敲除小鼠胚胎心脏流出道内第二生心区和心脏神

BACKGROUND:Cx43 plays an important role in human congenital heart disease. However, there is still no consistent conclusion about the formation mechanism of cardiac malformation in Cx43 knockout mouse embryos.

OBJECTIVE:To investigate the cardiac development defects and the migration and differentiation of progenitor cells of the second heart field and cardiac neural crest in Cx43 knockout mouse :Serial sections of Cx43 gene knockout homozygous (Cx43-/-) mouse and Cx43 wild-type (Cx43+/+) mouse embryos from embryonic day (ED) 10 to ED13 were made for immunohistochemical and immunofluorescent staining, and three-dimensional reconstruction of the AND CONCLUSION:(1) In Cx43 gene knockout mouse embryos at ED10-ED11, Isl1 positive second heart field cells in the ventral mesenchyme of the foregut extended through the area between the bilateral arch arteries to the dorsal wall of pericardial cavity. Meanwhile, Isl1 positive cells in the core mesenchyme of the branchial arches were continuous with those in the dorsal wall of pericardial cavity and the distal wall of the outflow tract. At ED13, the distribution of Isl1 positive cells was observed in the wall of the ascending aorta and pulmonary trunk as well as the wall of the left and right outflow tracts of the septated ventricles. However, compared with wild-type mouse embryos, fewer Isl1 positive second heart field cells were found in Cx43 gene knockout mouse embryos (P＜ 0.01). (2) During ED10 to ED11, Ap2α positive neural crest cells were still found in the wall of the arch artery and the dorsal and ventral walls of the aortic sac in Cx43 gene knockout mouse embryos, but the number of neural crest cells was less than that of wild-type mouse embryos (P＜ 0.01).(3) These results indicate that the migration path and distribution pattern of Isl1 positive second heart field cells and Ap2α positive cardiac neural crest cells are similar between the Cx43 gene knockout and wild-type mouse embryos, but the number of two kinds of migrating cells is reduced after Cx43 gene suggests that in addition to cardiac neural crest derived cells, the decrease of second heart field progenitor cells might be involved in the formation of outflow tract malformations in Cx43 knockout mouse embryos.

0 引言 Introduction

缝隙连接蛋白Connexin43(Cx43)广泛表达于心房和心室工作心肌、心室传导系统和心脏神经嵴细胞[1-5]，在心电传导、流出道正常发育和调节心脏神经嵴细胞的迁移行为中起着重要作用。Cx43基因敲除小鼠表现为心脏成襻延迟[3]、流出道梗阻及主动脉弓离断等[3，6]。已有研究证实敲除Cx43基因后p38 MAPK活性下调，FGF1表达增加，促进心肌细胞增生[7]，从而导致流出道梗阻。但有学者认为Cx43基因敲除小鼠死于右心室流出道梗阻也可由于心脏神经嵴细胞迁移异常所导致[6]。但特异性敲除神经嵴的Cx43基因，胚胎心脏并未发生形态异常[8]。因此，Cx43基因敲除小鼠胚胎心脏流出道畸形的发生机制有待进一步研究。

心脏神经嵴即耳板中部到第3体节水平的神经嵴，在参与心脏发育的过程中，需要Cx43缝隙连接的作用[9-10]。如上所述，特异性敲除神经嵴的Cx43基因，胚胎心脏并未发生形态异常，此结果提示可能是敲除Cx43后，其他因素影响了心脏神经嵴细胞的迁移从而导致流出道畸形的发生。另有研究揭示Cx43基因敲除小鼠胚胎心脏流出道畸形是由于流出道心肌分化异常所导致[11]。大量研究表明，流出道心肌来源于第二生心区，第二生心区的异常发育可引发流出道心肌分化异常[12-14]；另一方面，第二生心区可通过多种信号通路调节心脏神经嵴[15-19]，例如抑制第二生心区中Notch信号，可导致心脏神经嵴细胞向流出道迁移发生异常[15]。由此实验推测，第二生心区的异常发育可能是Cx43基因敲除小鼠胚胎发生心脏流出道畸形的一个原因。但目前，Cx43基因敲除小鼠胚胎第二生心区是否异常尚未见报道。

实验用免疫组织化学染色、荧光双染及三维重建的方法观察Cx43基因敲除后流出道的畸形与Isl1阳性第二生心区前体细胞和Ap2α阳性神经嵴细胞的迁移路径、分布模式，以确认Cx43基因敲除小鼠胚胎第二生心区发育是否异常，为进一步探讨Cx43基因敲除导致流出道发育异常的发生机制提供形态学依据。

1 材料和方法 Materials and methods

1.1 设计 细胞学体外实验。

1.2 时间及地点 于2018年4月至2019年12月在山西医科大学组织胚胎学实验室完成。

1.3 材料 Cx43转基因C57BL6小鼠共20只，体质量28-35 g。收集胚龄10-13 d Cx43+/+和Cx43-/-小鼠胚胎40只，每个胚龄胚胎收集5只，所使用小鼠均来自山西医科大学，动物许可证号：SCXK(晋)2015-0001。所有动物操作均符合山西医科大学伦理委员会对动物实验的要求和规定。

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