先天性巨结肠RET基因和PHOX2B基因多态性和肠神经元发育不良病理特点及与EC关系研究
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摘要
第一部分先天性巨结肠RET基因和PHOX2B基因多态性研究
研究背景
先天性巨结肠(Hirschsprung's disease，HD)，是小儿外科常见疾病，在高加索人，非洲黑人、亚洲黄种人的发病率分别为万分之1.5、2.1、2.8，亚洲人种发病率最高。HD是一种神经嵴源性疾病，当胚胎发育过程中神经嵴细胞迁徙、分化发生停顿或发生细胞凋亡，均会导致远端肠管的神经支配异常而导致HD。近年来通过遗传连锁分析和基因敲除等技术，发现有以下基因的突变与HD的发病有关：编码有关酪氨酸激酶通路基因(包括RET原癌基因，GDNF基因，GFRα-1基因等)，编码血管内皮素信号通路的基因(包括EDNRB基因，EDN3基因和ECE-1基因)，编码转录因子的基因(如SOX10基因和PHOX2B基因)等。其中RET基因表达对肠神经元细胞的发育十分关键，在HD的发病中最为重要。
但对HD进行大量RET基因突变研究发现，仅有20％左右的散发性和50％左右家族性HD存在突变，而且缺乏类似肿瘤相关的热点突变，另外HD相关RET突变有不完全外显(incomplete penetrance)，呈突变效应性别相关性。更为重要的是，在家族性病例，遗传连锁分析定位遗传位点在10q11.2，却仍缺乏RET编码区突变。随着SNPs疾病相关性研究的进展，对HD的RET基因研究重点逐渐转移到多态性的研究上。尚未证实单个SNPs导致HD相关RET功能改变，但可能有精确的SNPs组合(单倍型)决定调节意义。在高加索人种，发现有数个RET编码区SNPs与散发性HD相关：A45A(c135G＞(?)；exon 2)和L769L(c2307T＞(?)；exon 13)。对RET基因启动子的研究发现，在RET编码序列上游的启动子的两种多态性-5G＞A和-A＞C与HD相关。目前对RET基因的研究，均局限于编码区的突变及SNPs研究，虽然也有少量非编码区(内含子和启动子)的研究，但缺乏相应的功能研究。
PHOX2B基因表达研究证明，PHOX2B编码的同源域蛋白与多种非肾上腺素能神经元的发育有关，因此推断PHOX2B基因的改变可能与自主神经发育异常性疾病相关。在小鼠胚胎学研究发现，一旦成肠细胞进入到前肠间叶，即有PHOX2B蛋白表达，并贯穿肠神经元发育的始终。PHOX2B基因突变纯合子小鼠全胃肠道缺乏神经元，与HD病变相似，同时，该突变的小鼠无RET蛋白表达，因此推测PHOX2B基因可能与HD有关。Garcia-Barcelo通过对91例单纯HD研究，发现PHOX2B 1364 A＞G多态性与HD有关。目前对PHOX2B基因与临床疾病的关系研究尚处在起步阶段，PHOX2B基因与HD的研究也仅有个别报道，PHOX2B基因是否参与HD的发病机制仍有待进一步的研究。
根据Hapmap组织统计数据，不同种族人群的SNPs分布频率有明显差异，RET基因和PHOX2B基因SNPs也是如此，这可能是不同种族人群HD发病率有明显差别的原因之一。本课题拟通过对浙江汉族人群HD患者和正常人群的对照研究，分析RET基因和PHOX2B基因SNPs与HD发病之间的关系，不同类型HD与基因SNPs关系，进一步推断主要的HD相关单倍型，并对照其他种族人群已有数据，分析浙江汉族人群的SNPs分布特点。同时应用免疫组化法，检测RET基因启动子多态性与RET蛋白表达之间的关系。
材料和方法
1．病例收集及标准
(1)病例组：全部病例均为本院的浙江籍先天性巨结肠患儿，共123例。根据2005年第四届国际巨结肠与相关神经嵴源性疾病会议的巨结肠诊断标准，重新阅片确定。
(2)分组及标准：将巨结肠分为短段型与长段型两种。
短段型HD：病变仅局限于乙状结肠远端，乙状结肠直肠交界及直肠。
长段型HD：病变范围包括乙状结肠近端，升结肠及累及范围更广者。
(3)正常对照组：全部正常对照组均来自本院正常体检小儿，排除消化道和神经嵴相关畸形，共194例。
全部病例及对照组抽取外周血2ml，3.8％枸橼酸钠抗凝。
本研究经浙江大学医学院附属儿童医院伦理委员会批准，全部患儿及对照组均获得家长知情同意。
2．PCR反应及测序
对全部样本进行DNA提取后，根据选取的SNPs，合成引物，PCR扩增，经2％琼脂糖电泳证实PCR产物后，将PCR产物进一步纯化、测序。
3．免疫组化
根据RET启动子多态性，分别选取相应患儿病理组织切片，观察神经元，确定含正常神经元蜡块，再行C-Ret免疫组化染色，将该切片镜下所有神经元按免疫组化反应等级均予计数。
4．统计方法
(1)用Hardy-Weinberg平衡定律来检验样本人群中基因频率与基因型频率是否平衡。
(2)基因和疾病的相关性分析：使用x2方检验(Chi squared)，相关性以比值比(odds ratios，ORs)及其95％可信区间(confidence intervals，CIs)表示，以P值＜0.05确定为统计学差异有显著性。
(3)单倍型频率用PHASE软件计算。
(4)免疫组化结果用非参数统计中的两样本等级和检验(Mann-Whitney Test)。
结果
1．各位点基因型与HD风险
RET基因-5G＞A，-1A＞C，c135G＞A，c2307T＞G四个位点的少见基因型AA，CC，AA，GG在HD组频率明显高于对照组(P＜0.05)。
PHOX2B基因1364A＞G两组间未发现有基因型差异。
2．各位点等位基因频率与HD风险
RET基因-5A，-1C，c135A，c2307G在HD组等位基因频率明显高于对照组(P＜0.05)。
PHOX2B基因1364A＞G等位基因分布未发现显著性事件。
3．各位点等位基因频率与HD类型关系
RET基因各位点均未发现等位基因频率在不同类型间的差异。
4．单倍型与HD
RET基因启动子单倍型-5A／-1C占HD组82.5％，较对照组差异有显著性(P＜0.05)。
RET基因四位点(-5，-1，c135，c2307)单倍型ACAG占HD组75.2％，较对照组差异有显著性(P＜0.05)。
5．不同种族人群之间各等位基因频率
RET基因-5A、c135A和c2307G等位基因频率明显高于高加索和非洲人种(P＜0.05)
6．RET基因启动子多态性与RET蛋白表达关系
RET基因启动子基因型-5AA／-1CC较其他基因型RET蛋白表达降低(P＜0.05)。
启动子单倍型-5A／-1C纯合子较-5A／-1C杂和子RET蛋白表达降低(P＜0.05)。
结论
1．RET基因-5G＞A，-1A＞C，c135G＞A，c2307T＞G四个位点多态性与浙江汉族人群HD明显相关，但与HD类型无明显相关。
2．RET基因-5G＞A，-1A＞C，c135G＞A，c2307T＞G四个位点单倍型ACAG是浙江汉族人群的HD相关核心单倍型。
3．浙江汉族人群的RET基因-5、c135和c2307位点的少见等位基因频率较其他人种有明显增高。
4．RET启动子多态性对启动子功能有影响，与RET蛋白表达相关。
5．未发现PHOX2B基因多态性与浙江汉族人群HD相关。
第二部分肠神经元发育不良病理特点及与EC关系研究
研究背景
近年来逐渐认识到一类临床表现与HD十分相似，但病理改变与HD明显不同的疾病，称之为巨结肠同源病(allied disorders of Hirschsprung's disease；ADHD)，主要包括肠神经元发育不良(Intestinal neuronal dysplasia；IND)、神经节细胞减少症(Hypoganglionosis)、神经节细胞未成熟症(Immaturity of ganglion cells)等。1994年Holschneider和Meier-Ruge将HD和ADHD两者统称为肠神经元异常(Neuronal intestinal malformations；NIM)，并进行了详细的病理分类。在ADHD中以IND最为常见，其病理特点主要有以下4项：①粘膜下和肌间神经丛增生，形成巨大神经节(giant ganglia)，粘膜下丛节细胞数目≥7个(正常为3～5个)，肌间丛面积≥正常的3倍，常伴有神经元不成熟现象；②粘膜固有层和环肌副交感神经纤维乙酰胆碱酯酶(Acetylcholinesterase，Ache)活性增高：③粘膜固有层和粘膜肌层分散的平滑肌纤维之间有孤立的神经节细胞；④肌间神经丛交感神经发育不全或无发育。
混合型IND的临床表现几乎与HD一样，单纯型IND的发病主要集中在年幼儿童，而在新生儿期较少表现。临床表现为渐进性的便秘，腹胀或有顽固性／发作性的吸收异常。部分患儿经保守治疗或随年龄增长能逐渐好转，部分患儿则呈进行性加重，需手术治疗。
针对HD的辅助检查中，包括钡剂灌肠、直肠肛管测压，IND常缺乏特征性的改变而无法准确应用。目前国内对1ND的认识仍不足，一般根据临床表现或结合钡剂灌肠检查就予诊断并进一步手术治疗，随着经肛门手术的广泛开展和手术年龄提前至新生儿和婴幼儿期，单凭临床表现和钡灌肠可误将部分单纯型IND诊断为HD而手术治疗。
虽然IND作为一种明确的病理改变已经得到广泛认同，对IND是否作为一种独立的疾病一直存在争议，分歧的焦点在于IND是一种原发性疾病还是继发性改变，两者都有相应的证据支持。IND的诊断标准仍存在争议，Meier-Ruge提出了改良的粘膜活检来诊断IND，但行多处肠壁的全层病检，HE染色或Ache染色，对神经元发育不良较难判断的，加行神经元相关免疫组化检测的方法较为公认。
对IND的手术指征和切除标准仍不确定，大部分的单纯型IND可以根据病情适当的保守治疗(如改变饮食结构，应用缓泻剂等)和临床观察，随生长发育部分患儿不手术也可以自行好转甚至自愈，在一定条件下(如肠造瘘)，IND病变可以自行再发育为成熟的神经元细胞而恢复功能。但对严重腹胀，小肠结肠炎患儿仍需及时手术治疗，局限型IND，可以将病变肠段全部切除，以达到较好的手术效果；而对广泛性病变，仍缺乏切除范围的明确标准，可先行回肠造瘘术，营养情况改善后再行进一步根治术，有报道切除小部分病变结肠到全部结肠(甚至部分小肠)。
临床常见部分患儿巨结肠术后恢复良好，而部分患儿术后一段时间内仍有反复EC，甚至需要再次肠造瘘。术后EC是否与伴发IND病变有关?本课题拟通过对IND与HD的临床和病理资料的统计，研究术后EC和神经元形态、分布的关系。虽然对IND的光镜下病理特点有较多报道，但发育不良神经元的超微结构特点仍缺乏研究，这也是本课题的研究目的。同时，并通过对IND的NSE、Cathepsin-D和S-100的免疫组化染色，分析对IND的镜下免疫组化特点和有效的诊断手段。
材料和方法
1．IND的神经元分布与EC特点
(1)研究对象及临床资料收集：收集2001年～2004年本院诊断的巨结肠手术治疗的患儿，共347例。进行随访(≥6月)，评价患儿术后排便，腹胀情况，术后EC发生情况(以随访时间≥6月，发生≥2次EC为反复EC)；是否有再次手术。
(2)组织切片阅片、分类：根据患儿的病理号，调取所有患儿病理切片，每个病理号一般有4～6张切片，分别标记为A、B、C、D、E、F等，对应取材部位从大体标本痉挛段—移行段—扩张段—切缘，其中首尾编号分别对应痉挛段和切缘。重新阅片后，参照Holschneider提出的有关神经元异常的病理诊断标准进行分类。
2．免疫组化染色
根据组织切片结果，调取20例IND病变组织蜡块，同时取5例HD患儿神经元正常肠段组织和2例美克尔憩室肠切除边缘组织，分别进行NSE、Cathepsin-D和S-100免疫组化检测。
3．透射电镜检查
(1)研究对象及取材：巨结肠术中切除肠段立即取材，切取小块肠壁组织，在2.5％戊二醛液体覆盖下，进一步切割组织尽可能小，估计保留肌间组织和两侧部分肌层。样本置2.5％戊二醛4℃保存，根据病理结果，将所需样本分类：神经元正常肠段和IND病变肠段。
(2)戊二醛—锇酸双重固定，脱水，浸透，包埋，固化，预切片定位，超薄切片，电镜观察、拍片、记录。
4．数据处理
统计所有HD、IND和HD／IND病变患儿临床资料，病理结果，采用x~2检验行统计学分析，P＜0.05认为差异有统计学意义。
结果
1．IND的神经元分布与EC特点
(1)临床随访结果：获有效随访(≥6月)324例。根据组织类型，对其中HD210例，IND38例，HD伴IND(HD／IND)45例共293例患儿作为本课题研究对象。术后反复EC共41例，再次手术8例。
(2)病理观察结果：IND和HD／IND的异常神经元分布与典型HD不同，在扩张段和切缘都有较高比例的异常神经元分布(P＜0.01)。
(3)IND与EC关系：IND和IND／HD组的术后反复EC发生率高于典型HD组(P＜0.01)。切缘IND病变组术后反复EC发生率明显高于切缘神经元正常组(P＜0.01)。经肛门手术与经腹手术的术后反复EC发生率分别为18.1％和8.7％(P＜0.05)。
(4)再次手术与病理类型：有8例患儿术后因仍有反复腹胀、便秘或严重的EC再次行手术治疗，5例为切缘有IND(其中4例HD／IND，1例IND)，有3例为HD第一次术后切缘仍无神经元细胞。
2．IND的免疫组化特点
NSE免疫组化结果：粘膜下神经丛和肌间神经丛均有显色，神经丛胶质细胞、神经元细胞和神经纤维均呈阳性反应。IND病变肠段粘膜下及肌间均有巨神经丛，丛内各成分细胞均呈阳性反应，神经丛内有大量大小不等细胞，为胶质细胞与发育不良神经元，其中可见部分细胞胞体较大。
Cathepsin-D免疫组化结果：神经丛内仅神经元细胞胞浆呈阳性反应，神经纤维和胶质细胞均呈阴性反应。IND病变可见巨神经丛内有大量神经元细胞，形态较正常小，胞浆少，染色较深，细胞核较正常小，染色深，核仁有或不明显。
S-100免疫组化结果：神经丛内神经纤维和胶质细胞呈阳性反应，神经元细胞反应呈阴性。IND病变可见巨神经丛内布满大小不一的缺损区，缺损区中央可见发育不良细胞核，神经纤维增生不明显。
3．IND透射电镜下特点
正常神经元细胞直径约20～30gm，细胞形态较不规则，胞浆丰富，胞浆内可见清晰的线粒体、光面内质网和粗面内质网，伴有大量的神经内分泌囊泡结构，囊泡内可见递质颗粒。细胞核直径约7～8μm，细胞核较为规则，核膜完整。核仁明显，核内染色质疏松，分布均匀。
IND病变神经元细胞直径约10～15μm，细胞形态较为规则，胞浆较正常神经元明显减少，胞浆内可见清晰的线粒体、光面内质网和粗面内质网，但神经内分泌囊泡结构很少或没有。细胞核直径约4～5μm，圆或椭圆，核膜完整，往往局部有不规则突起，核仁有或没有，核染色质分布不均，有边聚现象。
结论
1．IND的病变神经元分布与肉眼病变肠段分布有不平衡性，术后EC发生率高，切缘仍有IND病变以及经肛门手术是术后反复EC的危险因素，IND的切除范围仍有不确定性。
2．NSE、Cathepsin-D和S-100三种免疫组化具有互补性，可以分别显示IND的神经丛结构，神经丛内神经元结构数量，神经丛神经纤维增生情况，从而能够对IND进行准确的判断。
3．电镜下，IND病变神经元结构与正常不同，胞浆内神经内分泌囊泡的减少或缺乏，伴有核染色质边聚现象。
Part Ⅰ The Study of the Polymorphisms of RET gene and PHOX2B gene on Hirschsprung's Disease
Introduction
Hirschsprung's disease (HD), or congenital aganglionosis, is a neurocristopathy characterized by the absence of ganglion cells in submucosal and myenteric plexuses along a variable length of the gastrointestinal tract. The incidence varies among different ethnic groups, with 1.5,2.1, and 2.8 per 10,000 live births in Caucasians, Africans, and Asians, respectively. HD appeared to be a multifactorial malformation, several genes involved in HD genetic aetiology including RET, GDNF, GFRα-1, EDNRB, EDN3, ECE-1, SOX10 and PHOX2B. The RET gene plays a crucial role in neural crest cell development in the bowel wall and has been identified as the most important one of HD associated genes.
Linkage analyses of multiplex HD families revealed that the RET gene locus at 10q11.2 is the major gene locus for Hirschsprung's disease, however, RET mutations in codon region were detected in only 20% of sporadic and 50% of familial cases of HD, with low, sex dependent penetrance and lack of genotype-phenotype correlation. This support that the existence of more
RET variants or other modified genes in HD.
To date, several RET polymorphisms were found associated with the HD in Caucasians, but no study of RET promoter has been published on the genetics of HD in the Chinese mainland population and no study of functional analysis of RET promoter's haplotypes polymorphism.
PHOX2B is a transcription factor involved in the development of the noradrenergic nervous system, so it may associated with malformations of neural-crest origin such CCHS and HD. Only one study showed that the SNP of PHOX2B 1364 A>G( intron 2) was associated with increased risk of HD but no more studies about the relationship between PHOX2B and HD.
Materials and Methods
Patients and controls
This study consisted of 123 HD patients of Zhejiang Han ethnic Chinese. All patients were histologically confirmed with the criteria of the fourth International Symposium on Hirschsprung's disease and related neurocristopathies. Matched control were unselected, unrelated of Zhejiang Han ethnic Chinese without HD or other congenital malformation.
Polymorphism Analysis
Genomic DNA was extracted from blood samples, genotypes were analyzed using PCR and direct sequencing,
Immunohistochemical stainning
According to the genotypes of RET promoter, forty tissue of normal ganglion segment of HD cases embedded in paraffin were cut with 5-μm-thick sections and immunohistochemical stained with C-Ret. The number of neuronal cells were examined.
Statistical Analysis
Chi square tests were performed to determine whether each SNP was in Hardy-Weinberg equilibrium within each group.
The associations between SNPs and risk of HD were estimated by ORs and their 95% CIs, tested by x~2 tests. Data were considered significant at a level of P<0.05.
Mann-Whitney Test was used for analysis the data of immunohistochemical staining.
Haplotypes and their frequencies were estimated by means of the PHASE software.
Results
Association between genotypes and the risk of HD
Increased risk of HD was observed in homozygous genotypes of the RET allele -5AA, -1CC, cl35AA or c2307GG when compared with the other genotypes(P<0.05). No increased risk of HD was found in PHOX2B 1364A>G.
Association between frequency of allele and the risk of HD
Increased risk of HD was observed in allele of RET -5A, -1C, cl35A, c2307G when compared with the other allele(P<0.05). In PHOX2B allele 1364A>G, no increased risk of HD was found.
Association between frequency of allele and the types of HD.
No associated risk was found between frequency of allele and the types of HD.
Association between haplotypes frequencies and the risk of HD
The haplotype -5A/-1C of RET promoter was the most frequently one in HD (82.5%). The difference in promoter haplotype frequencies was highly significant between the HD and controls(P<0.001). The haplotype ACAG of four RET SNPs account for 75.2% of the HD, and the difference between the two distribution was highly significant(P<0.001).
Frequency of allele in different ethnic people
The frequencies of HD-associated allele of RET -5A, c135A, c2307G in Chinese was higher than in the Caucasians or Africans significantly(.P<0.05).
Functional analysis of RET promoter SNPs
The decreased express of RET was observed in promoter phenotype -5AA/-1CC or in homozygous of haplotype -5A/-1C than in others(P<0.05).
Conclusions
The RET SNPs -5G>A, -1A>C, c135G>A, c2307T>G is associated with HD and the haplotype ACAG is the core one in Zhejiang Han ethnic Chinese. It is no relationships
between frequency of allele and the types of HD.
The frequencies allele of RET -5A, cl35A, c2307G in controls is higher than in Caucasians or Africans.
The different RET promoter haplotypes or genotypes associated with express of RET
It is no relationships between SNPs PHOX2B1364A>G and HD in Zhejiang Han ethnic Chinese.
Part Ⅱ The Study of Pathological and Enterocolitis Features of Intestinal Neuronal Dysplasia
Introduction
In past few years, a group of disease named Allied Disorders of Hirschsprung's Disease (ADHD) were found, which show the clinical features similar to Hirschsprung's disease (HD). ADHD consist of Intestinal Neuronal Dysplasia (IND), Hypoganglionosis and Immaturity of ganglion cells. IND is the most frequently kind of all ADHD. The pathological features include presence of giant ganglia of the submucosal plexuses and myenteric plexuses, giant ganglia containing more than seven nerve cells, increase of Ache activity in nerve fibres of the lamina propria, ectopic ganglion cells in the colonic circular muscle and lamina propria, deficiency of sympathetic innervation in the muscle. Depending on the length of the neuronal dysplasia intestine, IND B may have a localized or disseminated form, it may occur in an isolated pure form or in combination with HD.
IND as a cause for severe chronic constipation remains controversial. The congenital origin of IND B is supposed; nevertheless, IND were found secondary to intestinal obstruction or
inflammatory disease. The precise etiology remains unknown, and, to date, no specific diagnostic test exists other than morphology. The criteria for diagnosis is still uncertain, the most approbatory technique is laparoscopic full-thickness intestinal biopsy or resected surgical specimens with HE stain or Ache stain, additional immunohistochemical staining is used to identify the dysplasia nerve cells.
IND has no unified concept of treatment. There is an opinion that majority of patients with IND can be treated successfully with conservative treatment such as diet, laxatives and prokinetic drugs, however, in some severe cases a transient enterostomy or a segmental resection is unavoidable.
The postoperative bowel function is well in most of megacolon, but part of them is complicated with recurrent postoperative enterocolitis (EC). But now there are still few studies about the relationship between morphological findings and postoperative EC. The features of IND under optic microscopy is well known, but there is still no picture of dysplasia neurons under electron microscopy.
Materials and Methods
Clinical features of IND
The data of clinical features of 293 cases with HD, IND and HD/IND were collect. The postoperative bowel function were followed. The histopathologic features of all cases were rechecked according to Holschneider's criteria.
Immunohistochemical stainning
Twenty cases of IND tissue embedded in paraffin and 7 with normal neurons were cut with 5-μm-thick sections and immunohistochemical stained with NSE、Cathepsin-D and S-100. The morphology of plexuses, neurons and neuroglia cells were examined.
Transmission electron microscopy examining
Fresh tissue of IND and normal colon were shaped and followed a series of process. The morphological of dysplasia and normal neurons were examined by TEM.
Statistical analysis.
All data were analyzed by x~2 test. Data were considered significant at a level of P<0.05.
Results
Neurons distribution and EC features of IND
In the distension segment of colon, the frequencies of dysganglion is 11.9%, 75.6% and 81.6% in HD, HD/IND and IND respectively (P<0.01).
Incidences of EC in cases with the residual IND margins and with the normal margins were 38.2% and 8.7% respectively (P<0.01).
Eight cases underwent another procedure because of severe persistent constipation or EC after operation, including 5 cases with IND of the proximal segment (4 HD/IND, 1 IND), 3 cases with the proximal segment aganglion.
Histopathologic features of IND with immunohistochemical staining
With immunohistochemical stained with NSE, the whole plexuses are positive. Giant ganglia with a amount of cells in different size (dysplasia neurons and neuroglia cells) were showed in IND sections.
With immunohistochemical stained with Cathepsin-D, only the cytoplasm of neurons is positive. In IND sections, the picture of dysplasia neurons can be distinguished from the normal easily with a smaller cell and cytoblast diameter, a distinct increase of number, less and stain thickened cytoplasm, nucleolus exit or not.
With immunohistochemical stained with S-100, the nerve fibres and neuroglia cells are positive. In IND sections, we can see the giant ganglia present with a number of stain-deficiency area in different size.
Morphological features of IND under TEM
In normal neurons, the membrane of cells and nucleus is intact, the cell organs such as mitochondria and endoplasmic reticulum and ribosome are clear, the nucleolus are distinct, the chromatin is loosen, the cytoplasm is filled with neuroendocrine vesicle. In dysplasia neurons, whereas, neuroendocrine vesicle in cytoplasm is deficient, chromatin is collected near the nuclear
membrane, cell shape shrunk, smaller cytoblast and less cytoplasm.
Conclusions
Neurons distribution of IND is inconsistent with macropathology. Recurrent postoperative EC is more likely occur in patients with IND, especially in margins with residual IND. However, the extension of excision about IND is uncertain and need to be further studied.
IND can be diagnosed easily with immunohistochemical stained with NSE、 Cathepsin-D and S-100 showing different part of plexuses.
Dysplasia neurons is different from the normal. Neuroendocrine vesicle in cytoplasm is deficient, chromatin is collected near the nuclear membrane.
研究背景
先天性巨结肠(Hirschsprung's disease，HD)，是小儿外科常见疾病，在高加索人，非洲黑人、亚洲黄种人的发病率分别为万分之1.5、2.1、2.8，亚洲人种发病率最高。HD是一种神经嵴源性疾病，当胚胎发育过程中神经嵴细胞迁徙、分化发生停顿或发生细胞凋亡，均会导致远端肠管的神经支配异常而导致HD。近年来通过遗传连锁分析和基因敲除等技术，发现有以下基因的突变与HD的发病有关：编码有关酪氨酸激酶通路基因(包括RET原癌基因，GDNF基因，GFRα-1基因等)，编码血管内皮素信号通路的基因(包括EDNRB基因，EDN3基因和ECE-1基因)，编码转录因子的基因(如SOX10基因和PHOX2B基因)等。其中RET基因表达对肠神经元细胞的发育十分关键，在HD的发病中最为重要。
但对HD进行大量RET基因突变研究发现，仅有20％左右的散发性和50％左右家族性HD存在突变，而且缺乏类似肿瘤相关的热点突变，另外HD相关RET突变有不完全外显(incomplete penetrance)，呈突变效应性别相关性。更为重要的是，在家族性病例，遗传连锁分析定位遗传位点在10q11.2，却仍缺乏RET编码区突变。随着SNPs疾病相关性研究的进展，对HD的RET基因研究重点逐渐转移到多态性的研究上。尚未证实单个SNPs导致HD相关RET功能改变，但可能有精确的SNPs组合(单倍型)决定调节意义。在高加索人种，发现有数个RET编码区SNPs与散发性HD相关：A45A(c135G＞(?)；exon 2)和L769L(c2307T＞(?)；exon 13)。对RET基因启动子的研究发现，在RET编码序列上游的启动子的两种多态性-5G＞A和-A＞C与HD相关。目前对RET基因的研究，均局限于编码区的突变及SNPs研究，虽然也有少量非编码区(内含子和启动子)的研究，但缺乏相应的功能研究。
PHOX2B基因表达研究证明，PHOX2B编码的同源域蛋白与多种非肾上腺素能神经元的发育有关，因此推断PHOX2B基因的改变可能与自主神经发育异常性疾病相关。在小鼠胚胎学研究发现，一旦成肠细胞进入到前肠间叶，即有PHOX2B蛋白表达，并贯穿肠神经元发育的始终。PHOX2B基因突变纯合子小鼠全胃肠道缺乏神经元，与HD病变相似，同时，该突变的小鼠无RET蛋白表达，因此推测PHOX2B基因可能与HD有关。Garcia-Barcelo通过对91例单纯HD研究，发现PHOX2B 1364 A＞G多态性与HD有关。目前对PHOX2B基因与临床疾病的关系研究尚处在起步阶段，PHOX2B基因与HD的研究也仅有个别报道，PHOX2B基因是否参与HD的发病机制仍有待进一步的研究。
根据Hapmap组织统计数据，不同种族人群的SNPs分布频率有明显差异，RET基因和PHOX2B基因SNPs也是如此，这可能是不同种族人群HD发病率有明显差别的原因之一。本课题拟通过对浙江汉族人群HD患者和正常人群的对照研究，分析RET基因和PHOX2B基因SNPs与HD发病之间的关系，不同类型HD与基因SNPs关系，进一步推断主要的HD相关单倍型，并对照其他种族人群已有数据，分析浙江汉族人群的SNPs分布特点。同时应用免疫组化法，检测RET基因启动子多态性与RET蛋白表达之间的关系。
材料和方法
1．病例收集及标准
(1)病例组：全部病例均为本院的浙江籍先天性巨结肠患儿，共123例。根据2005年第四届国际巨结肠与相关神经嵴源性疾病会议的巨结肠诊断标准，重新阅片确定。
(2)分组及标准：将巨结肠分为短段型与长段型两种。
短段型HD：病变仅局限于乙状结肠远端，乙状结肠直肠交界及直肠。
长段型HD：病变范围包括乙状结肠近端，升结肠及累及范围更广者。
(3)正常对照组：全部正常对照组均来自本院正常体检小儿，排除消化道和神经嵴相关畸形，共194例。
全部病例及对照组抽取外周血2ml，3.8％枸橼酸钠抗凝。
本研究经浙江大学医学院附属儿童医院伦理委员会批准，全部患儿及对照组均获得家长知情同意。
2．PCR反应及测序
对全部样本进行DNA提取后，根据选取的SNPs，合成引物，PCR扩增，经2％琼脂糖电泳证实PCR产物后，将PCR产物进一步纯化、测序。
3．免疫组化
根据RET启动子多态性，分别选取相应患儿病理组织切片，观察神经元，确定含正常神经元蜡块，再行C-Ret免疫组化染色，将该切片镜下所有神经元按免疫组化反应等级均予计数。
4．统计方法
(1)用Hardy-Weinberg平衡定律来检验样本人群中基因频率与基因型频率是否平衡。
(2)基因和疾病的相关性分析：使用x2方检验(Chi squared)，相关性以比值比(odds ratios，ORs)及其95％可信区间(confidence intervals，CIs)表示，以P值＜0.05确定为统计学差异有显著性。
(3)单倍型频率用PHASE软件计算。
(4)免疫组化结果用非参数统计中的两样本等级和检验(Mann-Whitney Test)。
结果
1．各位点基因型与HD风险
RET基因-5G＞A，-1A＞C，c135G＞A，c2307T＞G四个位点的少见基因型AA，CC，AA，GG在HD组频率明显高于对照组(P＜0.05)。
PHOX2B基因1364A＞G两组间未发现有基因型差异。
2．各位点等位基因频率与HD风险
RET基因-5A，-1C，c135A，c2307G在HD组等位基因频率明显高于对照组(P＜0.05)。
PHOX2B基因1364A＞G等位基因分布未发现显著性事件。
3．各位点等位基因频率与HD类型关系
RET基因各位点均未发现等位基因频率在不同类型间的差异。
4．单倍型与HD
RET基因启动子单倍型-5A／-1C占HD组82.5％，较对照组差异有显著性(P＜0.05)。
RET基因四位点(-5，-1，c135，c2307)单倍型ACAG占HD组75.2％，较对照组差异有显著性(P＜0.05)。
5．不同种族人群之间各等位基因频率
RET基因-5A、c135A和c2307G等位基因频率明显高于高加索和非洲人种(P＜0.05)
6．RET基因启动子多态性与RET蛋白表达关系
RET基因启动子基因型-5AA／-1CC较其他基因型RET蛋白表达降低(P＜0.05)。
启动子单倍型-5A／-1C纯合子较-5A／-1C杂和子RET蛋白表达降低(P＜0.05)。
结论
1．RET基因-5G＞A，-1A＞C，c135G＞A，c2307T＞G四个位点多态性与浙江汉族人群HD明显相关，但与HD类型无明显相关。
2．RET基因-5G＞A，-1A＞C，c135G＞A，c2307T＞G四个位点单倍型ACAG是浙江汉族人群的HD相关核心单倍型。
3．浙江汉族人群的RET基因-5、c135和c2307位点的少见等位基因频率较其他人种有明显增高。
4．RET启动子多态性对启动子功能有影响，与RET蛋白表达相关。
5．未发现PHOX2B基因多态性与浙江汉族人群HD相关。
第二部分肠神经元发育不良病理特点及与EC关系研究
研究背景
近年来逐渐认识到一类临床表现与HD十分相似，但病理改变与HD明显不同的疾病，称之为巨结肠同源病(allied disorders of Hirschsprung's disease；ADHD)，主要包括肠神经元发育不良(Intestinal neuronal dysplasia；IND)、神经节细胞减少症(Hypoganglionosis)、神经节细胞未成熟症(Immaturity of ganglion cells)等。1994年Holschneider和Meier-Ruge将HD和ADHD两者统称为肠神经元异常(Neuronal intestinal malformations；NIM)，并进行了详细的病理分类。在ADHD中以IND最为常见，其病理特点主要有以下4项：①粘膜下和肌间神经丛增生，形成巨大神经节(giant ganglia)，粘膜下丛节细胞数目≥7个(正常为3～5个)，肌间丛面积≥正常的3倍，常伴有神经元不成熟现象；②粘膜固有层和环肌副交感神经纤维乙酰胆碱酯酶(Acetylcholinesterase，Ache)活性增高：③粘膜固有层和粘膜肌层分散的平滑肌纤维之间有孤立的神经节细胞；④肌间神经丛交感神经发育不全或无发育。
混合型IND的临床表现几乎与HD一样，单纯型IND的发病主要集中在年幼儿童，而在新生儿期较少表现。临床表现为渐进性的便秘，腹胀或有顽固性／发作性的吸收异常。部分患儿经保守治疗或随年龄增长能逐渐好转，部分患儿则呈进行性加重，需手术治疗。
针对HD的辅助检查中，包括钡剂灌肠、直肠肛管测压，IND常缺乏特征性的改变而无法准确应用。目前国内对1ND的认识仍不足，一般根据临床表现或结合钡剂灌肠检查就予诊断并进一步手术治疗，随着经肛门手术的广泛开展和手术年龄提前至新生儿和婴幼儿期，单凭临床表现和钡灌肠可误将部分单纯型IND诊断为HD而手术治疗。
虽然IND作为一种明确的病理改变已经得到广泛认同，对IND是否作为一种独立的疾病一直存在争议，分歧的焦点在于IND是一种原发性疾病还是继发性改变，两者都有相应的证据支持。IND的诊断标准仍存在争议，Meier-Ruge提出了改良的粘膜活检来诊断IND，但行多处肠壁的全层病检，HE染色或Ache染色，对神经元发育不良较难判断的，加行神经元相关免疫组化检测的方法较为公认。
对IND的手术指征和切除标准仍不确定，大部分的单纯型IND可以根据病情适当的保守治疗(如改变饮食结构，应用缓泻剂等)和临床观察，随生长发育部分患儿不手术也可以自行好转甚至自愈，在一定条件下(如肠造瘘)，IND病变可以自行再发育为成熟的神经元细胞而恢复功能。但对严重腹胀，小肠结肠炎患儿仍需及时手术治疗，局限型IND，可以将病变肠段全部切除，以达到较好的手术效果；而对广泛性病变，仍缺乏切除范围的明确标准，可先行回肠造瘘术，营养情况改善后再行进一步根治术，有报道切除小部分病变结肠到全部结肠(甚至部分小肠)。
临床常见部分患儿巨结肠术后恢复良好，而部分患儿术后一段时间内仍有反复EC，甚至需要再次肠造瘘。术后EC是否与伴发IND病变有关?本课题拟通过对IND与HD的临床和病理资料的统计，研究术后EC和神经元形态、分布的关系。虽然对IND的光镜下病理特点有较多报道，但发育不良神经元的超微结构特点仍缺乏研究，这也是本课题的研究目的。同时，并通过对IND的NSE、Cathepsin-D和S-100的免疫组化染色，分析对IND的镜下免疫组化特点和有效的诊断手段。
材料和方法
1．IND的神经元分布与EC特点
(1)研究对象及临床资料收集：收集2001年～2004年本院诊断的巨结肠手术治疗的患儿，共347例。进行随访(≥6月)，评价患儿术后排便，腹胀情况，术后EC发生情况(以随访时间≥6月，发生≥2次EC为反复EC)；是否有再次手术。
(2)组织切片阅片、分类：根据患儿的病理号，调取所有患儿病理切片，每个病理号一般有4～6张切片，分别标记为A、B、C、D、E、F等，对应取材部位从大体标本痉挛段—移行段—扩张段—切缘，其中首尾编号分别对应痉挛段和切缘。重新阅片后，参照Holschneider提出的有关神经元异常的病理诊断标准进行分类。
2．免疫组化染色
根据组织切片结果，调取20例IND病变组织蜡块，同时取5例HD患儿神经元正常肠段组织和2例美克尔憩室肠切除边缘组织，分别进行NSE、Cathepsin-D和S-100免疫组化检测。
3．透射电镜检查
(1)研究对象及取材：巨结肠术中切除肠段立即取材，切取小块肠壁组织，在2.5％戊二醛液体覆盖下，进一步切割组织尽可能小，估计保留肌间组织和两侧部分肌层。样本置2.5％戊二醛4℃保存，根据病理结果，将所需样本分类：神经元正常肠段和IND病变肠段。
(2)戊二醛—锇酸双重固定，脱水，浸透，包埋，固化，预切片定位，超薄切片，电镜观察、拍片、记录。
4．数据处理
统计所有HD、IND和HD／IND病变患儿临床资料，病理结果，采用x~2检验行统计学分析，P＜0.05认为差异有统计学意义。
结果
1．IND的神经元分布与EC特点
(1)临床随访结果：获有效随访(≥6月)324例。根据组织类型，对其中HD210例，IND38例，HD伴IND(HD／IND)45例共293例患儿作为本课题研究对象。术后反复EC共41例，再次手术8例。
(2)病理观察结果：IND和HD／IND的异常神经元分布与典型HD不同，在扩张段和切缘都有较高比例的异常神经元分布(P＜0.01)。
(3)IND与EC关系：IND和IND／HD组的术后反复EC发生率高于典型HD组(P＜0.01)。切缘IND病变组术后反复EC发生率明显高于切缘神经元正常组(P＜0.01)。经肛门手术与经腹手术的术后反复EC发生率分别为18.1％和8.7％(P＜0.05)。
(4)再次手术与病理类型：有8例患儿术后因仍有反复腹胀、便秘或严重的EC再次行手术治疗，5例为切缘有IND(其中4例HD／IND，1例IND)，有3例为HD第一次术后切缘仍无神经元细胞。
2．IND的免疫组化特点
NSE免疫组化结果：粘膜下神经丛和肌间神经丛均有显色，神经丛胶质细胞、神经元细胞和神经纤维均呈阳性反应。IND病变肠段粘膜下及肌间均有巨神经丛，丛内各成分细胞均呈阳性反应，神经丛内有大量大小不等细胞，为胶质细胞与发育不良神经元，其中可见部分细胞胞体较大。
Cathepsin-D免疫组化结果：神经丛内仅神经元细胞胞浆呈阳性反应，神经纤维和胶质细胞均呈阴性反应。IND病变可见巨神经丛内有大量神经元细胞，形态较正常小，胞浆少，染色较深，细胞核较正常小，染色深，核仁有或不明显。
S-100免疫组化结果：神经丛内神经纤维和胶质细胞呈阳性反应，神经元细胞反应呈阴性。IND病变可见巨神经丛内布满大小不一的缺损区，缺损区中央可见发育不良细胞核，神经纤维增生不明显。
3．IND透射电镜下特点
正常神经元细胞直径约20～30gm，细胞形态较不规则，胞浆丰富，胞浆内可见清晰的线粒体、光面内质网和粗面内质网，伴有大量的神经内分泌囊泡结构，囊泡内可见递质颗粒。细胞核直径约7～8μm，细胞核较为规则，核膜完整。核仁明显，核内染色质疏松，分布均匀。
IND病变神经元细胞直径约10～15μm，细胞形态较为规则，胞浆较正常神经元明显减少，胞浆内可见清晰的线粒体、光面内质网和粗面内质网，但神经内分泌囊泡结构很少或没有。细胞核直径约4～5μm，圆或椭圆，核膜完整，往往局部有不规则突起，核仁有或没有，核染色质分布不均，有边聚现象。
结论
1．IND的病变神经元分布与肉眼病变肠段分布有不平衡性，术后EC发生率高，切缘仍有IND病变以及经肛门手术是术后反复EC的危险因素，IND的切除范围仍有不确定性。
2．NSE、Cathepsin-D和S-100三种免疫组化具有互补性，可以分别显示IND的神经丛结构，神经丛内神经元结构数量，神经丛神经纤维增生情况，从而能够对IND进行准确的判断。
3．电镜下，IND病变神经元结构与正常不同，胞浆内神经内分泌囊泡的减少或缺乏，伴有核染色质边聚现象。
Part Ⅰ The Study of the Polymorphisms of RET gene and PHOX2B gene on Hirschsprung's Disease
Introduction
Hirschsprung's disease (HD), or congenital aganglionosis, is a neurocristopathy characterized by the absence of ganglion cells in submucosal and myenteric plexuses along a variable length of the gastrointestinal tract. The incidence varies among different ethnic groups, with 1.5,2.1, and 2.8 per 10,000 live births in Caucasians, Africans, and Asians, respectively. HD appeared to be a multifactorial malformation, several genes involved in HD genetic aetiology including RET, GDNF, GFRα-1, EDNRB, EDN3, ECE-1, SOX10 and PHOX2B. The RET gene plays a crucial role in neural crest cell development in the bowel wall and has been identified as the most important one of HD associated genes.
Linkage analyses of multiplex HD families revealed that the RET gene locus at 10q11.2 is the major gene locus for Hirschsprung's disease, however, RET mutations in codon region were detected in only 20% of sporadic and 50% of familial cases of HD, with low, sex dependent penetrance and lack of genotype-phenotype correlation. This support that the existence of more
RET variants or other modified genes in HD.
To date, several RET polymorphisms were found associated with the HD in Caucasians, but no study of RET promoter has been published on the genetics of HD in the Chinese mainland population and no study of functional analysis of RET promoter's haplotypes polymorphism.
PHOX2B is a transcription factor involved in the development of the noradrenergic nervous system, so it may associated with malformations of neural-crest origin such CCHS and HD. Only one study showed that the SNP of PHOX2B 1364 A>G( intron 2) was associated with increased risk of HD but no more studies about the relationship between PHOX2B and HD.
Materials and Methods
Patients and controls
This study consisted of 123 HD patients of Zhejiang Han ethnic Chinese. All patients were histologically confirmed with the criteria of the fourth International Symposium on Hirschsprung's disease and related neurocristopathies. Matched control were unselected, unrelated of Zhejiang Han ethnic Chinese without HD or other congenital malformation.
Polymorphism Analysis
Genomic DNA was extracted from blood samples, genotypes were analyzed using PCR and direct sequencing,
Immunohistochemical stainning
According to the genotypes of RET promoter, forty tissue of normal ganglion segment of HD cases embedded in paraffin were cut with 5-μm-thick sections and immunohistochemical stained with C-Ret. The number of neuronal cells were examined.
Statistical Analysis
Chi square tests were performed to determine whether each SNP was in Hardy-Weinberg equilibrium within each group.
The associations between SNPs and risk of HD were estimated by ORs and their 95% CIs, tested by x~2 tests. Data were considered significant at a level of P<0.05.
Mann-Whitney Test was used for analysis the data of immunohistochemical staining.
Haplotypes and their frequencies were estimated by means of the PHASE software.
Results
Association between genotypes and the risk of HD
Increased risk of HD was observed in homozygous genotypes of the RET allele -5AA, -1CC, cl35AA or c2307GG when compared with the other genotypes(P<0.05). No increased risk of HD was found in PHOX2B 1364A>G.
Association between frequency of allele and the risk of HD
Increased risk of HD was observed in allele of RET -5A, -1C, cl35A, c2307G when compared with the other allele(P<0.05). In PHOX2B allele 1364A>G, no increased risk of HD was found.
Association between frequency of allele and the types of HD.
No associated risk was found between frequency of allele and the types of HD.
Association between haplotypes frequencies and the risk of HD
The haplotype -5A/-1C of RET promoter was the most frequently one in HD (82.5%). The difference in promoter haplotype frequencies was highly significant between the HD and controls(P<0.001). The haplotype ACAG of four RET SNPs account for 75.2% of the HD, and the difference between the two distribution was highly significant(P<0.001).
Frequency of allele in different ethnic people
The frequencies of HD-associated allele of RET -5A, c135A, c2307G in Chinese was higher than in the Caucasians or Africans significantly(.P<0.05).
Functional analysis of RET promoter SNPs
The decreased express of RET was observed in promoter phenotype -5AA/-1CC or in homozygous of haplotype -5A/-1C than in others(P<0.05).
Conclusions
The RET SNPs -5G>A, -1A>C, c135G>A, c2307T>G is associated with HD and the haplotype ACAG is the core one in Zhejiang Han ethnic Chinese. It is no relationships
between frequency of allele and the types of HD.
The frequencies allele of RET -5A, cl35A, c2307G in controls is higher than in Caucasians or Africans.
The different RET promoter haplotypes or genotypes associated with express of RET
It is no relationships between SNPs PHOX2B1364A>G and HD in Zhejiang Han ethnic Chinese.
Part Ⅱ The Study of Pathological and Enterocolitis Features of Intestinal Neuronal Dysplasia
Introduction
In past few years, a group of disease named Allied Disorders of Hirschsprung's Disease (ADHD) were found, which show the clinical features similar to Hirschsprung's disease (HD). ADHD consist of Intestinal Neuronal Dysplasia (IND), Hypoganglionosis and Immaturity of ganglion cells. IND is the most frequently kind of all ADHD. The pathological features include presence of giant ganglia of the submucosal plexuses and myenteric plexuses, giant ganglia containing more than seven nerve cells, increase of Ache activity in nerve fibres of the lamina propria, ectopic ganglion cells in the colonic circular muscle and lamina propria, deficiency of sympathetic innervation in the muscle. Depending on the length of the neuronal dysplasia intestine, IND B may have a localized or disseminated form, it may occur in an isolated pure form or in combination with HD.
IND as a cause for severe chronic constipation remains controversial. The congenital origin of IND B is supposed; nevertheless, IND were found secondary to intestinal obstruction or
inflammatory disease. The precise etiology remains unknown, and, to date, no specific diagnostic test exists other than morphology. The criteria for diagnosis is still uncertain, the most approbatory technique is laparoscopic full-thickness intestinal biopsy or resected surgical specimens with HE stain or Ache stain, additional immunohistochemical staining is used to identify the dysplasia nerve cells.
IND has no unified concept of treatment. There is an opinion that majority of patients with IND can be treated successfully with conservative treatment such as diet, laxatives and prokinetic drugs, however, in some severe cases a transient enterostomy or a segmental resection is unavoidable.
The postoperative bowel function is well in most of megacolon, but part of them is complicated with recurrent postoperative enterocolitis (EC). But now there are still few studies about the relationship between morphological findings and postoperative EC. The features of IND under optic microscopy is well known, but there is still no picture of dysplasia neurons under electron microscopy.
Materials and Methods
Clinical features of IND
The data of clinical features of 293 cases with HD, IND and HD/IND were collect. The postoperative bowel function were followed. The histopathologic features of all cases were rechecked according to Holschneider's criteria.
Immunohistochemical stainning
Twenty cases of IND tissue embedded in paraffin and 7 with normal neurons were cut with 5-μm-thick sections and immunohistochemical stained with NSE、Cathepsin-D and S-100. The morphology of plexuses, neurons and neuroglia cells were examined.
Transmission electron microscopy examining
Fresh tissue of IND and normal colon were shaped and followed a series of process. The morphological of dysplasia and normal neurons were examined by TEM.
Statistical analysis.
All data were analyzed by x~2 test. Data were considered significant at a level of P<0.05.
Results
Neurons distribution and EC features of IND
In the distension segment of colon, the frequencies of dysganglion is 11.9%, 75.6% and 81.6% in HD, HD/IND and IND respectively (P<0.01).
Incidences of EC in cases with the residual IND margins and with the normal margins were 38.2% and 8.7% respectively (P<0.01).
Eight cases underwent another procedure because of severe persistent constipation or EC after operation, including 5 cases with IND of the proximal segment (4 HD/IND, 1 IND), 3 cases with the proximal segment aganglion.
Histopathologic features of IND with immunohistochemical staining
With immunohistochemical stained with NSE, the whole plexuses are positive. Giant ganglia with a amount of cells in different size (dysplasia neurons and neuroglia cells) were showed in IND sections.
With immunohistochemical stained with Cathepsin-D, only the cytoplasm of neurons is positive. In IND sections, the picture of dysplasia neurons can be distinguished from the normal easily with a smaller cell and cytoblast diameter, a distinct increase of number, less and stain thickened cytoplasm, nucleolus exit or not.
With immunohistochemical stained with S-100, the nerve fibres and neuroglia cells are positive. In IND sections, we can see the giant ganglia present with a number of stain-deficiency area in different size.
Morphological features of IND under TEM
In normal neurons, the membrane of cells and nucleus is intact, the cell organs such as mitochondria and endoplasmic reticulum and ribosome are clear, the nucleolus are distinct, the chromatin is loosen, the cytoplasm is filled with neuroendocrine vesicle. In dysplasia neurons, whereas, neuroendocrine vesicle in cytoplasm is deficient, chromatin is collected near the nuclear
membrane, cell shape shrunk, smaller cytoblast and less cytoplasm.
Conclusions
Neurons distribution of IND is inconsistent with macropathology. Recurrent postoperative EC is more likely occur in patients with IND, especially in margins with residual IND. However, the extension of excision about IND is uncertain and need to be further studied.
IND can be diagnosed easily with immunohistochemical stained with NSE、 Cathepsin-D and S-100 showing different part of plexuses.
Dysplasia neurons is different from the normal. Neuroendocrine vesicle in cytoplasm is deficient, chromatin is collected near the nuclear membrane.
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