CT-1C末端多肽对昆明小鼠心肌肌小节α-Actin、α-Actinin及UCP_2表达的影响

Influence of C-terminal polypeptide of cardiotrophin-1 on expression of sarcomaricα-Actin,α-Actinin and UCP_2 in myocardium of Kunming mice

目的:观察心脏营养素-1(CT-1)慢性作用所诱导的小鼠重构心肌中,肌小节收缩性蛋白α-Actin、细胞骨架蛋白α-Actinin及线粒体解偶联蛋白-2(UCP2)的表达情况。方法:实验组昆明小鼠腹腔注射CT-1C末端肽(carboxy-terminal polypeptide of cardiotrophin-1,CT-1-CP)1、2、3、4周(每组10只,雌雄各半)后,对照组小鼠(10只,雌雄各半)腹腔注射生理盐水4周后,摘取小鼠心脏标本,石蜡包埋,切5μm厚切片,采用SABC检测肌小节结构蛋白α-Actin、α-Actinin与UCP2在小鼠心肌中的表达情况;同时采用Western blot检测小鼠心肌组织中3种蛋白质的相对表达量。结果:免疫组化结果显示,α-Actin的阳性颗粒主要集中于细胞核的周围,α-Actinin则趋于向肌节的横纹处汇聚,而UCP2则较均匀地散布于肌细胞浆中。结合Western blot相对灰度的比较分析,在对照组,α-Actin的表达水平略高于α-Actinin和UCP2,但3者之间并无明显的差异(WB:F=0.249,P>0.05)。注射CT-1-CP后,α-Actin的表达基本呈逐渐减弱的趋势,但对照组与4个注射组之间并无明显差异(χ2=7.386,P>0.05);与之相反,α-Actinin的表达则呈逐渐增强的趋势,阳性细胞数的百分比和阳性颗粒的染色强度都逐渐增多,而且各组间呈现出明显差异(χ2=21.977,P<0.01);UCP2的表达则在1周后增强,2周后达最高值,随后出现降低,4周后降至接近对照组的水平。结论:CT-1-CP的慢性作用可导致肌小节不同结构蛋白的比例发生改变,α-Actin的表达减少,α-Actinin的表达增多;而线粒体UCP2的表达达到一定峰值后即开始降低。

Objective.. To determine the expressions of sarcomeric contractile protein a-Actin, the specialised cytoskeletal protein a Actinin and the mitochondrial uncoupling protein-2 （UCP2） during the process of myocardial remodeling induced by chronic exposure to cardiotrophin-1 in Kunming mice. Meth- ods..The samples of heart tissues were excised after the Kunming mice （4 groups, n~ 10, 5 male of each group） have received intraperitoneal injection of carboxy-terminal polypeptide of cardiotrophin-1 （CT-I- CP, 500/~g/kg ~ day） for 1, 2, 3and 4 weeks respectively, and then the half of each sample was embedded in paraffin for preparing tissue section, and the other half was used to prepare the protein samples. The control group （n~ 10, 5 male） was injected by physiological saline for 4 weeks and the heart samples were treated identically. The immunohistochemical method of strep avidin-biotin complex （SABC） combined with western blotting were used to detect the distribution and expression of the proteins of sarcomeric a- Actin, a-Actinin and mitochondrial UCP2 in myocardium. Results. The results of IHC showed that the brown signals of a-Actin mainly gathered surrounding the nuclei and that of a-Actinin had a tendency to gather to cross striae. The signals of UCP2 were scattered rather evenly in the plasma. The expression of a-Actin were higher than that of a-Actinin and UCP2 in the control group（WB.. F= 0. 249, P）0.05）and gradually decreased after exposing to CT-1-CP, which was detected by the analysis of scoring of immunos- taining and the comparison of relative grayscale in western blot band, but there were no significant differ- ence between the control group with the CT-1-CP-treated groups （）2 =7. 386, P^0.05）. On the contra- ry, the expression of a-Actinin was gradually increased and differed significantly between these groups （）C2 =21. 977,P^0.01）. The expression of UCP2 first was increased and then decreased. Conclusions.. Long- term exposure to CT-1-CP could lead to the alteration in ratio of different sarcomerie structure proteins, namely the expression of a-Actin decreased and ^-Actinin increased. Simultaneously the expression of UCP2 is increased firstly and then decreased after its expression reaches peak value.