本文選題：紫紺型 + 心病　； 參考：《第三軍醫(yī)大學》2014年碩士論文

【摘要】：背景： 紫紺型先天性心臟病威脅人類生命與健康，了解其病理生理機制，有助于促進臨床診療與預后。紫紺型先天性心臟病患者經(jīng)歷著慢性缺氧的病理生理過程，而心肌如何在慢性缺氧應激條件下，完成凋亡、自噬、分泌、代謝、重構等結構功能方面的適應性變化，其具體機制尚未完全闡明。 細胞信號抑制子-3(Suppressor of cell signaling-3，SOCS3)作為Janus激酶-信號轉導與轉錄激活子-3(Janus kinase-Signal transducer and activator of transcription-3，JAK-STAT3)信號通路下游分子，負反饋調控該通路活性，維持細胞信號穩(wěn)態(tài)。我們前期研究發(fā)現(xiàn)，相對于非紫紺型先心病患者，紫紺型先心病患者心肌IL-6-JAK-STAT3激活，SOCS3轉錄和翻譯水平上調，且SOCS3蛋白水平的升高依賴于轉錄水平升高；而缺氧心肌細胞中過表達SOCS3使IL-6-JAK-STAT3信號通路活性抑制，細胞凋亡增加。提示適度表達的SOCS3，對于細胞內IL-6-JAK-STAT3信號穩(wěn)態(tài)至關重要，在心肌慢性缺氧適應過程起到重要調控作用，但并不清楚缺氧心肌中何種因素參與了SOCS3的轉錄調控。之前腫瘤細胞中的研究顯示，SOCS3基因啟動子甲基化參與其轉錄調控，影響蛋白表達，從而調控JAK-STAT3通路活性，維持腫瘤細胞增殖與抗凋亡。因此，我們推測在慢性缺氧心肌中，SOCS3啟動子低甲基化可能參與了其轉錄表達調控，調控JAK-STAT3信號通路活性，參與心肌慢性缺氧適應信號通路平衡。本研究從表觀遺傳學角度探討心肌慢性缺氧適應的分子機制，為圍手術期心肌保護分子靶點尋找提供理論依據(jù)。 目的： 探討紫紺型先心病患兒心肌組織SOCS3基因啟動子甲基化水平及其可能的機制。 方法： 收集紫紺型先天性心臟病(紫紺組，n=18)和非紫紺型先天性心臟病(非紫紺組，n=16)患者術中切除的肥厚的右心室心肌組織。提取心肌組織全基因組DNA，重亞硫酸鹽修飾后，，甲基化特異性聚合酶鏈反應(Methylation specific PCR，MSP)、重亞硫酸鹽修飾測序(Bisulfite Sequencing PCR，BSP)檢測心肌組織中SOCS3基因啟動子CpG島甲基化差異。 Western blotting檢測心肌組織中DNA甲基化轉移酶1(DNA methyltransferase1，DNMT1)、DNA甲基化轉移酶3A(DNA methyltransferase3A，DNMT3A)、DNA甲基化轉移酶3B(DNA methyltransferase3B，DNMT3B)蛋白表達，qRT-PCR檢測心肌組織中DNMT1、DNMT3A、DNMT3B mRNA表達。 結果： 1.紫紺組心肌組織SOCS3基因啟動子CpG島呈部分甲基化（甲基化頻率為54.287±16.744%），非紫紺組無明顯甲基化(甲基化頻率為0.000±0.000%)。 2.紫紺組中DNMT3A蛋白表達較非紫紺組增高(0.407±0.469v.s0.160±0.034，P＜0.05)， DNMT1(0.084±0.115v.s0.081±0.085, P0.05)、 DNMT3B(0.054±0.012v.s0.052±0.093，P＞0.05)蛋白表達在紫紺組及非紫紺組間無顯著性差異。 3.紫紺組中DNMT1(0.548±0.553v.s0.920±0.456, P0.05)、DNMT3A(0.555±0.395v.s0.756±0.240，P0.05)、DNMT3B (0.473±0.509v.s1.018±0.749，P0.05) mRNA水平較非紫紺組明顯降低。 4.紫紺組中DNMT3A蛋白水平與SOCS3基因啟動子甲基化程度顯著正相關(r=0.359，P=0.047)。 結論： 紫紺型先心病患者心肌組織中，SOCS3基因啟動子呈部分甲基化，DNMT3A蛋白水平增高，且DNMT3A蛋白水平與SOCS3基因啟動子甲基化程度顯著正相關。DNMT3A蛋白可能通過酶促反應，以及招募、錨定含有甲基化CpGs的核小體，參與建立和維持了SOCS3啟動子CpG島甲基化模式。
[Abstract]:Background:
Cyanotic congenital heart disease, which threatens human life and health, and understands its pathophysiological mechanism, helps to promote clinical diagnosis and prognosis. Patients with cyanotic congenital heart disease experience the pathophysiological process of chronic anoxia, and how the myocardium can complete apoptosis, autophagy, secretion, metabolism, remodeling and other structural functions under the condition of chronic hypoxia stress. The specific mechanism of adaptive changes has not yet been fully elucidated.
The cell signal suppressor -3 (Suppressor of cell signaling-3, SOCS3) is the downstream molecule of the Janus kinase signal transduction and transcriptional activator -3 (Janus kinase-Signal transducer and) signaling pathway. Negative feedback regulates the activity of the pathway and maintains the cell signal homeostasis. In patients with non cyanotic congenital heart disease, IL-6-JAK-STAT3 activation, SOCS3 transcriptional and translation levels are up-regulated in patients with cyanotic congenital heart disease, and the increase of SOCS3 protein level depends on the increase of transcriptional level, while the overexpression of SOCS3 in anoxic cardiomyocytes inhibits the activity of IL-6-JAK-STAT3 signaling pathway and increases the apoptosis, suggesting a moderately expressed SOCS3. It is important for the homeostasis of intracellular IL-6-JAK-STAT3 signal and plays an important role in the regulation of chronic hypoxia adaptation, but it is not clear what factors in the hypoxia myocardium are involved in the transcription regulation of SOCS3. Previous studies in tumor cells showed that the promoter methylation of SOCS3 gene involved in its transcriptional regulation and influence protein expression. Therefore, we speculate that hypomethylation of SOCS3 promoter in chronic hypoxia myocardium may participate in the regulation of transcriptional expression, regulate the activity of JAK-STAT3 signaling pathway and participate in the pathway balance of chronic hypoxia adaptation signal in the myocardium of chronic anoxic myocardium. This study explored the heart from the epigenetic point of view. The molecular mechanism of chronic hypoxia adaptation provides a theoretical basis for finding molecular targets of myocardial protection during perioperative period.
Objective:
Objective to investigate the methylation level of SOCS3 gene promoter and its possible mechanism in children with cyanotic congenital heart disease.
Method錛

本文編號：1780102