肛门直肠畸形大鼠盆底肌胚胎发育过程中细胞凋亡的研究

Apoptosis during the development of pelvic floor muscle in ARM rats

目的研究肛门直肠畸形鼠盆底肌胚胎发育过程巾的凋亡现象，探索凋亡存盆底肌异常发育中的可能致病作用。方法体重250-300g的Wistar大白鼠74只随机分组，雌雄午夜合笼后次晨雌鼠阴道涂片，显微镜下见到精子即确认已交配，此日计妊娠第0天。ETU组：52只大鼠于妊娠第10天经胃管注入125mg／kg的乙烯硫脲。正常对照组：22只大鼠于妊娠第10天经胃管注入等量的生理盐水。妊娠第16、17、19、21天刮宫取胎，经连续石蜡切片及提取盆底横纹肌复合体，应用TUNEL染色及DNA Ladder方法检测盆底肌的凋亡，免疫组织化学及RT-PCR方法分析Bcl-2／Bax在盆底肌中的表达。结果①正常对照组共孕雄性胎鼠108只，未见畸形发生。ETU致畸组共得雄性胎鼠247只，肛门直肠畸形173只，其中伴发脊柱裂或脊膜膨出21只（12．1％：21／173），3．5％（6／173）伴有肢体畸形（肢体缺如或马蹄内翻足）；②在正常组巾，孕17d后出现TUNEL阳性染色，散在分布于横纹肌复合体与球海绵体肌交界区及直肠后两侧横纹肌复合体汇合处；畸形组，孕16d已有TUNEL阳性染色，大量的阳性染色细胞主要分布在横纹肌复合体的背侧；两组DNA电泳时出现均出现DNA梯度，以畸形组明显；③在两组中，孕17d时开始出现Bax及Bcl-2的表达，孕19d及孕21d表达增强。与正常组相比，Bcl-2表达下调（分别为0．44±0．06比0．49±0．07，0．69±0．09比0.90±0．14，0.51±0．07比0．71±0．11），孕19d及21d时差异有统计学意义。而Bax基因在畸形组中表达上调（分别为0．39±0．05比0．31±0．05，0．47±0．1）6比0．37±0．1）6，0．40±0．05比0．37±0．05），孕17d及19d时差异有统计学意义。RT-PCR的结果提示畸形组Pax表达显著增强而Bcl2表达减弱，差异有统计学意义（P〈0．05）。结论肛门直肠畸形鼠盆底肌存在异常凋亡及Bax／gcl-2表达紊乱，过早、过度及异位的凋亡可能是肛门直肠畸形时盆底肌发育异常机制之一；Bax／Bcl-2在盆底肌中的时空表达，提示其在盆底肌凋亡过程中的重要调节作用。

Objective To investigate the role of apoptosis during the embryogenesis of pelvic floor muscle （PFM） in ARM rat. Methods ARM rat embryos were induced by injecting the E10 pregnant rats with ethylenethiourea （ETU）. Normal and ARM rat embryos from E16 to E21 were seriabseetioned transversely or sag ittally,and striated muscle compleres （SMCs） were dissected out and snap frozen. TUNEI. staining and DNA Ladder analysis were performed to identify the apoptosis, expression of Bax/Bcl-2 were confirmed with immunohistochemical staining and RTPCR analysis. Results Hypoplastic and dimrdered SMC sling shifted significantly cephalad, ventrally, and converged inferior to the rectourethral fistula, infiltrated with connective tissue in ARM embryos. In normal group, TUNEL positive cells became evident on E17, sporadic positive staining mainly localized in two areas, the junction area between SMC and M. bulbocarvernosus, and posterior to the rectum where bilateral SMC converged. In ARM group,massive positive staining nuclei were observed from E16 to E21, and were mainly distributed in the dorsal part of the SMC. Electrophoresis of DNA samples yielded a ＂ladder＂ pattern of nigration both in normal and in ARM group from E17 to E21 ; the ladders were stronger in ARM group. In both groups, the expressions of Bax/Bob2 were detectable on El 7, the immunoreactivity increased on E19 and E21. Compared with normal group, the expression of Bax were increased, while Bcl-2 were declined in ARM group. Significant up-regulation of Bax rnRNA levels and down-regulation of Bcl 2 mRNA levels were observed in ARM group. Conclusions In the current study, abnormal apoptosis and disturbed expression of Bax/Bcl 2 were identified during SMC development in ARM rats. It suggests that excessive and dislocated apoptosis might contribute to the malformation of SMC in ARM rats. The temporo spatial expressions of Bax/Bcl-2 indicated their important role in the regulation of apoptosis of SMC.