【作者】 张超；

【导师】 吴泰华；

【作者基本信息】 大连医科大学 ， 内科学， 2011， 硕士

【摘要】 背景：福山型先天性肌营养不良综合征(Fukuyama Congenital Muscular Dystrophy, FCMD)是一类常见的先天性肌营养不良综合征(Congenital Muscular Dystrophies, CMDs),属于常染色体隐性遗传病,在日本人群中发病率最高,仅次于杜氏肌营养不良综合征(Duchenne Muscular Dystrophy, DMD)。以进行性肌营养不良伴广泛的神经系统先天畸形为特点。多于出生后6个月以内发病,临床表现为肌张力低下,抬头、端坐延迟,面肌受累明显,呼吸肌也可受累,仅少数患儿可独立行走,常有假肥大和关节挛缩,精神发育迟滞。血肌酐CK常增加10-60倍,多在2-23岁死亡。现已通过定位克隆的方法,确定与FCMD发病的基因位于9q31,基因产物为461个氨基酸长度的蛋白质分子,分子量为53.7KDa,命名为fukutin,以发现本基因的科学家Fukuyama来命名。目前fukutin的具体功能尚未被确认,科学假设认为是一种糖基转移酶,与细胞外基质的形成有关,含有N端的疏水序列和C端的糖基化位点。转染试验表明fukutin和一种高尔基分泌蛋白共转运至细胞外。但其具体功能与基因缺失后致病机制仍未清楚。目的：本实验以心肌为研究对象,通过对FCMD小鼠模型的心功能的心脏超声学和组织病理学观察,设计体内体外试验,研究FCMD小鼠模型的心脏自相对正常的状态到凋亡状态的过程中心肌纤维及肌膜上相关蛋白质变化,探讨与肌纤维坏死的分子机制。方法：本实验以fukutin基因为靶基因,利用Cre-Lox重组酶,结合心肌中的MCK因子,制作条件基因敲除的小鼠,即心肌内fukutin敲除的小鼠,并进行杂交,在产生的F1子代小鼠中选择纯和的双显性和双隐性小鼠,即fukutin+/+(WT)及fukutin-/-(KO)小鼠模型。分别分离两组F1小鼠杂交后的F2代新生小鼠的心肌细胞,利用Gq蛋白激动剂诱导心肌肥大,观察两组对诱导剂的反应,在此过程中,利用免疫荧光定位,查找相关蛋白质分子NCX1是否发生变化,以寻找心肌纤维凋亡原因；取F2代中KO与WT两组成年小鼠的心肌标本,进行冰冻切片和免疫染色及荧光观察,比较对比两组中假定蛋白质分子的不同,结合体内试验结果,以确定与凋亡有关的分子。结果：1、经不同浓度的去氧肾上腺素诱导48小时后,KO组的新生小鼠的心肌细胞未发生明显的形态学变化(P>0.05),而WT组则明显发生代偿反应,心肌细胞面积增大(P<0.05)。而诱导72小时后,KO组的心肌细胞死亡,WT组仍有明显代偿反应。2、以10um/L的去氧肾上腺素诱导心肌肥大,48小时候观察肌节形成,发现在KO组中无规则的肌节形成,而WT组心肌细胞则可形成规则的肌节结构。3、通过对新生心肌细胞和成年小鼠心肌纤维NCX1的免疫定位观察,发现WT组中,NCX1明显位于肌膜,而在KO组,NCX1并未定位于肌膜。结论：1、fukutin KO的小鼠心肌细胞不能对Gq蛋白偶联受体激动剂去上肾上腺素产生代偿反应(P<0.05)。2.在fukutin KO情况下,荧光显像结果示NCX1并未定位于肌膜,提示FCMD心肌细胞凋亡的原因为钙超载。更多还原

【Abstract】 Backgroud:Fukuyama congenital dystrophy (FCMD) is one common congenital mucular dystrophies. It is one of the most common autosomal recessive disorders in the Japanese population, following the Duchenne muscular dystrophy (DMD). FCMD is characterized by congenital muscular dystrophy in combination with cortical dysgenesis, and most of the patients get illness within 6 months after birth. Patients show hypotonia, motor development delay, facial muscle is obvious, especially the cheek. Respiratory distress is not so often, and pseudohypertrophy becomes evident in the cheeks and calves in early infancy, as well as joint contracture. The blood CK of patients is also 10-60 times higher than normal children, and most of the patients die between 2-23 years old.Now the FCMD gene was identified in the 9q31, and the product of this gene is fukutin, after Fukuyama-type. Fukutin is a protein of 461 amino acids with molecular weight of 53.7KDa. This protein has an N-terminal hydrophobic signal sequence and one putative N-glycosilation site, and it is thought to be a glycosyltransferase but the function of fukutin and the molecular mechanism of FCMD is now unclear. Transfection experiments indicated that the the fukutin protein is transferred to an extracellular region, co-locolizing with a Golgi protein.Objective:Use the myocardium as the object in this experiment, to check the changes of related protein of the membrane of myofibers, to identify the molecular mechanism of cardiac muscle degeneration and necrosis in FCMD.Methods:Take the FCMD gene in the cardiomyofibers as the target gene, make conditional FCMD gene knockout mice with a Cre-Lox recombinase, then hybrid them to get F1. Choose the homozygous FCMD gene+/+ and FCMD gene-/-groups in the F1. The F2 is fukutin+/+(WT) and fukutin-/-(KO) contrast groups in the heart. Induce the hypertrophic response of neonatal cardiomyocytes (1-3 days) of WO and KO groups with a Gq-receptor agonist to observe the difference of the KO and WT types, and check the related proteins with immunofluorescence. Make a contrast of adult WT and KO groups of cardiac muscle to affirm the conclusion.Results:1. After 48 hours induction of different concentration of Gq-receptor agonist, fukutin KO group did not show an obvious change of cytomorphology (P>0.05), However, in WT group, we saw the compensatory response:the cell area enlarged obviously (P<0.05). After 72 hours induction, all the cardiomyocytes of KO group became cell death and WT groups still show the compensatory response.2. Use 10um/L Gq-receptor agonist phenylephrine to induce the hypertrophy of neonatal cardiomyocytes of both groups, after 48hours, clear sarcomere is detected in the WT group while in KO group, we did not see the regular sarcomere.3. The immunofluorescence indicates that NCX1 is not located in the membrane of cardiomyocytes of fukutin KO mice.Conclusions:1. The neonatal cardiomyocytes of fukutin KO mice cannot response to the Gq-receptor agonist.2. Immnofluorescence shows that NCX1 is not localized in the membrane of the cardiomyofiber which indicates that the reason of cardiac muscle necrosis is Ca+ overload.更多还原