本文選題：ENH + Cypher��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：肌節(jié)的Z線是一個高密度的蛋白復(fù)合體。Z線具有維持肌細(xì)胞正常的骨架結(jié)構(gòu)、細(xì)胞信號傳導(dǎo)和收縮功能的作用。在Z線中存在Cypher和Enigma homolog protein (ENH)兩種Enigma家族的亞型蛋白。ENH和Cypher高度同源,分別具有維持z線結(jié)構(gòu)、參與細(xì)胞內(nèi)信號調(diào)節(jié)和維持肌細(xì)胞正常收縮功能的重要作用。但Cypher和ENH同時變異或缺失后對于小鼠心臟發(fā)育過程有何影響目前未知。 本實驗利用Cypher和ENH雙重敲除的小鼠模型。觀察Cypher和ENH被同時敲除后新生小鼠胚胎期心肌發(fā)育過程的變化情況。通過觀察小鼠心臟組織發(fā)育過程中的組織形態(tài)變化、病理特征、心肌細(xì)胞Z線結(jié)構(gòu)變化以及Z線結(jié)構(gòu)內(nèi)相關(guān)蛋白的表達(dá)情況,為進(jìn)一步解釋上述兩種基因敲除后,小鼠模型心臟發(fā)育過程的變化機制提供證據(jù)；同時也為研究人類相關(guān)基因變異所導(dǎo)致的疾病奠定基礎(chǔ)。 方法 基因敲除小鼠模型建立與鑒定：將一段含有ENH外顯子3的序列插入小鼠ENH基因的第3個內(nèi)含子中,特異性的敲除小鼠的ENH基因。另外,將一段含β-galactosidase (LacZ)的序列插入小鼠Cypher基因的翻譯起始位點ATG之后,從而干擾小鼠Cypher基因的表達(dá)。通過兩條品系的小鼠雜交得到Cypher和ENH雙重敲除的小鼠。取成年小鼠尾尖組織或小鼠胚胎的新鮮胎膜,使用PCR技術(shù)篩選Cypher和ENH基因被敲除的小鼠。 小鼠成活率觀察：分別取不同時間節(jié)點的小鼠胚胎,觀察不同基因型小鼠的存活情況。 小鼠胚胎心臟大體情況觀察：取新鮮小鼠胚胎進(jìn)行全胚對比并拍照。選擇不同基因型的小鼠胚胎制成石蠟組織塊后切片并進(jìn)行HE染色。使用顯微鏡下觀察小鼠胚胎心臟大體情況并拍照。 小鼠胚胎心臟超微結(jié)構(gòu)觀察：取新鮮小鼠胚胎。固定后行超薄切片,使用透射電鏡觀察小鼠胚胎心肌的超微結(jié)構(gòu)并拍照。 小鼠胚胎心臟內(nèi)蛋白表達(dá)水平和位置檢測：取新鮮小鼠胚胎。制成冷凍組織塊后切片或全胚固定,使用免疫染色技術(shù)和相關(guān)的特異性抗體檢測小鼠胚胎或心臟切片內(nèi)的特異性蛋白表達(dá)和分布情況。然后使用熒光顯微鏡或共聚焦顯微鏡觀察胚胎染色情況并拍照。取新鮮小鼠胚胎。急凍后收集蛋白,使用免疫蛋白印跡技術(shù)檢測小鼠胚胎內(nèi)相關(guān)蛋白的表達(dá)情況。使用酵母雙雜交技術(shù)及鄰位連接技術(shù)檢測蛋白之間的相互作用。 成年小鼠心臟內(nèi)特異性RNA量檢測：使用斑點分析技術(shù)對出生后6周成年小鼠心臟內(nèi)不同的RNA進(jìn)行檢測和半定量分析。 結(jié)果 小鼠胚胎被雙重敲除Cypher和ENH后在心臟發(fā)育早期即出現(xiàn)心室壁變薄、心臟擴大及死亡,同時有Z線結(jié)構(gòu)不完整及肌節(jié)成熟障礙。對小鼠胚胎心臟的實驗發(fā)現(xiàn)Z線內(nèi)α-Actinin和細(xì)肌絲排列結(jié)構(gòu)紊亂Cypher與α-Actinin存在相互作用,Cypher的LIM結(jié)構(gòu)域還可以與Integrin的細(xì)胞膜內(nèi)結(jié)構(gòu)域產(chǎn)生相互作用。但小鼠胚胎心肌Z線內(nèi)其它蛋白和粗肌絲的排列及表達(dá)情況未受Cypher和ENH缺失的影響。 結(jié)論 Cypher和ENH是兩個參與小鼠胚胎心臟發(fā)育的重要基因,并在小鼠胚胎心肌細(xì)胞的成熟過程中發(fā)揮相互補償?shù)淖饔�。單敲除Cypher或ENH都不會導(dǎo)致小鼠胚胎死亡,而雙敲除Cypher和ENH則會引起小鼠胚胎早期(E10.5)死亡,并伴有一系列的胚胎與心臟發(fā)育異常,如胚胎發(fā)育延緩、心室腔擴大及心室壁變薄。在小鼠胚胎心肌發(fā)育過程中,Cypher和ENH可能通過與α-Actinin的相互作用,在小鼠胚胎心肌細(xì)胞發(fā)育的過程中起到維持Z線和細(xì)肌絲成分正常組裝的作用。我們還發(fā)現(xiàn)CypherL與CypherS都參與了小鼠胚胎早期心臟發(fā)育。CypherL與ENH雙敲除小鼠死于胚胎發(fā)育較早階段(E12.5),CypherS與ENH雙敲除小鼠則可以活到出生后。這提示在后續(xù)的心臟發(fā)育過程中,CypherS的作用可以被CypherL替代,反之則不行,這可能是由于Cypher蛋白的LIM結(jié)構(gòu)域可以與Integrin的細(xì)胞膜內(nèi)結(jié)構(gòu)相結(jié)合,從而介導(dǎo)肌節(jié)成熟過程中的某些細(xì)胞內(nèi)調(diào)節(jié)機制。
[Abstract]:The Z line of the myocytes is a high density protein complex.Z line having the function of maintaining the normal skeletal structure, signal transduction and contraction function of the muscle cells. In the Z line, there are two subtypes of Enigma family of Cypher and Enigma homolog protein (ENH) in the same source, which maintain the structure of the Z line and participate in the intracellular letter, respectively. Number regulates and maintains the important function of normal contractile function of muscle cells. However, the effect of Cypher and ENH variation or deletion on the development of the heart in mice is unknown.
In this experiment, the mouse model of Cypher and ENH double knockout was used to observe the changes of myocardial development in the embryonic stage of neonatal mice after the simultaneous knockout of Cypher and ENH. By observing the changes of tissue morphology, pathological features, the alteration of the Z line structure of cardiac myocytes and the expression of related protein in the Z line structure by observing the development of the cardiac tissue in mice. In addition, it provides evidence for further explanation of the change mechanism of the mouse model heart development process after the two genes are knocked out, and it also lays the foundation for the study of the disease caused by human related gene mutation.
Method
The gene knockout mouse model was established and identified: a sequence containing ENH exon 3 was inserted into the third introns of the mouse ENH gene to specifically knock out the ENH gene of mice. In addition, the sequence of a segment containing beta -galactosidase (LacZ) was inserted into the ATG of the starting site of the mouse Cypher gene, thereby interfering with the mouse Cypher gene. Cypher and ENH double knockout mice were obtained through two strains of mouse hybridization. The fresh fetal membranes of the tail tip tissue of adult mice or mouse embryos were taken, and the PCR technique was used to screen the Cypher and ENH gene knockout mice.
The survival rate of mice was observed: the mice embryos at different time points were taken separately to observe the survival of different genotype mice.
The overall observation of the mouse embryo heart: the whole embryo was compared and photographed in the fresh mouse embryo. The mouse embryos of different genotypes were selected to be made into paraffin tissue and then sliced and stained with HE. Under the microscope, the general condition of the mouse embryo heart was observed and photographed.
Ultrastructure observation of mouse embryo heart: take fresh mouse embryos. After fixed, ultrathin sections were fixed. Ultrastructure of mouse embryonic myocardium was observed by transmission electron microscope and photographed.
Detection of protein expression level and location in the heart of mouse embryos: fresh mouse embryos were taken. Frozen tissue blocks were made into slices or whole embryos were fixed. Specific protein expression and distribution in mouse embryos or heart slices were detected by immunostaining and related specific antibodies. Then fluorescence microscopy or confocal microscopy was used. The embryo of the embryo was observed and taken. The fresh mouse embryos were taken. The protein was collected after the freeze. The expression of the related protein in the mouse embryo was detected by the immunoblotting technique. The interaction between the protein and the yeast two hybrid technique and the adjacent connection technique were used to detect the protein.
Detection of specific RNA in the heart of adult mice: dot blot analysis was used to detect and quantify RNA in the heart of adult mice 6 weeks after birth.
Result
The mouse embryo was double knocked out of Cypher and ENH in the early stage of the heart development, the ventricular wall thinning, the heart enlargement and death, the Z line structure incomplete and the myocal maturity barrier. In the mouse embryo heart experiment, the Z line alpha -Actinin and the musculus myofacal arrangement disorder Cypher were interacted with the alpha -Actinin, and the LIM structure of Cypher The domain can also interact with the intracellular domain of the cell membrane of Integrin, but the arrangement and expression of other proteins and musculus filaments in the Z line of mouse embryonic myocardium are not affected by the deletion of Cypher and ENH.
conclusion
Cypher and ENH are two important genes involved in the development of mouse embryo heart, and play the role of mutual compensation in the maturation of mouse embryonic cardiac myocytes. Single knockout Cypher or ENH will not lead to death of mouse embryos, while double knockout of Cypher and ENH will cause the early stage of mouse embryo (E10.5) death, accompanied by a series of embryos and hearts. In the development process of mouse embryonic myocardium, Cypher and ENH may play a role in maintaining the normal assembly of the Z line and the filament components during the development of mouse embryonic cardiac myocytes during the development of mouse embryonic myocardium. We also found that CypherL and CypherS are both in the process of mouse embryonic cardiac development. .CypherL and ENH double knockout mice died of early embryonic development (E12.5), and CypherS and ENH double knockout mice could survive to birth. This suggests that the effect of CypherS can be replaced by CypherL during subsequent cardiac development, which may be due to the LIM structure of Cypher protein. The domain can be combined with the intracellular membrane structure of Integrin, thereby mediating some intracellular regulatory mechanisms in the process of sarcomere maturation.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

【共引文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王亮;董如廣;朱兵;何昆侖;;肌聯(lián)蛋白與心臟舒張功能的關(guān)系[J];中華保健醫(yī)學(xué)雜志;2008年02期

2 余志斌;;肌節(jié)Z盤:心肌細(xì)胞的信號轉(zhuǎn)導(dǎo)中心[J];心臟雜志;2012年02期

相關(guān)博士學(xué)位論文 前1條

1 王亮;慢性缺血性舒張期心力衰竭動物模型的建立及藥物干預(yù)的研究[D];中國人民解放軍軍醫(yī)進(jìn)修學(xué)院;2008年

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本文編號：1966871