)于細(xì)胞正常功能的維持起到重要作用,尤其是在心血管系統(tǒng)中,經(jīng)L型鈣離子通道(L-type calcium channel,LTCC)Cav1.2介導(dǎo)的鈣離子內(nèi)流影響心肌細(xì)胞興奮-收縮耦聯(lián)(excitation-contraction coupling,ECC)、平滑肌收縮等重要生理功能。在此前的研究中我們發(fā)現(xiàn),鈣離子通道α亞基中可變剪接外顯子9*與通道電生理性質(zhì)有關(guān)。半乳糖凝集素1(Galectin-1,Gal-1)作為一種半乳糖結(jié)合蛋白,能夠在血管中通過與不含9*的Cav1.2可變剪接體Cav1.2 CM△9*結(jié)合發(fā)揮調(diào)節(jié)血管收縮的作用。然而,Gal-1在心肌細(xì)胞中與Cav1.2的作用尚不明確。目的:本實(shí)驗(yàn)探究Gal-1與外顯子9*在心肌肥大過程中的表達(dá)變化,闡明Gal-1在心肌肥大過程中的作用。方法:運(yùn)用Western blot和RT-PCR檢測在不同周齡WKY/SHR大鼠和胸主動(dòng)脈縮窄術(shù)后大鼠心臟中Gal-1與外顯子9*的表達(dá)情況;運(yùn)用全細(xì)胞膜片鉗檢測分離的原代心肌細(xì)胞以及表達(dá)不同可變剪接亞型Cav1.2通道的HEK293細(xì)胞鈣電流,分別觀察過表達(dá)Gal-1對(duì)其的影響;使用Fluo-4動(dòng)態(tài)監(jiān)測心肌細(xì)胞內(nèi)鈣離子濃度變化;使用real-time PCR檢測過表達(dá)Gal-1對(duì)異丙腎上腺素(Isoproterenol,ISO)誘導(dǎo)的原代心肌細(xì)胞肥大的影響;使用免疫熒光染色檢測過表達(dá)Gal-1對(duì)ISO誘導(dǎo)的肥大心肌細(xì)胞表面積的影響;使用KN93、H89及ISO等藥物研究Gal-1對(duì)CaMKII-HDAC4通路的影響。結(jié)果:我們發(fā)現(xiàn)1)Gal-1在心肌肥大組織中表達(dá)明顯增高,同時(shí)它與肥大心肌中外顯子9*的表達(dá)升高有相關(guān)性;2)在乳鼠原代心肌細(xì)胞中過表達(dá)Gal-1能夠降低鈣電流,能夠緩解短時(shí)程的由胞外刺激所引發(fā)的細(xì)胞內(nèi)鈣濃度異常升高;此外,3)過表達(dá)Gal-1能夠減少由ISO或Bay K8644引起的CaMKII-HDAC4通路的激活從而抑制肥大基因的表達(dá),減小細(xì)胞表面積,抑制心肌肥大。Gal-1可能能夠作為治療心肌肥大的一個(gè)靶點(diǎn)。結(jié)論:Gal-1能夠通過與不含剪接外顯子9*的Cav1.2 α1C亞基Ⅰ-Ⅱ loop結(jié)合發(fā)揮降低[Ca2+]i水平的功能,繼而影響δCaMKII-HDAC4通路,最終導(dǎo)致肥大相關(guān)基因轉(zhuǎn)錄降低,改善了心肌肥大過程。Gal-1在肥大心臟中表達(dá)上調(diào)可能是病理過程中的一種代償機(jī)制。
[Abstract]:Background: pathological myocardial hypertrophy caused by chronic pressure overload is a common myocardial disease. Intracellular calcium homeostasis plays an important role in the maintenance of normal cell function, especially in the cardiovascular system. Calcium influx mediated by L-type calcium channel (L-type calcium channel) affects the excitation-contractile coupling of cardiomyocytes, smooth muscle contraction and other important physiological functions. In previous studies, we have found that the variable splicing exon 9 * in a subunit of calcium channel is related to the electrophysiological properties of the channel. As a galactose-binding protein, galactosamine 1 Galectin-1 (Gal-1) can regulate vasoconstriction by binding with Cav1.2 alternative splice Cav1.2 CM9 *, which does not contain 9 *. However, the role of Gal-1 and Cav1.2 in cardiomyocytes is unclear. Aim: to investigate the expression of Gal-1 and exon 9 * in the process of myocardial hypertrophy and to elucidate the role of Gal-1 in the process of myocardial hypertrophy. Methods: Western blot and RT-PCR were used to detect the expression of Gal-1 and exon 9 * in the hearts of WKY / SHR rats and rats after thoracic aortic coarctation. The effects of Gal-1 expression on calcium current in isolated primary cardiomyocytes and HEK293 cells expressing different alternative splicing subtypes of Cav1.2 channel were detected by whole-cell patch clamp method, and the changes of intracellular calcium concentration in cardiac myocytes were monitored dynamically by Fluo-4. The effects of overexpression of Gal-1 on primary cardiomyocyte hypertrophy induced by isoprenaline isoproterenolISO and the effect of overexpression of Gal-1 on the surface area of ISO-induced hypertrophic cardiomyocytes were detected by real-time PCR and immunofluorescence staining. The effects of Gal-1 on CaMKII-HDAC4 pathway were studied by using KN93 H89 and ISO. Results: we found that the expression of Gal-1 was significantly increased in hypertrophic myocardium, and it was correlated with the expression of exon 9 * in hypertrophic myocardium.) overexpression of Gal-1 in primary neonatal rat cardiomyocytes decreased calcium current. The overexpression of Gal-1 reduced the activation of CaMKII-HDAC4 pathway induced by ISO or Bay K8644, which inhibited the expression of hypertrophic genes and reduced the surface area of cells. Inhibition of myocardial hypertrophy. Gal-1 may be a target for the treatment of myocardial hypertrophy. Conclusion: Genus Gal-1 can reduce [Ca 2] I level by binding to Cav1.2 偽 1C subunit 鈪，

本文編號(hào)：2000976