【摘要】：心臟發(fā)育的過程是極其復雜的,涉及不同時間和空間的一系列胚胎發(fā)育相關(guān)基因的順序表達。心臟結(jié)構(gòu)和功能對心臟發(fā)育相關(guān)基因的擾動相當敏感。在心臟發(fā)育相關(guān)基因網(wǎng)絡中,任何一個基因突變都可能導致先天性心臟病的發(fā)生。因此,先天性心臟病遺傳學病因的探索仍然是預防和治療該病的關(guān)鍵。LYRM1(LYR motif containing 1)基因是郭錫熔教授課題組發(fā)現(xiàn)并克隆得到的一條人類新基因。令人驚訝的是,多組織表達譜分析發(fā)現(xiàn)LYRM1基因除了在人類脂肪組織中高豐度表達外,在心臟組織中同樣呈現(xiàn)高豐度表達。另外,研究表明LYRM1是LYR超家族復合體1的成員,而LYR蛋白主要是線粒體蛋白質(zhì),影響線粒體內(nèi)穩(wěn)態(tài)。郭錫熔教授課題小組成員發(fā)現(xiàn)LYRM1在3T3-L1前體脂肪細胞的增值、凋亡及分化過程均扮演重要角色,且具有損傷3T3-L1成熟脂肪細胞線粒體功能作用。鑒于心臟和脂肪均來源于中胚層,來源于相同胚層的組織具有相似的結(jié)構(gòu)與功能,因此提示LYRM1也可能參與心臟發(fā)生發(fā)育的生理過程。本研究小組探索了LYRM1基因過表達在P19細胞向心肌樣細胞分化過程的作用,結(jié)果顯示LYRM1基因過表達雖然不影響P19細胞向心肌樣細胞的分化,但是能顯著誘導P19細胞的增殖、明顯抑制P19細胞凋亡。因此,我們推測LYRM1基因在心臟發(fā)育中可能占有重要作用。為進一步探討心臟發(fā)育過程中LYRM1基因的功能角色,我們選擇P19細胞作為研究對象,運用RNA干擾技術(shù)使LYRM1基因表達沉默,進而探索LYRM1基因表達沉默對P19細胞增殖、凋亡和分化的影響,以及對P19誘導分化的心肌樣細胞線粒體功能的影響。深度分析LYRM1基因?qū)π募蛹毎蛲龅挠绊懠暗蛲鲎饔门c線粒體功能之間的關(guān)系,進一步論證在胚胎心臟發(fā)育過程中LYRM1基因的重要作用及致畸的可能機制,這將為先天性心臟病的病理生理學的基礎研究提供新線索。第一部分LYRM1基因表達沉默對P19細胞增殖凋亡及分化的影響目的：探索LYRM1基因表達沉默對P19細胞增殖、凋亡及分化的影響。方法：設計LYRM1干擾靶序列,構(gòu)建LYRM1基因短發(fā)夾狀RNA的慢病毒載體pgLV-U6-puro-LYRM1-shRNA,對照組為pgLV-U6-puro-NC-shRNA.將pgLV-U6-puro-LYRM1-shRNA或pgLV-U6-puro-NC-shRNA轉(zhuǎn)染到HEK-293T細胞并獲得病毒上清,感染P19細胞并用嘌呤霉素篩選得到穩(wěn)定轉(zhuǎn)染的P19細胞。定量PCR檢測LYRM1基因沉默干擾效果；細胞計數(shù)盒(CCK-8)檢測其對P19細胞增殖的影響;流式細胞儀檢測細胞周期、分析Caspase-3的活性及檢測細胞凋亡；倒置顯微鏡觀察P19細胞向心肌樣細胞分化過程中細胞形態(tài)的改變；定量PCR及Westem blot檢測心肌細胞分化過程中的心肌標志物基因(GATA4和Nkx2-5)的表達水平。結(jié)果:1) LYRM1基因沉默明顯抑制P19細胞增殖,表現(xiàn)為：促進細胞進入G1期,受阻于S期；2)LYRM1基因沉默顯著促進P19細胞凋亡；3)沉默LYRM1基因的P19細胞分化過程中,細胞邊緣模糊,不規(guī)整。心肌標志物GATA4和Nkx2-5的基因表達水平均在心肌細胞分化第6天和第10天顯著下降,蛋白表達水平在分化第6天前均無明顯變化,但第10天明顯下降。結(jié)論:LYRM1基因表達沉默具有抑制P19細胞的增值、促進其凋亡以及抑制P19細胞向心肌樣細胞分化的作用,提示LYRM1基因在胚胎心臟發(fā)育過程可能占有重要作用。第二部分LYRM1基因表達沉默對P19細胞誘導分化的心肌樣細胞線粒體功能的影響目的：探討LYRM1基因表達沉默對P19細胞誘導分化的心肌樣細胞線粒體功能的影響。方法：穩(wěn)定轉(zhuǎn)染LYRM1基因沉默病毒的P19細胞株,經(jīng)1%DMSO誘導分化成為心肌樣細胞。以誘導分化第10天的心肌樣細胞為研究對象,采用熒光定量PCR技術(shù)檢測線粒體DNA拷貝數(shù)；ATP檢測試劑盒檢測細胞ATP含量；采用DCFDA (2,7-Dichlorofluorescein diacetate, DCFDA)熒光探針、熒光顯微鏡、流式細胞儀檢測細胞活性氧(reactive oxygen species, ROS)的改變；應用JC-1(Mitochondrial membrane potential assay kit with JC-1)熒光探針、熒光顯微鏡、流式細胞儀檢測線粒體膜電位的變化。結(jié)果：1) LYRM1基因沉默對心肌樣細胞的線粒體DNA拷貝數(shù)無明顯影響；2) LYRM1基因沉默的心肌樣細胞中ATP含量顯著降低；3) LYRM1基因沉默顯著增加心肌樣細胞中ROS的產(chǎn)生；4) LYRM1基因沉默明顯降低心肌樣細胞線粒體膜電位水平。結(jié)論：]LYRM1基因沉默具有損傷心肌樣細胞線粒體功能作用,提示LYRM1基因在心肌樣細胞線粒體功能維持過程中可能扮演重要角色。
[Abstract]:The process of heart development is extremely complex, involving the sequential expression of a series of embryonic development-related genes in different time and space. Cardiac structure and function are sensitive to the disturbance of heart development-related genes. Any gene mutation in the heart development-related gene network can lead to congenital heart disease. The LYRM1 (LYR motif containing 1) gene is a new human gene discovered and cloned by Professor Guo Xirong's research group. Surprisingly, multiple tissue expression profiling analysis revealed that LYRM1 gene is highly expressed in human adipose tissue in addition to human adipose tissue. In addition, LYRM1 is a member of LYR superfamily complex 1, and LYR proteins are mainly mitochondrial proteins that affect mitochondrial homeostasis. Professor Guo Xirong's team members found that LYRM1 plays an important role in the proliferation, apoptosis and differentiation of 3T3-L1 preadipocytes. LYRM1 may also be involved in the physiological process of cardiogenesis and development. Our team explored the over-expression of LYRM1 gene in P19 cells, which is myocardially fine. The results showed that LYRM1 gene overexpression did not affect the differentiation of P19 cells into cardiomyocyte-like cells, but could significantly induce the proliferation of P19 cells and inhibit the apoptosis of P19 cells. Because of the function of P19 cells, we used RNA interference to silence LYRM1 gene expression, and then explored the effects of LYRM1 gene silencing on the proliferation, apoptosis and differentiation of P19 cells, and on the mitochondrial function of P19-induced differentiated cardiomyocytes. The relationship between apoptosis and mitochondrial function, the important role of LYRM1 gene in embryonic heart development and the possible mechanism of teratogenesis will provide new clues for the basic study of pathophysiology of congenital heart disease. Objective: To explore the effects of LYRM1 gene silencing on proliferation, apoptosis and differentiation of P19 cells. Methods: LYRM1 interference target sequence was designed to construct a lentiviral vector pgLV-U6-puro-LYRM1-shRNA with short hairpin RNA of LYRM1 gene. The control group was pgLV-U6-puro-NC-shRNA. HEK-293T cells were infected with virus supernatant and purinomycin was used to screen stable transfected P19 cells.Quantitative PCR was used to detect the effect of LYRM1 gene silencing interference.Cell counting kit (CCK-8) was used to detect its effect on the proliferation of P19 cells.Flow cytometry was used to detect the cell cycle,Caspase-3 activity and apoptosis. Results: 1) LYRM1 gene silencing significantly inhibited the proliferation of P19 cells, which could promote the cells to enter G1 phase and be blocked by S phase. LYRM1 gene silencing significantly promoted P19 cell apoptosis; 3) LYRM1 gene silencing in P19 cell differentiation process, cell margin blurred, irregular. Myocardial markers GATA4 and Nkx2-5 gene expression levels were significantly decreased on the 6th and 10th day of cardiomyocyte differentiation, protein expression levels were not significantly changed before the 6th day of differentiation, but the 1st day. Conclusion: LYRM1 gene silencing can inhibit the proliferation of P19 cells, promote their apoptosis and inhibit the differentiation of P19 cells into cardiomyocyte-like cells, suggesting that LYRM1 gene may play an important role in the development of embryonic heart. AIM: To investigate the effect of LYRM1 gene silencing on mitochondrial function of cardiomyocyte-like cells induced by P19 cells. METHODS: P19 cells stably transfected with LYRM1 gene silencing virus were induced to differentiate into cardiomyocyte-like cells by 1% DMSO. Mitochondrial DNA copy number was detected by quantitative PCR, ATP assay kit, DCFDA (2,7-Dichlorofluorescein diacetate) fluorescence probe, fluorescence microscope and flow cytometry were used to detect the changes of reactive oxygen species (ROS) and JC-1 (Mitochondrial membrane potential assay k). It with JC-1) fluorescence probe, fluorescence microscope and flow cytometry were used to detect the changes of mitochondrial membrane potential. Results: 1) LYRM1 gene silencing had no significant effect on the mitochondrial DNA copy number of cardiomyocyte-like cells; 2) ATP content in LYRM1 gene silenced cardiomyocyte-like cells decreased significantly; 3) LYRM1 gene silencing significantly increased ROS content in cardiomyocyte-like cells. Conclusion: LYRM1 gene silencing can impair the mitochondrial function of cardiomyocytes, suggesting that LYRM1 gene may play an important role in the maintenance of mitochondrial function of cardiomyocytes.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R54

【參考文獻】

相關(guān)期刊論文 前8條

1 賀晶;高凌根;;馬凡綜合征的分子遺傳學研究進展[J];中華保健醫(yī)學雜志;2015年05期

2 趙鶴;羅毅;;法洛四聯(lián)癥相關(guān)基因研究進展[J];中華胸心血管外科雜志;2013年09期

3 鄧展?jié)?章文文;易龍;;法洛四聯(lián)癥與FOG2基因研究進展[J];中國產(chǎn)前診斷雜志(電子版);2013年03期

4 秦廣寧;羅毅;;法洛四聯(lián)癥相關(guān)基因研究進展[J];中國醫(yī)藥;2011年07期

5 周平;漆洪波;;先天性心臟病的基因研究[J];實用婦產(chǎn)科雜志;2011年04期

6 王棟;劉迎龍;;先天性心臟病宮內(nèi)微創(chuàng)治療進展[J];心肺血管病雜志;2011年01期

7 陳福坤;胡德亮;劉W毲，

本文編號：2186467