Junctophilin 1在小鼠胚胎干细胞定向分化心肌细胞及大鼠胚胎心肌发生中的表征  

Expression of Junctophilin 1 during cardiogenesis of mouse embryonic stem cells and rat embryos

作　　者：梁星光[1,2] 吴博文[1] 张维晨[3] 周立民[1] 朱丹雁[1] 楼宜嘉[1] 

机构地区：[1]浙江大学药学院心脑血管与肝脏药理研究室,浙江杭州310058 [2]浙江大学医学院附属第一医院,浙江杭州310003 [3]浙江大学竺可桢学院SRTP学生,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2012年第4期359-365,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助课题(81173135,30973600);浙江省自然科学基金重点资助项目(LZ12H31001);浙江省重点科技创新团队基金(2010R50047)

摘　　要：目的:评价膜连接蛋白Junctophilin 1(JP1)亚型在哺乳动物心肌发生过程中,基因转录和蛋白表达特征,为心肌发育生物学及心脏再生医学相关生命现象本质提供实验依据。方法:小鼠胚胎干(embryonic stem,ES)细胞相继制成单细胞悬液和悬滴,收集拟胚体转至培养皿中悬浮分化培养。每隔2 d收集细胞供分子生物学评价,并于心肌分化成熟期(第17天),免疫荧光成像原位检测JP1与心肌小节蛋白α-Actinin和Troponin-T是否呈共表达,流式细胞术定量确认JP1阳染率。另以大鼠胎心为研究对象,大鼠受精后第14天起,每隔2 d取胎心直至分娩新生鼠,供RT-PCR和免疫印迹实验用。结果:ES细胞分化过程中,JP1在基因转录层面全程表达,至第11天达高峰。蛋白层面呈一过性表达,在第9天达高峰,此后迅速下降,至分化培养第15天消失,分化成熟期未见表达。大鼠胎心表达趋势与ES细胞分化过程基本一致,但JP1蛋白表达持续在出生后。在分化终端,流式细胞术显示JP2和肌小节蛋白双染率达16.59%,而未见JP1阳染细胞。结论:JP1基因在小鼠ES细胞定向分化心肌细胞全程均有转录,但JP1仅在分化早期有一过性表达。大鼠胎心JP1表达趋势与ES细胞分化过程基本相一致。Objective： To investigate the expression of Junctophilin 1（JP1） in cardiogenesis of mammalian. Methods： Cardiac differentiation of embryonic stem cells（ESCs） was generated by hanging drop method.Fetal heart was obtained from the rats aged d 14-20 of gestation.The expression of JP1and JP2 during cardiogenesis of ESCs and rat embryos was analyzed by RT-PCR or Western blotting.Immunofluorescence staining was employed to reveal the distribution of JP1 and JP2 in embryoid body（EB）,probing for merging of JP1and JP2 and cardiac sarcomeric α-Actinin or Troponin-T.Percentage of JP1and JP2-positive staining cells was analyzed quantitatively by FCS on d17. Results： JP1 mRNA was up-regulated at the early stage（d 5-11） and then decreased.The expression of JP1 protein was up-regulated at the early stage（d 7-9）,then decreased gradually and disappeared after d 15.While JP2 gene and protein expression increased in a time-dependent manner during cardiogenesis of rat embryos.The results of immunofluorescence staining showed that there was a parallel co-localization of JP2 with Troponin-T or α-Actinin on d17,while JP1 failed to express in the sarcomeric positive area at the same time point.Furthermore,FCS analysis showed that about 16.59% of cells were JP2-positive,while no cells were stained positively for JP1 in d17 EBs. Conclusions： JP1 gene is expressed during the whole process of cardiogenesis,while JP1 protein only appears on the early stage.The expression of JP1 in cardiogenesis of ESCs is consistent with that of rat embryos.

关 键 词：Junctophilin 1 胚胎干细胞 连接蛋白类 心肌/生长和发育 心肌/细胞学 细胞分化 定向分化 大鼠胚胎 心肌发生 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]