Chen Qing Jiang, Jia Hui Min, Zhang Shi Wei, Zhang Shu Cheng, Bai Yu Zuo, Yuan Zheng Wei, Wang Wei Lin
Department of Pediatric Surgery, Shengjing Hospital, China Medical University, Shenyang 110004, PR China.
J Pediatr Surg. 2009 Oct;44(10):1884-91. doi: 10.1016/j.jpedsurg.2009.02.004.
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 maldevelopment of SMC in ARM, apoptosis was investigated during pelvic floor muscle development in rat embryos with ARM.
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.
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 E17; 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 E16 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.
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.
大便失禁和便秘仍是肛门直肠畸形（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，根据ARM的复杂程度，肌肉复合体发育异常的程度不同。为了探究ARM中SMC发育异常的机制，我们研究了ARM大鼠胚胎盆底肌肉发育过程中的细胞凋亡情况。
在胚胎第10天（E10）用乙硫脲处理孕鼠，诱导大鼠胚胎发生肛门直肠畸形。将E16至E21的正常和ARM大鼠胚胎进行横向或矢状连续切片，解剖出SMC并速冻。进行TdT介导的dUTP缺口末端标记（TUNEL）染色和DNA梯状分析以鉴定细胞凋亡，并通过免疫组织化学染色和逆转录-聚合酶链反应（RT-PCR）分析确认Bax/Bcl-2的表达。
ARM胚胎中，发育不全且紊乱的SMC吊带向头侧、腹侧移位，在直肠尿道瘘下方汇合并浸润结缔组织。在正常组中，TUNEL阳性细胞在E17时变得明显；散在的阳性染色主要位于以下两个区域：SMC与球海绵体肌之间的交界区域以及双侧SMC汇合处直肠后方。在ARM组中，从E16至E21观察到细胞核大量阳性染色，主要分布在SMC的背侧部分。从E17至E21，正常组和ARM组的DNA样本电泳均产生“梯状”迁移模式，ARM组的梯状条带更强。在两组中，E17时均可检测到Bax/Bcl-2的表达，E19和E21时免疫反应性增加。与正常组相比，ARM组中Bax的表达增加，而Bcl-2的表达下降。在ARM胚胎中观察到Bax信使核糖核酸（mRNA）水平显著上调，Bcl-2 mRNA水平下调。
在本研究中，在ARM胚胎的SMC发育过程中鉴定出异常凋亡和Bax/Bcl-2表达紊乱。提示早熟、过度和错位的凋亡可能是ARM大鼠中SMC发育异常的根本发病机制。Bax/Bcl-2的时空表达表明它们可能在SMC凋亡的调节中起重要作用。
