肛门直肠畸形泄殖腔凋亡及肠管Cajal间质细胞的研究
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摘要
肛门直肠畸形泄殖腔凋亡及肠管Cajal间质细胞的研究
目的
肛门直肠畸形(anorectal malformation，ARM)是小儿外科常见的先天性消化道畸形，发生率1／5000-1／1500，是世界卫生组织常规监测的先天畸形之一。其病理改变复杂，术后并发症多，严重影响患者的身心健康发展，是国内外研究者一直高度关注的先天畸形之一。
肛门直肠正常胚胎发育的研究已有百余年的历史，但它的具体发育演变规律仍存在许多争议。在研究中发现尿直肠隔的下降过程，泄殖腔构型的变化和肛膜的裂解均与凋亡密切相关。目前国内外对肛门直肠畸形胚胎发育过程中泄殖腔细胞凋亡的研究很少，这引起了我们的关注。我们选用乙烯硫脲(ethylenethioureal，ETU)致畸wistar大鼠产生肛门直肠畸形胎鼠，研究其胚胎发育的关键时期—胚胎13天到16天泄殖腔细胞凋亡的动态分布情况及控制细胞凋亡的二个重要基因bcl-2，bax在泄殖腔的表达情况。
20世纪80年代，随着后矢状入路肛门直肠成形术的应用，肛门直肠畸形术后便失禁的发生率明显降低，而便秘的发生率明显升高，甚至一些便秘患者需要多次手术治疗，严重影响患者的生活质量。近年来的研究显示Cajal间质细胞(interstitial cells of Cajal，ICC)是胃肠肌肉的起搏细胞，能产生生理性慢波，控制胃肠道的收缩和蠕动活动。一些胃肠动力疾病ICC发育异常，是否肛门直肠畸形也存在ICC发育异常，目前的研究很少。我们拟通过乙烯硫脲致畸动物模型研究胚胎末期ARM整个肠管ICC的发育情况并进一步利用ARM患者直肠末端标本来研究直肠末端ICC的表达情况，为ARM术后便秘的诊断、治疗提供一些理论依据。
方法
1、利用TUNEL染色技术检测正常胎鼠、泄殖腔畸形胎鼠胚胎13天-16天泄殖腔凋亡细胞的动态分布情况。
2、利用SABC染色方法研究正常胎鼠、泄殖腔畸形胎鼠胚胎13天-16天泄殖腔bcl-2，bax基因的表达情况。
3、利用SABC染色方法检测胚胎21天正常胎鼠、ARM胎鼠小肠、结肠和直肠ICC的发育情况。
4、利用RT-PCR方法研究28例ARM患者直肠末端c-kit基因和SCF基因的表达情况。
结果
1、肛门直肠畸形泄殖腔凋亡细胞的动态分布情况：
正常胎鼠胚胎13天生殖结节已形成，泄殖腔的轮廓清晰，在泌尿生殖窦和直肠间可见尿直肠隔，尾沟已形成。尿直肠隔上皮层和间质区域可见凋亡细胞。随着胚胎发育，生殖结节逐渐向腹尾侧延伸，尿直肠隔逐渐向下延伸并向腹尾侧迁移，尾沟逐渐加深，泄殖腔膜向背尾侧旋转。尿直肠隔间质的凋亡细胞逐渐增多并向下延伸，腹侧间质比背侧间质凋亡细胞明显；直肠背侧间质可见大量凋亡细胞。胚胎14天，直肠末端和未来肛门开口处的泄殖腔膜开始出现凋亡细胞，此期为细胞凋亡发挥作用的关键时期。胚胎15天，尿直肠隔与泄殖腔膜融合，尿直肠隔间质内的凋亡细胞一直向下延伸到融合部位。胚胎16天，肛膜裂解，直肠与外界相通。
泄殖腔发育畸形胎鼠与正常胎鼠相比，生殖结节的发育、尿直肠隔的下降和泄殖腔构型的变化均不同程度的延迟。尿直肠隔间质、直肠背侧间质和泄殖腔膜的凋亡细胞均明显减少。胚胎15天，尿直肠隔未与泄殖腔膜融合，直肠末端与泄殖腔膜的距离仍较远。胚胎16天，肛膜未裂解，产生不同类型的ARM畸形。
2、肛门直肠畸形泄殖腔bcl-2，bax基因的表达情况：
正常胎鼠胚胎13天尿直肠隔上皮层bax抗体染色阳性细胞占细胞总数的80％以上；尿直肠隔间质染色阳性细胞很少，不超过总数的10％。随着胚胎发育，上皮层阳性细胞逐渐减少；而间质，胚胎14天阳性细胞明显增多，在未来肛门开口的泄殖腔膜上皮层可见阳性细胞。胚胎15、16天间质内的阳性细胞较少。与正常胎鼠相比，泄殖腔畸形胎鼠胚胎13.5天、14天上皮层和间质区域bax抗体阳性细胞明显减少。
正常胎鼠bcl-2抗体染色阳性细胞在尿直肠隔上皮层胚胎13天最明显，后逐渐减少。而在尿直肠隔间质区域阳性细胞不超过10％，各期无明显变化。与正常胎鼠相比，泄殖腔畸形胎鼠胚胎13.5天尿直肠隔上皮层和间质区域bcl-2抗体阳性细胞比例明显增加。
3、肛门直肠畸形肠管Cajal间质细胞的表达情况：
小肠、结肠和直肠c-kit抗体阳性细胞主要分布于肌间神经丛，肌间可见少量阳性细胞。
正常胎鼠，胚胎21天小肠、结肠和直肠80％以上的肌间神经丛周围可见大量c-kit抗体阳性细胞；ARM畸形胎鼠除小肠80％以上肌间神经丛周围有大量阳性细胞外，结肠和直肠阳性细胞明显减少，仅50％或50％以下的肌间神经丛周围有阳性细胞。
4、肛门直肠畸形直肠末端c-kit和SCF的表达情况：
c-kit mRNA的表达在正常对照组、高位ARM组、中位ARM组、低位ARM组、后天性肛瘘组分别为1.79±0.22、0.67±0.19、0.89±0.17、1.57±0.25、1.68±0.18。与正常对照组相比较，高位ARM组、中位ARM组有意义(P＜0.05)；后天性肛瘘组、低位ARM组无意义(P＞0.05)。
SCF mRNA的表达在正常对照组、高位ARM组、中位ARM组、低位ARM组、后天性肛瘘组分别为1.72±0.19、0.65±0.16、0.98±0.19、1.61±0.24、1.69±0.14。与正常对照组相比较，高位、中位ARM组有意义(P＜0.05)；后天性肛瘘组、低位ARM组无意义(P＞0.05)。
结论
1、在肛门直肠的胚胎发育过程中，尿直肠隔的下降、融合过程、泄殖腔的构型变化和肛膜的裂解均与细胞凋亡密切相关。泄殖腔畸形胎鼠尿直肠隔间质、直肠背侧间质和泄殖腔膜的凋亡细胞明显减少，阻碍了泄殖腔的正常胚胎发育过程，可能是导致ARM产生的重要机制之一。
2、在泄殖腔发育过程中，尿直肠隔上皮细胞、间质细胞表达bcl-2、bax基因。并且这两种基因相互作用共同调节泄殖腔细胞的凋亡，保证泄殖腔的正常发育。而肛门直肠畸形胎鼠尿直肠隔上皮细胞、间质细胞bcl-2、bax基因表达异常，导致泄殖腔细胞凋亡的异常，是产生ARM畸形的原因之一。
3、ARM胎鼠结肠和直肠胚胎后期ICC的发育异常可能影响其生后完善的ICC网络的建立，导致胃肠动力功能紊乱，可能是ARM术后便秘的原因之一。
4、高、中位ARM患者直肠末端c-kit和SCF表达的减少是先天性的，并影响ICC的发育和功能，可能是术后便秘产生的分子生物学证据之一。
The study of Cloacal Apoptosis and Intestinal Interstitial Cells of Cajal in Anorectal Malformation
Objective
Anorectal malformation(ARM) are very common pediatric surgical disorders affecting 1 in 5,000 to 1 in 1,500 live births. The spectrum of these abnormalities is very complicate and the complications are very much which affect pediatric health.
Despite a long history of normal embryological research, the developmental processes of anorectum remain contentious. In embryological developments, apoptosis are deeply involved in the descending of urogenital septum(URS), the configuration transformation of cloaca and the rupture of cloacal membrane. Presently, the role of apoptosis in cloaca remains unclear in the embryological developments of ARM. In this study, Wistar rats were treated by ethylenethioureal(ETU) and gave birth to embryos with ARM. We studied the spatiotemporal distribution of cloacal apoptotic cells as well as the cloacal expression of apoptotic genes bcl-2 and bax in abnormal embryos of gestation day 13 to 16.
In 1980s, posterior sagital anorectoplasty provided an optimal access to reconstruct musclecomplex in ARM. The rate of faecal incontinence descended significantly, while the rate of constipation increased apparently. Some patients with severe constipation needed one or more operations, which affected their quality of life. Recently, interstitial cells of Cajal(ICC) were regarded as electrical pacemaker and mediated enteric neurotransmission. Abnormal development of ICC lain in some gastrointestinal functional diseases. The development of ICC in ARM are unclear. We studied the entire intestinal development of ICC in rat embryos with ARM and the expression of genes c-kit and SCF in the terminal rectum of patients with ARM.
Methods
1、To detect the spatiotemporal distribution of apoptosis in cloaca of normal and abnormal rat embryos from gestation day 13 to 16 with TUNEL staining.
2、To detect the spatiotemporal expression of gene bcl-2 and gene bax in cloaca of normal and abnormal rat embryos from gestation day 13 to 16 with SABC staining.
3、To detect the expression of ICC in intestine of normal and abnormal rat embryos on gestation day 21 with SABC staining.
4、To detect the expression of gene c-kit and gene SCF in terminal rectum of patients with ARM with RT-PCR.
Results
1、Spatiotemporal distribution of cloacal apoptotic cells in ARM On embryonic day(E) 13, the genital tubercle had developed and the cloaca appeared clearly. The URS divided cloaca into urogenital sinus and rectum. The tail groove had formed. There were apoptotic cells in the epithelial layers and mesenchyme region of URS. Following the development, genital tubercle growed gradually and shifted ventrocaudally. The tail groove deepened gradually and cloacal membrane rotated dorsalcaudally. Apoptotic cells increased and descended in the mesenchyme region of URS. Many apoptotic cells appeared in the dorsal mesenchyme of the rectum. On E14, apoptotic cells began to appear at the terminal rectum and the future anal orifice part of cloacal membrane. On E15, URS fused with cloacal membrane and apoptosis in URS descended to the fusion position. Anal membrane ruptured and rectum communicated with the outside on E16.
In embryos with abnormal cloaca, the development of genital tubercle, the descent of URS and the configuration transformation of cloaca dalayed. Apoptotic cells decreased apparently in URS, the dorsal part of rectum and the cloacal membrane. On E15, the fusion of URS and cloacal membrane did not appear. On E16, anal membrane did not rupture which resulted in ARM.
2、Spatiotemporal expression of gene bcl-2 and gene bax in the cloaca of ARM
On E13, over 80% of epithelial cells in URS were bax positive cells. Less than 10% of mesenchyme cells in URS were bax positive cells. Follwing the development, positive cells in epithelial cells decreased. But on E14, positive cells in mesenchyme increased significantly and positive cells appeared at the future anal orifice part of cloacal menbrane. On E15 and E16, positive cells of mesenchyme region of URS were few. Comparing with normal embryos, bax positive cells were fewer in the epithelial layers and mesenchyme region of URS in abnormal cloacal embryos on E13.5 and E14.
On E13, bcl-2 positive cells were more apparent in the epithelial layers of URS. Then they decreased gradually. Positive cells in mesenchyme region of URS were similar from E13 to E16, less than 10%. But in abnormal cloaca, bcl-2 positive cells were abundant in the epithelial layers and mesenchyme region of URS on E13.5.
3、The expression of ICC in the entire intestine of ARM
c-kit positive cells distributed mostly around the myenteric plexus region of the small intestine, colon and rectum. There were a few positive cells in the muscularis.
In normal embryos of E21,there were abundant c-kit positive cells around over 80% of the myeteric plexus in the small intestine, colon and rectum. In embryos with ARM, positive cells in small intestine were similar, but positive cells were fewer in colon and rectum compared with normal embryos.
4、The expression of c-kit and SCF in terminal rectum of patients with ARM
The expression level of c-kit mRNA in control group、high ARM、intermedia ARM、low ARM and acquired anal fistula were 1.79±0.22、0.67±0.19、0.89±0.17、1.57±0.25、1.68±0.18, respectively. There were significant difference of expression level in high and interrnadia ARM groups (P＜0.05) and no significant difference in low ARM and acquired anal fistula groups (P＞0.05) compared with control group. The expression level of SCF mRNA in control group、high ARM、intermedia ARM、low ARM and acquired anal fistula were 1.72±0.19、0.65±0.16、0.98±0.19、1.61±0.24、1.69±0.14, respectively. There were significant difference of expression level in high and intermadia ARM groups (P＜0.05 ) and no significant difference in low ARM and acquired anal fistula groups (P＞0.05) compared with control group.
Conclusions
1、In anorectal embryological developments, apoptosis were deeply involved in descend and fusion of URS, the configuration transformation of cloaca and the rupture of cloacal membrane. Apoptotic cells decreased apparently in URS, the dorsal part of rectum and the cloacal membrane with ARM embryos. These hampered the normal anorectal embryological development and might result in ARM.
2、The gene bcl-2 and gene bax were involved in cloacal apoptosis. The abnormal expression of gene bcl-2 and gene bax in the epithelial layers and mesenchyme region of URS in malformation embryos might result in abnormal apoptosis.
3、On E21, abnormal development of ICC in the colon and rectum with ARM embryos might affect the building of neonatal ICC net. This might be one of the reasons resulting in post-operative constipation.
4、The expression level of c-kit mRNA and SCF mRNA decreased in high and intermadia ARM groups. These might be congenital and damage the development and functions of ICC. This phenomenon might be one of the biomolecular evidences which resulted in post-operative constipation.
目的
肛门直肠畸形(anorectal malformation，ARM)是小儿外科常见的先天性消化道畸形，发生率1／5000-1／1500，是世界卫生组织常规监测的先天畸形之一。其病理改变复杂，术后并发症多，严重影响患者的身心健康发展，是国内外研究者一直高度关注的先天畸形之一。
肛门直肠正常胚胎发育的研究已有百余年的历史，但它的具体发育演变规律仍存在许多争议。在研究中发现尿直肠隔的下降过程，泄殖腔构型的变化和肛膜的裂解均与凋亡密切相关。目前国内外对肛门直肠畸形胚胎发育过程中泄殖腔细胞凋亡的研究很少，这引起了我们的关注。我们选用乙烯硫脲(ethylenethioureal，ETU)致畸wistar大鼠产生肛门直肠畸形胎鼠，研究其胚胎发育的关键时期—胚胎13天到16天泄殖腔细胞凋亡的动态分布情况及控制细胞凋亡的二个重要基因bcl-2，bax在泄殖腔的表达情况。
20世纪80年代，随着后矢状入路肛门直肠成形术的应用，肛门直肠畸形术后便失禁的发生率明显降低，而便秘的发生率明显升高，甚至一些便秘患者需要多次手术治疗，严重影响患者的生活质量。近年来的研究显示Cajal间质细胞(interstitial cells of Cajal，ICC)是胃肠肌肉的起搏细胞，能产生生理性慢波，控制胃肠道的收缩和蠕动活动。一些胃肠动力疾病ICC发育异常，是否肛门直肠畸形也存在ICC发育异常，目前的研究很少。我们拟通过乙烯硫脲致畸动物模型研究胚胎末期ARM整个肠管ICC的发育情况并进一步利用ARM患者直肠末端标本来研究直肠末端ICC的表达情况，为ARM术后便秘的诊断、治疗提供一些理论依据。
方法
1、利用TUNEL染色技术检测正常胎鼠、泄殖腔畸形胎鼠胚胎13天-16天泄殖腔凋亡细胞的动态分布情况。
2、利用SABC染色方法研究正常胎鼠、泄殖腔畸形胎鼠胚胎13天-16天泄殖腔bcl-2，bax基因的表达情况。
3、利用SABC染色方法检测胚胎21天正常胎鼠、ARM胎鼠小肠、结肠和直肠ICC的发育情况。
4、利用RT-PCR方法研究28例ARM患者直肠末端c-kit基因和SCF基因的表达情况。
结果
1、肛门直肠畸形泄殖腔凋亡细胞的动态分布情况：
正常胎鼠胚胎13天生殖结节已形成，泄殖腔的轮廓清晰，在泌尿生殖窦和直肠间可见尿直肠隔，尾沟已形成。尿直肠隔上皮层和间质区域可见凋亡细胞。随着胚胎发育，生殖结节逐渐向腹尾侧延伸，尿直肠隔逐渐向下延伸并向腹尾侧迁移，尾沟逐渐加深，泄殖腔膜向背尾侧旋转。尿直肠隔间质的凋亡细胞逐渐增多并向下延伸，腹侧间质比背侧间质凋亡细胞明显；直肠背侧间质可见大量凋亡细胞。胚胎14天，直肠末端和未来肛门开口处的泄殖腔膜开始出现凋亡细胞，此期为细胞凋亡发挥作用的关键时期。胚胎15天，尿直肠隔与泄殖腔膜融合，尿直肠隔间质内的凋亡细胞一直向下延伸到融合部位。胚胎16天，肛膜裂解，直肠与外界相通。
泄殖腔发育畸形胎鼠与正常胎鼠相比，生殖结节的发育、尿直肠隔的下降和泄殖腔构型的变化均不同程度的延迟。尿直肠隔间质、直肠背侧间质和泄殖腔膜的凋亡细胞均明显减少。胚胎15天，尿直肠隔未与泄殖腔膜融合，直肠末端与泄殖腔膜的距离仍较远。胚胎16天，肛膜未裂解，产生不同类型的ARM畸形。
2、肛门直肠畸形泄殖腔bcl-2，bax基因的表达情况：
正常胎鼠胚胎13天尿直肠隔上皮层bax抗体染色阳性细胞占细胞总数的80％以上；尿直肠隔间质染色阳性细胞很少，不超过总数的10％。随着胚胎发育，上皮层阳性细胞逐渐减少；而间质，胚胎14天阳性细胞明显增多，在未来肛门开口的泄殖腔膜上皮层可见阳性细胞。胚胎15、16天间质内的阳性细胞较少。与正常胎鼠相比，泄殖腔畸形胎鼠胚胎13.5天、14天上皮层和间质区域bax抗体阳性细胞明显减少。
正常胎鼠bcl-2抗体染色阳性细胞在尿直肠隔上皮层胚胎13天最明显，后逐渐减少。而在尿直肠隔间质区域阳性细胞不超过10％，各期无明显变化。与正常胎鼠相比，泄殖腔畸形胎鼠胚胎13.5天尿直肠隔上皮层和间质区域bcl-2抗体阳性细胞比例明显增加。
3、肛门直肠畸形肠管Cajal间质细胞的表达情况：
小肠、结肠和直肠c-kit抗体阳性细胞主要分布于肌间神经丛，肌间可见少量阳性细胞。
正常胎鼠，胚胎21天小肠、结肠和直肠80％以上的肌间神经丛周围可见大量c-kit抗体阳性细胞；ARM畸形胎鼠除小肠80％以上肌间神经丛周围有大量阳性细胞外，结肠和直肠阳性细胞明显减少，仅50％或50％以下的肌间神经丛周围有阳性细胞。
4、肛门直肠畸形直肠末端c-kit和SCF的表达情况：
c-kit mRNA的表达在正常对照组、高位ARM组、中位ARM组、低位ARM组、后天性肛瘘组分别为1.79±0.22、0.67±0.19、0.89±0.17、1.57±0.25、1.68±0.18。与正常对照组相比较，高位ARM组、中位ARM组有意义(P＜0.05)；后天性肛瘘组、低位ARM组无意义(P＞0.05)。
SCF mRNA的表达在正常对照组、高位ARM组、中位ARM组、低位ARM组、后天性肛瘘组分别为1.72±0.19、0.65±0.16、0.98±0.19、1.61±0.24、1.69±0.14。与正常对照组相比较，高位、中位ARM组有意义(P＜0.05)；后天性肛瘘组、低位ARM组无意义(P＞0.05)。
结论
1、在肛门直肠的胚胎发育过程中，尿直肠隔的下降、融合过程、泄殖腔的构型变化和肛膜的裂解均与细胞凋亡密切相关。泄殖腔畸形胎鼠尿直肠隔间质、直肠背侧间质和泄殖腔膜的凋亡细胞明显减少，阻碍了泄殖腔的正常胚胎发育过程，可能是导致ARM产生的重要机制之一。
2、在泄殖腔发育过程中，尿直肠隔上皮细胞、间质细胞表达bcl-2、bax基因。并且这两种基因相互作用共同调节泄殖腔细胞的凋亡，保证泄殖腔的正常发育。而肛门直肠畸形胎鼠尿直肠隔上皮细胞、间质细胞bcl-2、bax基因表达异常，导致泄殖腔细胞凋亡的异常，是产生ARM畸形的原因之一。
3、ARM胎鼠结肠和直肠胚胎后期ICC的发育异常可能影响其生后完善的ICC网络的建立，导致胃肠动力功能紊乱，可能是ARM术后便秘的原因之一。
4、高、中位ARM患者直肠末端c-kit和SCF表达的减少是先天性的，并影响ICC的发育和功能，可能是术后便秘产生的分子生物学证据之一。
The study of Cloacal Apoptosis and Intestinal Interstitial Cells of Cajal in Anorectal Malformation
Objective
Anorectal malformation(ARM) are very common pediatric surgical disorders affecting 1 in 5,000 to 1 in 1,500 live births. The spectrum of these abnormalities is very complicate and the complications are very much which affect pediatric health.
Despite a long history of normal embryological research, the developmental processes of anorectum remain contentious. In embryological developments, apoptosis are deeply involved in the descending of urogenital septum(URS), the configuration transformation of cloaca and the rupture of cloacal membrane. Presently, the role of apoptosis in cloaca remains unclear in the embryological developments of ARM. In this study, Wistar rats were treated by ethylenethioureal(ETU) and gave birth to embryos with ARM. We studied the spatiotemporal distribution of cloacal apoptotic cells as well as the cloacal expression of apoptotic genes bcl-2 and bax in abnormal embryos of gestation day 13 to 16.
In 1980s, posterior sagital anorectoplasty provided an optimal access to reconstruct musclecomplex in ARM. The rate of faecal incontinence descended significantly, while the rate of constipation increased apparently. Some patients with severe constipation needed one or more operations, which affected their quality of life. Recently, interstitial cells of Cajal(ICC) were regarded as electrical pacemaker and mediated enteric neurotransmission. Abnormal development of ICC lain in some gastrointestinal functional diseases. The development of ICC in ARM are unclear. We studied the entire intestinal development of ICC in rat embryos with ARM and the expression of genes c-kit and SCF in the terminal rectum of patients with ARM.
Methods
1、To detect the spatiotemporal distribution of apoptosis in cloaca of normal and abnormal rat embryos from gestation day 13 to 16 with TUNEL staining.
2、To detect the spatiotemporal expression of gene bcl-2 and gene bax in cloaca of normal and abnormal rat embryos from gestation day 13 to 16 with SABC staining.
3、To detect the expression of ICC in intestine of normal and abnormal rat embryos on gestation day 21 with SABC staining.
4、To detect the expression of gene c-kit and gene SCF in terminal rectum of patients with ARM with RT-PCR.
Results
1、Spatiotemporal distribution of cloacal apoptotic cells in ARM On embryonic day(E) 13, the genital tubercle had developed and the cloaca appeared clearly. The URS divided cloaca into urogenital sinus and rectum. The tail groove had formed. There were apoptotic cells in the epithelial layers and mesenchyme region of URS. Following the development, genital tubercle growed gradually and shifted ventrocaudally. The tail groove deepened gradually and cloacal membrane rotated dorsalcaudally. Apoptotic cells increased and descended in the mesenchyme region of URS. Many apoptotic cells appeared in the dorsal mesenchyme of the rectum. On E14, apoptotic cells began to appear at the terminal rectum and the future anal orifice part of cloacal membrane. On E15, URS fused with cloacal membrane and apoptosis in URS descended to the fusion position. Anal membrane ruptured and rectum communicated with the outside on E16.
In embryos with abnormal cloaca, the development of genital tubercle, the descent of URS and the configuration transformation of cloaca dalayed. Apoptotic cells decreased apparently in URS, the dorsal part of rectum and the cloacal membrane. On E15, the fusion of URS and cloacal membrane did not appear. On E16, anal membrane did not rupture which resulted in ARM.
2、Spatiotemporal expression of gene bcl-2 and gene bax in the cloaca of ARM
On E13, over 80% of epithelial cells in URS were bax positive cells. Less than 10% of mesenchyme cells in URS were bax positive cells. Follwing the development, positive cells in epithelial cells decreased. But on E14, positive cells in mesenchyme increased significantly and positive cells appeared at the future anal orifice part of cloacal menbrane. On E15 and E16, positive cells of mesenchyme region of URS were few. Comparing with normal embryos, bax positive cells were fewer in the epithelial layers and mesenchyme region of URS in abnormal cloacal embryos on E13.5 and E14.
On E13, bcl-2 positive cells were more apparent in the epithelial layers of URS. Then they decreased gradually. Positive cells in mesenchyme region of URS were similar from E13 to E16, less than 10%. But in abnormal cloaca, bcl-2 positive cells were abundant in the epithelial layers and mesenchyme region of URS on E13.5.
3、The expression of ICC in the entire intestine of ARM
c-kit positive cells distributed mostly around the myenteric plexus region of the small intestine, colon and rectum. There were a few positive cells in the muscularis.
In normal embryos of E21,there were abundant c-kit positive cells around over 80% of the myeteric plexus in the small intestine, colon and rectum. In embryos with ARM, positive cells in small intestine were similar, but positive cells were fewer in colon and rectum compared with normal embryos.
4、The expression of c-kit and SCF in terminal rectum of patients with ARM
The expression level of c-kit mRNA in control group、high ARM、intermedia ARM、low ARM and acquired anal fistula were 1.79±0.22、0.67±0.19、0.89±0.17、1.57±0.25、1.68±0.18, respectively. There were significant difference of expression level in high and interrnadia ARM groups (P＜0.05) and no significant difference in low ARM and acquired anal fistula groups (P＞0.05) compared with control group. The expression level of SCF mRNA in control group、high ARM、intermedia ARM、low ARM and acquired anal fistula were 1.72±0.19、0.65±0.16、0.98±0.19、1.61±0.24、1.69±0.14, respectively. There were significant difference of expression level in high and intermadia ARM groups (P＜0.05 ) and no significant difference in low ARM and acquired anal fistula groups (P＞0.05) compared with control group.
Conclusions
1、In anorectal embryological developments, apoptosis were deeply involved in descend and fusion of URS, the configuration transformation of cloaca and the rupture of cloacal membrane. Apoptotic cells decreased apparently in URS, the dorsal part of rectum and the cloacal membrane with ARM embryos. These hampered the normal anorectal embryological development and might result in ARM.
2、The gene bcl-2 and gene bax were involved in cloacal apoptosis. The abnormal expression of gene bcl-2 and gene bax in the epithelial layers and mesenchyme region of URS in malformation embryos might result in abnormal apoptosis.
3、On E21, abnormal development of ICC in the colon and rectum with ARM embryos might affect the building of neonatal ICC net. This might be one of the reasons resulting in post-operative constipation.
4、The expression level of c-kit mRNA and SCF mRNA decreased in high and intermadia ARM groups. These might be congenital and damage the development and functions of ICC. This phenomenon might be one of the biomolecular evidences which resulted in post-operative constipation.
引文
1 Sasaki C, Yamaguchi K, Akita K, et al. Spatiotemporal distribution of apoptosis during normal cloacal development in mice. Anat Rec A Discov Mol Cell Evol Biol. 2004; 279:761-767.
2 Qi BQ, Beasley SW, Williams AK, et al. Apoptosis during regression of the tailgut and septation of the cloaca. J Pediatric Surg. 2000; 35:1556 - 1561.
3 Van der Putte SC. Normal and abnormal development of the anorectum. J Pediatr Surg. 1986; 21:434-440.
4 Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development. J Pediatr Surg. 1995; 30:1143-1147.
5 Nievelstein RAJ, van der Werff JFA, Verbeek FJ, et al. Normal and abnormal embryonic development of the anorectum in human embryos. Teratology. 1998; 57:70-78.
6 Paidas CN, Morreale RF, Holoski KH, et al. Septation and differentiation of the embryonic human cloaca. J Pediatr Surg. 1999; 34:877 - 884.
7 Qi BQ, Beasley SW, Williams AK, et al. Does the urorectal septum fuse with the cloacal membrane? J Urol. 2000; 164:2070 - 2072.
8 Qi BQ, Williams A, Beasley S, et al. Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo. J Pediatr Surg. 2000; 35:1810 - 1816.
9 Bai Y, Chen H, Yuan ZW, et al. Normal and abnormal embryonic development of the anorectum in rats. J Pediatr Surg. 2004; 39:587-90.
10 Haraguchi R, Mo R, Hui C, et al. Unique functions of Sonic hedgehog signaling during external genitalia development. Development. 2001; 128:4241 - 4250.
11 Kimmel SG, Mo R, Hui C-C, et al. New mouse models of congenital anorectal malformations. J Pediatr Surg. 2000; 35:227-231.
12 Sanders EJ, Khare MK, Ooi VC, et al. An experimental and morphological analysis of the tail bud mesenchyme of the chick embryo. Anat Embryol. 1986; 174:179 - 185.
13 Kubota Y, Shimotake T, Yanagihara J, et al. Development of anorectal malformations using etretinate. J Pediatr Surg. 1998; 21:434-440.
14 Penington EC, Hutson JM. The absence of lateral fusion in cloacal partition J Pediatr Surg. 2003; 38:1287-1295.
15 Penington EC, Hutson JM. The urethral plate-does it grow into the genital tubercle or within it? BJU Int. 2002; 89,:733-739.
16 Penington EC, Hutson JM. The cloacal plate: the missing link in anorectal and urogenital development. BJU Int. 2002; 89,726-32.
17 Qi BQ, Beasley SW, Frizelle FA.Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus. J Pediatr Surg. 2002; 37:1305-12.
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19 Langemeijer RATM, Molenaar JC. Continence after poserior sagittal anorectoplasty. J Pediatr Surg. 1991; 26: 587-590.
20 Meier-Ruge WA, Holschneider AM. Histopathologic observations of anorectal abnormalities in anal atresia. Pediatr Surg Int. 2000; 16: 2-7.
21 沈丽萍。小儿肛门直肠畸形术后排便障碍的原因及治疗。临床外科杂志．2002；10：56—58．
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36 Guarino N, Yoneda A, Shima H,et al. Selective neurotrophin deficiency in infantile hypertrophic stenosis. J Pediarr Surg. 2001; 36: 1280-1284.
2 Qi BQ, Beasley SW, Williams AK, et al. Apoptosis during regression of the tailgut and septation of the cloaca. J Pediatric Surg. 2000; 35:1556 - 1561.
3 Van der Putte SC. Normal and abnormal development of the anorectum. J Pediatr Surg. 1986; 21:434-440.
4 Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development. J Pediatr Surg. 1995; 30:1143-1147.
5 Nievelstein RAJ, van der Werff JFA, Verbeek FJ, et al. Normal and abnormal embryonic development of the anorectum in human embryos. Teratology. 1998; 57:70-78.
6 Paidas CN, Morreale RF, Holoski KH, et al. Septation and differentiation of the embryonic human cloaca. J Pediatr Surg. 1999; 34:877 - 884.
7 Qi BQ, Beasley SW, Williams AK, et al. Does the urorectal septum fuse with the cloacal membrane? J Urol. 2000; 164:2070 - 2072.
8 Qi BQ, Williams A, Beasley S, et al. Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo. J Pediatr Surg. 2000; 35:1810 - 1816.
9 Bai Y, Chen H, Yuan ZW, et al. Normal and abnormal embryonic development of the anorectum in rats. J Pediatr Surg. 2004; 39:587-90.
10 Haraguchi R, Mo R, Hui C, et al. Unique functions of Sonic hedgehog signaling during external genitalia development. Development. 2001; 128:4241 - 4250.
11 Kimmel SG, Mo R, Hui C-C, et al. New mouse models of congenital anorectal malformations. J Pediatr Surg. 2000; 35:227-231.
12 Sanders EJ, Khare MK, Ooi VC, et al. An experimental and morphological analysis of the tail bud mesenchyme of the chick embryo. Anat Embryol. 1986; 174:179 - 185.
13 Kubota Y, Shimotake T, Yanagihara J, et al. Development of anorectal malformations using etretinate. J Pediatr Surg. 1998; 21:434-440.
14 Penington EC, Hutson JM. The absence of lateral fusion in cloacal partition J Pediatr Surg. 2003; 38:1287-1295.
15 Penington EC, Hutson JM. The urethral plate-does it grow into the genital tubercle or within it? BJU Int. 2002; 89,:733-739.
16 Penington EC, Hutson JM. The cloacal plate: the missing link in anorectal and urogenital development. BJU Int. 2002; 89,726-32.
17 Qi BQ, Beasley SW, Frizelle FA.Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus. J Pediatr Surg. 2002; 37:1305-12.
18 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNAfragmentation. J Cell Biol. 1992; 119:493 - 501.
19 Mori C, Nakamura N, Kimura S, et al. Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation. Anat Rec. 1995; 242:103 - 110.
20 Miller SA, Briglin A. Apoptosis removes chick tail gut and remnant of the primitive streak. Dev Dynamic. 1996; 206:212 - 218.
21 Merei J, Farmer P, Hasthorpe S, et al. Timing and embryology of esophageal atresia and tracheo-oesophageal fistula. Anat Record. 1997; 249: 240-248.
22 Steller H. Mechanisms and genes of cellular suicide. Science. 1995; 267:1445 - 1449.
23 Diez-Pardo JA, Qi BQ, Navarro C, et al. A new rodent experimental model of oesophageal atresia and tracheo-oesophageal fistula: Preliminaryreport. J Pediatr Surg. 1996; 31:498-502.
24 Kayaba H, Hebiguchi T, Yoshino H, et al. Evaluation of anorectal functions of children with anorectal malformations using fecoflowmetry. J Pediatr Surg. 2002; 37:623-628.
25 Cho S, Moore SP, Fangman T. One hundred three cousecutive patients with anorectal malformations and their associated anomalies. Arch Pediatr Adolesc Med. 2001; 155:587-591.
26 Tsuji H, Okada A, Nakai H, et al. Follow-up studies of anorectal malformations after posterior sagittal anorectoplasty. J Pediatr Surg. 2002; 37:1529-1533.
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