【摘要】： 心臟病已經(jīng)成為威脅人類(lèi)健康和生命最為嚴(yán)重的疾病之一，近幾年，心血管疾病的死亡人數(shù)占到疾病死亡總?cè)藬?shù)的三分之一。為攻克這一頑癥，首先必須從分子水平上系統(tǒng)地研究控制心臟發(fā)育的基因及其相互調(diào)控關(guān)系以及導(dǎo)致該疾病的分子機(jī)理。 小鼠Lbh(Limb-bud-and-heart)基因在心臟和肌肉組織特異表達(dá)，在對(duì)Lbh轉(zhuǎn)基因小鼠模型的研究中發(fā)現(xiàn)該小鼠心臟表型異常，類(lèi)似人類(lèi)先天性心臟疾病的癥狀。人類(lèi)LBH基因與小鼠的Lbh基因在蛋白質(zhì)水平的相似性高達(dá)89％，由此推測(cè)它可能與人類(lèi)心臟疾病發(fā)生有關(guān)。對(duì)人LBH基因的深入研究對(duì)于探討人心臟發(fā)育的基因調(diào)控機(jī)制，對(duì)于心臟疾病的診斷和治療具有重大的理論和實(shí)踐意義。 生物體內(nèi)的許多簡(jiǎn)單的生理活動(dòng)都是在眾多蛋白質(zhì)的協(xié)調(diào)相互作用下進(jìn)行的，每個(gè)蛋白質(zhì)行使其功能都要通過(guò)和其他與其相互作用的蛋白質(zhì)來(lái)協(xié)調(diào)作用完成。研究一個(gè)蛋白質(zhì)功能的工作包括確定它與哪些蛋白質(zhì)發(fā)生作用，而鑒定這些與其發(fā)生作用的蛋白質(zhì)也可以為研究這個(gè)蛋白質(zhì)本身的功能提供很多的線(xiàn)索。酵母雙雜交技術(shù)簡(jiǎn)便，靈敏，高效，而且能夠大規(guī)模篩選活細(xì)胞內(nèi)的蛋白質(zhì)相互作用。它在基因功能的研究中得到了廣泛的應(yīng)用。目前，對(duì)于LBH基因還沒(méi)有做任何深入的研究，LBH的功能尚不清楚，利用酵母雙雜交技術(shù)篩選鑒定相互作用蛋白，從而研究該基因的可能功能作用，是一個(gè)簡(jiǎn)單而有效的方法。 為了研究LBH在心臟中的作用機(jī)制，本研究利用酵母雙雜交方法在成人心臟文庫(kù)中篩選鑒定到7個(gè)與LBH發(fā)生相互作用的蛋白質(zhì)，其中αB-crystallin(CRYAB)是一小分子熱休克蛋白，在心肌細(xì)胞表達(dá)非常豐富；作者用免疫共沉淀和Pull-Down驗(yàn)證了LBH與αB-crystallin確實(shí)發(fā)生相互作用。利用免疫熒光研究LBH和αB-crystallin在COS-7細(xì)胞的定位情況發(fā)現(xiàn)，，LBH主要定位在細(xì)胞核，細(xì)胞質(zhì)中也有一定量的表達(dá)，αB-crystallin主要在細(xì)胞質(zhì)中表達(dá)，二者共同轉(zhuǎn)入細(xì)胞時(shí)，αB-crystallin發(fā)生了入核現(xiàn)象，說(shuō)明細(xì)胞核中的二者相互作用，熒光報(bào)告基因分析發(fā)現(xiàn)LBH和αB-crystallin的共同表達(dá)協(xié)同抑制p53、p21轉(zhuǎn)錄活性；另外，Tcap是一種肌節(jié)蛋白，對(duì)心臟發(fā)育和心臟功能都有重要作用，并且其突變均心肌病密切相關(guān)。作者用免疫共沉淀和Pull-Down驗(yàn)證了LBH與Tcap確實(shí)發(fā)生相互作用。利用免疫熒光研究LBH和Tcap在COS-7細(xì)胞的定位情況發(fā)現(xiàn)，LBH主要定位在細(xì)胞核，細(xì)胞質(zhì)中也有一定量的表達(dá)，Tcap在核質(zhì)均有表達(dá)，當(dāng)二者共同轉(zhuǎn)入細(xì)胞時(shí)，二者在細(xì)胞核質(zhì)共定位表達(dá)情況一致。綜合目前的研究結(jié)果來(lái)看，LBH很可能通過(guò)與αB-crystallin、Tcap的相互作用參與心臟功能并且與心臟疾病相關(guān)聯(lián)。
[Abstract]:Heart disease has become one of the most serious diseases threatening human health and life. In recent years, the number of deaths from cardiovascular diseases accounts for 1/3 of the total number of deaths. In order to overcome this persistent disease, it is necessary to systematically study the genes that control cardiac development and their mutual regulation at the molecular level, as well as the molecular mechanism leading to the disease. Mouse Lbh (Limb-bud-and-heart) gene was specifically expressed in heart and muscle tissue. In the study of Lbh transgenic mouse model, it was found that the mouse heart phenotype was abnormal, which was similar to the symptoms of human congenital heart disease. The similarity between human LBH gene and mouse Lbh gene at protein level is as high as 89%, which suggests that it may be related to the occurrence of human heart disease. The further study of human LBH gene is of great theoretical and practical significance for exploring the gene regulation mechanism of human heart development and for the diagnosis and treatment of heart diseases. Many simple physiological activities in organisms are carried out under the coordinated interaction of many proteins, and each protein exercises its function by coordinating with other proteins that interact with it. The study of the function of a protein includes determining which proteins it interacts with, and identifying the proteins that interact with it can also provide a lot of clues to the study of the function of the protein itself. Yeast two-hybrid technique is simple, sensitive and efficient, and can screen protein interactions in living cells on a large scale. It has been widely used in the study of gene function. At present, no further research has been done on LBH gene, and the function of LBH is not clear. It is a simple and effective method to screen and identify the interacting protein by yeast two-hybrid technique, so as to study the possible functional effect of the gene. In order to study the mechanism of LBH in heart, seven proteins interacting with LBH were screened and identified by yeast two-hybrid method in adult heart library, in which 偽 B-crystallin (CRYAB) was a small molecule heat shock protein. The expression was very rich in cardiomyocytes. Immunoprecipitation and Pull-Down were used to verify the interaction between LBH and 偽 B-crystallin. The localization of LBH and 偽 B-crystallin in COS-7 cells was studied by immunofluorescence. It was found that LBH was mainly located in the nucleus and a certain amount of expression was also found in the cytoplasm. 偽 B-crystallin was mainly expressed in the cytoplasm. 偽 B-crystallin entered the nucleus, indicating the interaction between the two in the nucleus. Fluorescence reporter gene analysis showed that the co-expression of LBH and 偽 B-crystallin coordinated the inhibition of p53 and the transcriptional activity of p21. In addition, Tcap is a sarcoprotein, which plays an important role in cardiac development and cardiac function, and its mutation is closely related to cardiomyopathy. Immunoprecipitation and Pull-Down were used to verify the interaction between LBH and Tcap. The localization of LBH and Tcap in COS-7 cells was studied by immunofluorescence. It was found that LBH was mainly located in the nucleus, and there was also a certain amount of expression in cytoplasm, and Tcap was expressed in both nucleoplasms. when both of them were transferred into the cells, The co-localization and expression of the two in nucleus and cytoplasm were the same. Based on the results of current research, LBH is likely to be involved in cardiac function and associated with heart disease through the interaction with 偽 B 鈮

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