【摘要】：阿霉素(doxorubicin,DOX)是一種廣泛應(yīng)用的非常有效的抗腫瘤藥物,其心肌毒性是腫瘤治療中主要關(guān)注的問題。心肌細(xì)胞自噬在維持心肌功能中發(fā)揮重要作用,但自噬是把“雙刃劍”,關(guān)于自噬在DOX誘導(dǎo)心肌損傷中的作用以及一些心臟保護(hù)劑到底是通過促進(jìn)自噬還是抑制自噬發(fā)揮心肌保護(hù)作用還存在爭議。近幾年,促血小板生成素(thrombopoietin,TPO)被發(fā)現(xiàn)具有心肌保護(hù)作用,有望成為新的心臟保護(hù)藥物,但TPO是否通過調(diào)控自噬來保護(hù)心肌細(xì)胞尚未見報(bào)道。最新研究發(fā)現(xiàn)與TPO同源的促紅細(xì)胞生成素(erythropoietin,EPO)能夠調(diào)控上皮細(xì)胞的自噬,我們提出TPO也可能調(diào)控心肌細(xì)胞自噬。因此,本研究旨在(1)研究DOX和(或)TPO對(duì)心肌自噬的影響;(2)研究TPO調(diào)控心肌自噬的分子機(jī)制;(3)研究TPO作用下心肌自噬與凋亡的關(guān)系。我們將心肌細(xì)胞系H9C2分為Control、DOX、DOX+BFA、DOX+TPO四個(gè)組。以自噬抑制劑巴佛洛霉素A1(bafilomycin A,BFA)作為實(shí)驗(yàn)的陽性對(duì)照。各組細(xì)胞主要作如下檢測(cè):(1)CCK-8染色法檢測(cè)細(xì)胞存活率;(2)Western-blot和實(shí)時(shí)定量PCR檢測(cè)自噬和凋亡相關(guān)分子的表達(dá);(3)自噬小體檢測(cè);(4)共聚焦顯微鏡檢測(cè)LC3與LAMP1共定位。研究結(jié)果顯示,DOX處理使H9C2細(xì)胞的存活率明顯下降(p0.05),而加入TPO組相較于單加DOX組,細(xì)胞存活率升高(p0.05)。加入DOX后,H9C2細(xì)胞自噬小體數(shù)量明顯增多,而同時(shí)孵育TPO則使自噬小體數(shù)量減少(p0.05)。進(jìn)一步檢測(cè)自噬相關(guān)基因的表達(dá),我們發(fā)現(xiàn)與單DOX處理組相比,TPO使Beclin-1和LC3-Ⅱ的表達(dá)降低,而使p62表達(dá)升高,提示TPO能夠抑制DOX誘導(dǎo)的心肌細(xì)胞自噬。為了探討TPO對(duì)心肌自噬調(diào)控的機(jī)制,我們檢測(cè)了 GATA-4、ATG14L、Rubicon的表達(dá)以及LC3和LAMP1共定位。結(jié)果發(fā)現(xiàn),TPO可以增加H9C2細(xì)胞中GATA-4的表達(dá)。DOX使ATG14L、Rubicon的表達(dá)水平下降。與對(duì)照組相比,DOX可誘導(dǎo)LAMP1的蛋白水平增加,但加入TPO對(duì)LAMP1的蛋白水平無明顯影響(p0.001)。此外,TPO對(duì)LC3和LAMP1的共定位無影響。最后,我們研究TPO作用下心肌細(xì)胞自噬與凋亡的關(guān)系。與DOX組相比,TPO使H9C2細(xì)胞中caspase-3的mRNA和蛋白水平都有所減少。TPO還可上調(diào)Bcl-2蛋白水平,對(duì)Bcl-2 mRNA水平則沒有明顯的影響(p0.05)。綜上所述,TPO能夠減少DOX誘導(dǎo)的心肌細(xì)胞自噬小體的數(shù)量,抑制自噬相關(guān)分子的表達(dá),提示TPO能夠抑制DOX誘導(dǎo)的心肌細(xì)胞自噬;對(duì)TPO抑制自噬的分子機(jī)制進(jìn)行研究,發(fā)現(xiàn)TPO能夠促進(jìn)GATA-4的表達(dá),提示TPO有可能通過促進(jìn)GATA-4的表達(dá)抑制心肌細(xì)胞自噬;TPO和BFA都能夠使凋亡減少,提示DOX處理的心肌細(xì)胞中,抑制自噬能夠減少凋亡的發(fā)生。
[Abstract]:Doxorubicin (doxorubicin,DOX) is a widely used anti-tumor drug, and its myocardial toxicity is a major concern in the treatment of cancer. Autophagy plays an important role in maintaining myocardial function, but autophagy is a "double-edged sword". The role of autophagy in myocardial injury induced by DOX and whether some cardioprotective agents can promote autophagy or inhibit autophagy are still controversial. In recent years, thrombopoietin (thrombopoietin,TPO) has been found to have cardioprotective effects and is expected to become a new cardioprotective drug. However, it has not been reported whether TPO can protect cardiomyocytes by regulating autophagy. Recent studies have found that erythropoietin (erythropoietin,EPO) homologous with TPO can regulate autophagy in epithelial cells. We suggest that TPO may also regulate autophagy in cardiomyocytes. Therefore, the aim of this study was to (1) study the effects of DOX and / or TPO on myocardial autophagy, (2) study the molecular mechanism of TPO regulating myocardial autophagy, and (3) study the relationship between myocardial autophagy and apoptosis induced by TPO. We divided the myocardial cell line H9C2 into four groups of Control,DOX,DOX BFA,DOX TPO. The autophagy inhibitor bafilomycin A 1 (BFA) was used as the positive control. The main results were as follows: (1) CCK-8 staining was used to detect cell viability; (2) Western-blot and real-time quantitative PCR were used to detect the expression of autophagy and apoptosis-related molecules; (3) autophagy bodies were detected; (4) the co-localization of LC3 and LAMP1 was detected by confocal microscope. The results showed that the survival rate of H9C2 cells treated with DOX was significantly decreased (p0.05), while the survival rate of H9C2 cells treated with TPO was significantly higher than that of DOX alone (p0.05). After adding DOX, the number of autophagy bodies in H9C2 cells increased significantly, while the number of autophagy bodies decreased when TPO was incubated at the same time (p0.05). By further detecting the expression of autophagy-related genes, we found that TPO decreased the expression of Beclin-1 and LC3- 鈪，

本文編號(hào)：2445358