Cypher和ENH在小鼠早期心脏发育中的作用及其分子机制

发布时间：2018-06-02 02:16

本文选题：ENH + Cypher　；参考：《中南大学》2012年博士论文

【摘要】：肌节的Z线是一个高密度的蛋白复合体。Z线具有维持肌细胞正常的骨架结构、细胞信号传导和收缩功能的作用。在Z线中存在Cypher和Enigma homolog protein (ENH)两种Enigma家族的亚型蛋白。ENH和Cypher高度同源,分别具有维持z线结构、参与细胞内信号调节和维持肌细胞正常收缩功能的重要作用。但Cypher和ENH同时变异或缺失后对于小鼠心脏发育过程有何影响目前未知。本实验利用Cypher和ENH双重敲除的小鼠模型。观察Cypher和ENH被同时敲除后新生小鼠胚胎期心肌发育过程的变化情况。通过观察小鼠心脏组织发育过程中的组织形态变化、病理特征、心肌细胞Z线结构变化以及Z线结构内相关蛋白的表达情况,为进一步解释上述两种基因敲除后,小鼠模型心脏发育过程的变化机制提供证据；同时也为研究人类相关基因变异所导致的疾病奠定基础。方法基因敲除小鼠模型建立与鉴定：将一段含有ENH外显子3的序列插入小鼠ENH基因的第3个内含子中,特异性的敲除小鼠的ENH基因。另外,将一段含β-galactosidase (LacZ)的序列插入小鼠Cypher基因的翻译起始位点ATG之后,从而干扰小鼠Cypher基因的表达。通过两条品系的小鼠杂交得到Cypher和ENH双重敲除的小鼠。取成年小鼠尾尖组织或小鼠胚胎的新鲜胎膜,使用PCR技术筛选Cypher和ENH基因被敲除的小鼠。小鼠成活率观察：分别取不同时间节点的小鼠胚胎,观察不同基因型小鼠的存活情况。小鼠胚胎心脏大体情况观察：取新鲜小鼠胚胎进行全胚对比并拍照。选择不同基因型的小鼠胚胎制成石蜡组织块后切片并进行HE染色。使用显微镜下观察小鼠胚胎心脏大体情况并拍照。小鼠胚胎心脏超微结构观察：取新鲜小鼠胚胎。固定后行超薄切片,使用透射电镜观察小鼠胚胎心肌的超微结构并拍照。小鼠胚胎心脏内蛋白表达水平和位置检测：取新鲜小鼠胚胎。制成冷冻组织块后切片或全胚固定,使用免疫染色技术和相关的特异性抗体检测小鼠胚胎或心脏切片内的特异性蛋白表达和分布情况。然后使用荧光显微镜或共聚焦显微镜观察胚胎染色情况并拍照。取新鲜小鼠胚胎。急冻后收集蛋白,使用免疫蛋白印迹技术检测小鼠胚胎内相关蛋白的表达情况。使用酵母双杂交技术及邻位连接技术检测蛋白之间的相互作用。成年小鼠心脏内特异性RNA量检测：使用斑点分析技术对出生后6周成年小鼠心脏内不同的RNA进行检测和半定量分析。结果小鼠胚胎被双重敲除Cypher和ENH后在心脏发育早期即出现心室壁变薄、心脏扩大及死亡,同时有Z线结构不完整及肌节成熟障碍。对小鼠胚胎心脏的实验发现Z线内α-Actinin和细肌丝排列结构紊乱Cypher与α-Actinin存在相互作用,Cypher的LIM结构域还可以与Integrin的细胞膜内结构域产生相互作用。但小鼠胚胎心肌Z线内其它蛋白和粗肌丝的排列及表达情况未受Cypher和ENH缺失的影响。结论 Cypher和ENH是两个参与小鼠胚胎心脏发育的重要基因,并在小鼠胚胎心肌细胞的成熟过程中发挥相互补偿的作用。单敲除Cypher或ENH都不会导致小鼠胚胎死亡,而双敲除Cypher和ENH则会引起小鼠胚胎早期(E10.5)死亡,并伴有一系列的胚胎与心脏发育异常,如胚胎发育延缓、心室腔扩大及心室壁变薄。在小鼠胚胎心肌发育过程中,Cypher和ENH可能通过与α-Actinin的相互作用,在小鼠胚胎心肌细胞发育的过程中起到维持Z线和细肌丝成分正常组装的作用。我们还发现CypherL与CypherS都参与了小鼠胚胎早期心脏发育。CypherL与ENH双敲除小鼠死于胚胎发育较早阶段(E12.5),CypherS与ENH双敲除小鼠则可以活到出生后。这提示在后续的心脏发育过程中,CypherS的作用可以被CypherL替代,反之则不行,这可能是由于Cypher蛋白的LIM结构域可以与Integrin的细胞膜内结构相结合,从而介导肌节成熟过程中的某些细胞内调节机制。
[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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【共引文献】