Apoptosis during the development of pelvic floor muscle in anorectal malformation rats
Apoptosis during the development of pelvic floor muscle in anorectal malformation rats
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肛门直肠畸形大鼠盆底肌发育过程中的细胞凋亡
DOI：
10.1016/j.jpedsurg.2009.02.004
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发表时间：
2009-10-01
影响因子：
2.4
通讯作者：
Wang, Wei Lin
中科院分区：
文献类型：
--
作者：
Chen, Qing Jiang;Jia, Hui Min;Wang, Wei Lin
目的：大便失禁和便秘仍然是肛肠畸形（ARM）手术后的主要并发症。横纹肌复合体（SMC）是影响排便的最重要因素之一。先前的研究表明，不同程度的肌肉复杂发育不良依赖于ARM的复杂性。为了探讨ARM中SMC发育不良的机制，我们研究了ARM大鼠胚胎盆底肌发育过程中的细胞凋亡。方法：在胚胎第10天（E10），用乙烯硫脲处理妊娠大鼠，诱导大鼠胚胎肛肠畸形。对E16 ~ E21的正常和ARM大鼠胚胎进行横切面或矢状面连续切片，解剖SMCs并快速冷冻。采用TdT介导的dUTP Nick Ending Labeling （TUNEL）染色和DNA ladder分析鉴定细胞凋亡，免疫组织化学染色和逆转录-聚合酶链反应（RT-PCR）分析证实Bax/Bcl-2表达。结果：在ARM胚胎中，SMC发育不全和紊乱的吊索向头侧、腹侧移位，并在直肠尿道瘘管下方汇合，并浸润结缔组织。正常组e17可见tunel阳性细胞；散在阳性染色主要集中在SMC与球角骨肌交界处和双侧SMC会聚处直肠后2个区域。ARM组在e16 ~ E21可见大量核阳性染色，主要分布于SMC背侧。DNA样品电泳结果显示，正常组和ARM组从E17到E21呈阶梯状迁移，其中ARM组的阶梯更强。两组E17均可检测到Bax/Bcl-2的表达，E19和E21的免疫反应性增强。与正常组比较，ARM组细胞中Bax表达升高，Bcl-2表达降低。ARM胚胎中Bax信使RNA （mRNA）水平显著上调，Bcl-2 mRNA水平显著下调。结论：本研究在ARM胚胎SMC发育过程中发现了Bax/Bcl-2的异常凋亡和表达紊乱。提示细胞过早、过度和脱位凋亡可能是ARM大鼠SMC发育不良的根本发病机制。Bax/Bcl-2的时空表达表明它们可能在SMC细胞凋亡的调控中起重要作用。(C) 2009爱思唯尔公司版权所有。
Purpose: Fecal incontinence and constipation still remain as major postoperative complications after procedures for anorectal malformations (ARM). The striated muscle complex (SMC) is one of the most important factors that influence defecation. Previous studies have demonstrated different degrees of the muscle complex dysplasia dependent on the complexity of ARM. To explore the mechanisms of mal development of SMC in ARM, apoptosis was investigated during pelvic floor muscle development in rat embryos with ARM.Methods: Anorectal malformations in rat embryos were induced by treating pregnant rats with ethylenethiourea on the 10th embryonic day (E10). Normal and ARM rat embryos from E16 to E21 were serial-sectioned transversely or sagittally, and SMCs were dissected and snap frozen. TdT mediated dUTP Nick Ending Labeling (TUNEL) staining and DNA ladder analysis were performed to identify apoptosis and expression of Bax/Bcl-2 were confirmed with immunohistochemical staining and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis.Results: Hypoplastic and disordered SMC sling shifted cephalad, ventrally, and converged inferior to the rectourethral fistula and infiltrated connective tissue in ARM embryos. In the normal group, TUNEL-positive cells became evident on E 17; sporadic positive staining was mainly localized in 2 areas as follows: the junction area between SMC and bulbocarvernosus muscle and posterior to the rectum where bilateral SMC converged. In the ARM group, massive positive staining of nuclei was observed from E 16 to E21 and was mainly distributed in the dorsal part of the SMC. Electrophoresis of DNA samples yielded a "ladder" pattern of migration both in normal and the ARM group from E17 to E21, the ladders were stronger in the ARM group. In both groups, the expression of Bax/Bcl-2 was detectable on E17, the immunoreactivity increased on E19 and E21. Compared with the normal group, the expression of Bax was increased, whereas Bcl-2 was declined in the ARM group. Significant upregulation of Bax messenger RNA (mRNA) levels and downregulation of Bcl-2 mRNA levels were observed in ARM embryos.Conclusions: In the current study, abnormal apoptosis and disturbed expression of Bax/Bcl-2 were identified during SMC development in ARM embryos. It is suggested that precocious, excessive, and dislocated apoptosis might be a fundamental pathogenesis for the maldeveloped SMC in ARM rats. The temporospatial expressions of Bax/Bcl-2 indicate they may have an important role in the regulation of apoptosis of SMC. (C) 2009 Elsevier Inc. All rights reserved.