發(fā)布時間：2018-06-09 22:03

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

【摘要】：背景:由慢性壓力后負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,細胞內(nèi)鈣離子穩(wěn)態(tài)對于細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介導的鈣離子內(nèi)流影響心肌細胞興奮-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。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在心肌細胞中與Cav1.2的作用尚不明確。目的:本實驗探究Gal-1與外顯子9*在心肌肥大過程中的表達變化,闡明Gal-1在心肌肥大過程中的作用。方法:運用Western blot和RT-PCR檢測在不同周齡WKY/SHR大鼠和胸主動脈縮窄術(shù)后大鼠心臟中Gal-1與外顯子9*的表達情況;運用全細胞膜片鉗檢測分離的原代心肌細胞以及表達不同可變剪接亞型Cav1.2通道的HEK293細胞鈣電流,分別觀察過表達Gal-1對其的影響;使用Fluo-4動態(tài)監(jiān)測心肌細胞內(nèi)鈣離子濃度變化;使用real-time PCR檢測過表達Gal-1對異丙腎上腺素(Isoproterenol,ISO)誘導的原代心肌細胞肥大的影響;使用免疫熒光染色檢測過表達Gal-1對ISO誘導的肥大心肌細胞表面積的影響;使用KN93、H89及ISO等藥物研究Gal-1對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明顯增高,同時它與肥大心肌中外顯子9*的表達升高有相關性;2)在乳鼠原代心肌細胞中過表達Gal-1能夠降低鈣電流,能夠緩解短時程的由胞外刺激所引發(fā)的細胞內(nèi)鈣濃度異常升高;此外,3)過表達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,減小細胞表面積,抑制心肌肥大。Gal-1可能能夠作為治療心肌肥大的一個靶點。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,最終導致肥大相關基因轉(zhuǎn)錄降低,改善了心肌肥大過程。Gal-1在肥大心臟中表達上調(diào)可能是病理過程中的一種代償機制。
[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：2000976