Midkine-a通过限制心肌细胞总数的方式阻碍斑马鱼胚胎心脏生长  

Midkine-a inhibits zebrafish embryonic heart growth by limiting the cardiomyocyte pool

作　　者：陈静[1] Luo Jing Hitchcock Peter 

机构地区：[1]福建师范大学生命科学院,福州350117 [2]美国密歇根大学安娜堡分校克鲁格眼科中心,安娜堡48103

出　　处：《中国实验动物学报》2015年第5期458-465,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(No.30971106);福建省自然科学基金(No.2014J01304);教育部留学回国人员基金(教外司留【2010】1561号)

摘　　要：目的探讨一种可溶性的外分泌因子midkine-a在斑马鱼胚胎心脏发育过程中的功能。方法在整体胚胎上做midkine-a RNA的原位杂交实验;利用原有的转基因斑马鱼系Tg(pmidkine-a:EGFP),动态观察胚胎从出生到心脏发育成形这一段时间心脏荧光表达情况;将原有的转基因斑马鱼体系Tg(phsp:midkine-a:EGFP)胚胎进行热休克而过表达midkine-a,观察胚胎心脏表型;利用Tg(pcmlc2:dsRed)鱼系胚胎的心肌细胞核带有红色荧光,能进行单个心脏心肌细胞计数这一特点,将杂合的Tg(phsp:midkine-a:EGFP)鱼系与纯合的Tg(pcmlc2:dsRed)鱼系交配,以得到Tg(phsp:midkine-a:EGFP/pcmlc2:dsRed)的杂合胚胎,对其进行热休克而过表达midkinea,计算每个胚胎心脏内心肌细胞的总数;用吗啉寡聚核苷酸(morphonino,MO)阻碍新生胚胎内的midkine-a mRNA表达,观察胚胎心脏表型。结果原位杂交试验证实midkine-a在胚胎48 hpf(hour post fertilization,受精后,用来标记斑马鱼胚胎年龄)大时表达于心脏;转基因Tg(pmidkine-a:EGFP)胚胎在72 hpf时其EGFP表达于心脏;Tg(phsp:midkine-a:EGFP)胚胎在过表达midkine-a后心脏变小;吗啉寡聚核苷酸敲除midkine-a对胚胎心脏发育无影响;最后在Tg(phsp:midkine-a:EGFP/pcmlc2:dsRed)鱼系胚胎内过表达midkine-a导致其单个心脏内心肌细胞数目变少,与其小心脏外形吻合。结论 midkine-a在斑马鱼胚胎发育过程中表达于胚胎心脏;过表达midkine-a导致胚胎心脏内心肌细胞总数减少及心脏变小;敲除midkine-a则对胚胎心脏发育无影响。Objective To investigate the functional role of a soluble secretion factor midkine-a in the process of zebrafish embryonic heart development .Methods Whole-mount in situ hybridization was performed to detect whether midkine-a is expressed in the embryonic heart .In the transgenic embryonic heart of Tg （ pmidkine-a：EGFP） , the expres-sion of EGFP in the heart was monitored .Multiple heat shock treatments were applied to Tg （phsp：midkine-a：EGFP） em-bryos in order to overexpress midkine-a, and the phenotype of the heart was observed .The heterozygous Tg （ phsp：mid-kine-a：EGFP） fish were crossed with homozygous Tg （pcmlc2：dsRed） fish, which specifically expresses RFP in the nucle-oli of embryonic cardiomyocytes to facilitate myocyte number counting .To get Tg （ phsp：midkine-a：EGFP/pcmlc2：dsRed）embryos, and the total number of cardiomyocytes in their heart was counted and compared with the controls when the heat shock induced overexpression of midkine-a.Morphonino knocked out of midkine-a was performed to observe the heart phe-notype.Results Midkine-a was expressed in the zebrafish embryonic heart during development .Overexpression of mid-kine-a led to a smaller heart and reduced total number of cardiomyoctes in a single heart , which might be associated with the smaller heart phenotype .Morphonilo knocked out of midkine-a had no effect upon the heart development .Conclusions Midkine-a impedes zebrafish embryonic heart growth by limiting its cardiomyocyte pool .

关 键 词：斑马鱼胚胎 心脏发育 midkine-a 心肌细胞池 

分 类 号：Q95-33[生物学—动物学]