摘要
先天性巨结肠(Hirschsprung's disease, HD)又称结肠无神经节细胞症,是一种常见且严重的小儿先天性畸形,其发病率高达1/3000-1/5000,居小儿消化道畸形的第二位。其主要的病理变化为远端结肠肠壁内神经节细胞的缺如或功能异常,导致病变肠段持续性痉挛,成狭窄状态,粪便淤积于近端结肠,导致近端结肠代偿性肥厚、扩张。目前对先天性巨结肠(Hirschsprung's disease, HD)的病因研究表明多种基因因素和环境因素共同作用而导致疾病的形成。研究表明,多种基因参与调控肠壁内神经节细胞发育,目前已知的基因有RET基因,EDNRB基因与ED3基因,GDNF基因,Bcl-2基因等.这些基因的异常可能是HD发病的分子机制。与此同时,如细胞外基质,细胞粘附分子、缺氧和毒素、炎症等胚胎肠道神经发育的环境缺陷,也有可能参与了HD的发病。总之先天性巨结肠的病因是由遗传因素和环境因素共同参与作用,最终导致的一种多因子、多基因参与的先天畸形疾病。
Neurexin及Neuroligin蛋白分别为突出前跨膜吸附蛋白和突出后跨膜吸附蛋白,他们之间相互作用,相互连接,共同参与突触的构成,介导突触的连接。现在的研究表明,在中枢神经系统中,Neurexin与Neuroligin的相互作用已经被证实可以促进中枢神经系统中突触的形成及支配突触的发生。而肠神经系统是一个在结构和功能上与中枢神经系统相似的复杂与独立的胃肠动力与感觉系统。肠神经系统的功能也是需要通过神经突触来实现,而先天性巨结肠的病因为典型的肠神经系统发育障碍造成狭窄段神经节细胞的缺失。因此通过研究Neurexin与Neuroligin在先天性巨结肠不同肠段表量,进一步探索和分析先天性巨结肠神经节细胞却如的原因,可能为目前的研究工作提供一种新的理论依据。
Ghrelin(GHRL)是1999年被Kojmia等在大鼠胃中利用免疫组织化学方法发现的GHSR的内源性配体,是目前为止除生长激素释放激素和生长抑素外人们发现的第3个调节腺垂体生长激素分泌的内源性物质。Ghrelin是由28个氨基酸组成的活性肽,胃已被证明为合成和分泌Ghrelin的主要器官,同时其他消化器官如十二指肠、回肠、盲肠和结肠在表达Ghrelin的同时其浓度顺次逐渐减少另外在体内多种组织中广泛分布,胰、肾、肝、下丘脑、其余器官如淋巴组织,肾脏,肾上腺,心脏,垂体及丘脑下部,肿瘤组织等也有分泌也都表达Ghrelin.
现在的研究表明Ghrelin(GHRL)除了具有调节生长激素释放的作用外,应该具有更广泛的生物功能。Jeffery等的研究证实,Ghrelin可以通过刺激癌细胞增殖而参与肿瘤的发生的,包括前列腺癌、肝细胞癌及大鼠垂体的生长激素瘤细胞.目前已经有研究表明Ghrelin主要的生物学作用有以下几个方面1.促进多种激素释放(生长激素,催乳素,肾上腺素等)2.提高食欲,促进摄食,促进胃肠动力3.促进肿瘤细胞的增殖,抑制细胞凋亡。4.促进免疫细胞的增殖和分化5.刺激成骨细胞的增殖与分化等等。
众所周知先天性巨结肠(Hirschsprung's disease, HD)患儿的病变原因是结肠远端神经节细胞的缺失,为典型的神经细胞发育障碍。其主要病因为患儿在胚胎发育过程中神经嵴细胞发育或者分化障碍所造成的神经发育停顿,肌间神经节细胞的缺失。目前对于先天性巨结肠(Hirschsprung's discase, HD)的病因研究多是集中于神经干细胞本身发育过程中的某些基因改变或者是环境改变。但是究竟是什么原因导致的巨结肠病变部位的多种基因改变和肠道神经发育的环境缺陷?仍然有很漫长的路要走。
我们前期的研究已经证实(?)Neurexin及Neuroligin在动物和人的肠神经系统都有表达,并且已经证实Neurexin及Neuroligin分别为突触前和突触后跨膜蛋白,因此我们下一步需要探索Neurexin及Neuroligin蛋白在先天性巨结肠患儿不同的病变肠段的表达变化及临床意义。而且Kawamura等研究表明在小鼠胚胎期Ghrelin mRNA就已经表达,并且贯穿胚胎发育的全过程,而且具有刺激多种细胞增殖和分化的功能.因此我们认为Ghrelin可能通过影响包括Neurexin及Neuroligi(?)基因在内的多种基因改变或胚胎发育环境改变,从而自胚胎时期就参与结肠神经干细胞的发育和分化过程,造成神经干细胞分化障碍,从而引起巨结肠疾病的发生。因此我们设计一系列的实验来探讨这一问题。第一部分先天性巨结肠中Neurexin及Neuroligin蛋白在的表达变化及临床意义。
实验目的：
研究Neurexin及Neuroligin蛋白在先天性巨结肠患儿不同肠段表达变化及临床意义。
实验方法：
1.选择2009年10月-2012年6月入住我院病房诊断为先天性巨结肠的患儿共20例(3月-2.5岁),男12例,女8例,均采用经肛门巨结肠根治术手术。术中切除病变肠段至近端正常肠管处(术中该处肠管病理示：查见正常神经节细胞)
2.术后在切除肠段分出狭窄段、移行段及扩张段,分别取长约1cm肠段,PBS冲洗干净后放入EP管中剪碎加入细胞裂解液后进行Westernblot检测。因扩张段为正常肠管的继发性扩张,具有正常的神经节细胞,肠神经系统发育正常,因此多种文献报道,可以讲扩张段肠管作为正常对照组进行研究。因此我们将扩张段肠管作为正常对照组来进行研究.
实验结果：
先天性巨结肠患儿狭窄段几乎无Neurexin及Neuroligin蛋白表达,移行段表达量小于扩张段表达量。
实验结论：
Neurexin及Neuroligin蛋白在先天性巨结肠在各段的表达量,与神经节细胞的分布方式一致。呈正相关。说明Neurexin及Neuroligin作为突触前膜和突触后膜的跨膜蛋白,可能与肠神经系统神经节细胞发育过程中有关联,共同参与肠神经系统的发育和支配调节肠道运动的作用。
第二部分先天性巨结肠中生长素Ghrelin表达的表达变化及临床意义
I Ghrelin(GHRL)在先天性巨结肠患儿血清的表达及临床意义
目的：
研究Ghrelin(GHRL)在在先天性巨结肠患儿及正常对照儿童血清的表达,并分析它们的表达情况同先天性巨结肠畸形之间的关系。
方法：
1选择2009年10月-2012年6月入住我院病房诊断为先天性巨结肠的患儿共40例,男22例,女18例,为研究组；正常对照组儿童40例,男21例,女19例,为正常对照组。
2.研究组分别取术前1周内及术后1周内清晨空腹血3m1,同一时间取对照组清晨空腹血3ml,分别置于加入促凝管中,低温离心(3000r/min)10min后取上清液(血清)置于-70℃冰箱中待测Ghrelin。
3. Ghrelin的测定采用意大利西亚克公司Alisei全自动酶标分析仪,ELISA方法进行测定,酶免试剂盒由美国Phoenix公司提供(SZK15ghrelin)。
4.所有实验数据用SPSS13.0软件进行统计学处理,结果用均值±标准差(x±s)表示,两组间比较采用t检验,多组间比较用方差分析。P<0.05为差异有统计学意义
结果：
1.先天性巨结肠患儿年龄(7.40±1.28月)与正常对照年龄(7.45±0.98月)相比无统计学差异(P>0.05)
2.同样的年龄条件下,先天性巨结肠畸形患儿体重(7.25±0.38kg)和身高(63.724±1.97cm)较正常对照儿童体重(8.61±0.39kg)和身高(70.79±2.07cm)均略小,有统计学差异(P<0.05),说明先天性巨结肠患儿较正常对照组儿童瘦小。
3.同一时间段,术前1周内先天性巨结肠组血清中Ghrelin的水平(359.5±20.71ng/l)高于同一时间正常对照组血清中Ghrelin的水平(300.3±15.04ng/l)；术后1周内先天性巨结肠组血清中Ghrelin的水平(355.9±16.97ng/ml)高于同一时间正常对照组血清中Ghrelin的水平(298.8±15.34ng/ml),均有统计学差异(P<0.05)。
4.先天性巨结肠组内,术前1周内血清中Ghrelin的水平(359.5±20.71ng/l)与术后1周内血清中Ghrelin的水平(355.9±16.97ng/1),无明显统计学差异(P>0.05)；正常对照组内,同巨结肠组术前1周内一时间血清中Ghrelin的水平(300.3±15.04ng/ml)与同巨结肠术后1周内同一时间血清中Ghrelin的水平(298.8±15.34ng/l)，无明显统计学差异(P>0.05)
结论：
1.同样年龄条件下,先天性巨结肠患儿较正常对照组儿童瘦小,可能与其排便困难,营养吸收受限有关。
2.先天性巨结肠患儿血清中Ghrelin水平术前1周内及术后1周内均高于正常对照儿童血清中Ghrelin水平.先天性巨结肠患儿组(及正常对照组)术前1周内和术后1周内血清中Ghrelin水平无明显变化,说明切除肠管未能改变血清Ghrelin的水平,结肠分布的Ghrelin的作用可能为局部作用。
Ⅱ Ghrelin(GHRL)蛋白在先天性巨结肠中的表达及临床意义
试验目的：
研究Ghrelin(GHRL)蛋白在先天性巨结肠患儿不同肠段中的表达及临床意义。
试验方法：
1.选择2009年10月-2012年6月入住我院病房诊断为先天性巨结肠的患儿共40例,男22例,女18例,病理资料完整。年龄3月-12月。手术均采用经肛门巨结肠根治术,术后病理报告证实,根治术切除的结肠近端肠壁均可检测到神经节细胞,狭窄段无神经节细胞,移行段肌问神经节细胞较扩张段明显减少。
2.将手术后取出的标本分出狭窄段,移行段和扩张段一部分放置于甲醛中固定,甲醛液中固定好后将肠壁取出,梯度脱水,石蜡包埋,组织切片,厚度为4-5μm,先后经历脱蜡及水化,最后进行免疫组织化学染色(按说明说操作)。因扩张段具有正常的神经节细胞,肠神经系统发育正常,因此多种文献报道,可以讲扩张段肠管作为正常对照组进行研究。因此我们将扩张段肠管作为正常对照组来进行研究。
3.在光学显微镜下,仔细观察免疫组化染色后的结果,通过显微图像采集系统,仔细观察各组切片免疫组化的表达情况,将每张切片放在相同倍数下(20×10)观察,然后随机采取6个不同视野的图像,图像分析采用软image-proplus5.0件进行,测定的数据为反应阳性部位的积分光密度值(integral optical density, IOD)。
4.将手术后取出的标本在切除肠段中分出狭窄段、移行段及扩张段,分别取长约1cm肠段,PBS冲洗干净后放入EP管中剪碎加入细胞裂解液后进行Westemblot检测
5.将Westernblot检测胶片扫描分析。将胶片图像通过Quantity One-4.4.0软件进行图像分析,通过分别多次测定目的蛋白及Mark的平均光密度值,将图像资料转化为计数资料,进行统计
6.所有实验数据用SPSS13.0软件进行统计学处理,结果用均数±标准差s表示。对所得数据行方差分析和组间两两比较,P<0.05为差异有统计学意义。
实验结果：
1. GHRL蛋白主要分布于结肠的粘膜层内,染色为棕褐色(200倍光镜),核呈阴性。表达程度为狭窄段最少,移行段和扩张段逐渐增多。
2. Westemblot检测显示GHRL蛋白表达从狭窄段至扩张段逐渐增多.
3.利用图像分析软件对其免疫组化结果阳性染色面积平均光密度进行分析。狭窄段(0.188±0.009)、移行段(0.253±0.101)及扩张段(0.473±0.008)中GHRL的蛋白表达逐渐增多增多,具有统计学意义
4. Westemblot检测胶片扫描分析显示GHRL蛋白表达从狭窄段至扩张段逐渐增多.具有统计学意义.
实验结论：
Ghrelin蛋白表达主要分布于结肠的粘膜层的腺管上皮细胞内及细胞间,在不同肠段表达存在差异,说明Ghrelin细胞在巨结肠不同肠段的分布不同,这种不同可能造成了局部环境的改变,从而导致了不同肠段的功能存在差异,内环境的改变有可能自胚胎时期导致了病变肠段神经节细胞在发育或分化过程障碍,最终导致先天性巨结肠的产生。
Ⅲ Ghrelin(GHRL)蛋白对神经干细胞分化的影响
试验目的：
研究Ghrelin(GHRL)蛋白及其拮抗剂在神经干细胞分化中的影响及临床意义。
试验方法：
1.神经干细胞的原代培养及传代：采用山东大学实验动物中心提供的孕12-14天SD大鼠,复合麻醉后,取出胚胎,取胎鼠的大脑皮层组织剪碎后处理成单细胞悬液,接种于未包被的细胞培养瓶中,加入无血清的神经干细胞培养基进行神经干细胞培养。神经干细胞球培养成功后进行传代,经过2-3代培养后将神经干细胞球进行鉴定和分化实验。
2.神经干细胞的鉴定：将培养好的神经干细胞球打散成单个神经干细胞,接种于装有多聚赖氨酸包被玻片的5ml培养皿中,固定后进行神经干细胞特异性抗体Nestin免疫荧光检测。
3. Ghrelin(GHRL)蛋白对神经干细胞分化情况的影响：将分散好的单个神经干细胞,以5×105/ml的浓度接种于装有多聚赖氨酸包被好的玻片的5ml培养皿中,液体量以能将神经干细胞铺到盖玻片上即可。将接种好的培养皿分成4组(A,B,C,D),分别加入除无血清的神经干细胞培养基5ml外,分别加入(A)：10%胎牛血清；(B)10%胎牛血清,加Ghrelin (10-5mol/L);(C)10%胎牛血清,加Ghrelin特异性受体拮抗剂[D-Lys3]-GHRP-6(10-5mol/L);(D)10%胎牛血清,加Ghrelin (10-5mol/L),加Ghrelin特异性受体拮抗剂[D-Lys3]-GHRP-6(10-5mol/L)。
4.将分组好的(A)(B)(C)(D)四组细胞培养48h后观察分化情况,并且给予固定后进行神经元细胞特异性抗体MAP免疫荧光检测。
5.染色完成后,在荧光显微镜下,每个实验组分别随即选取5个高倍镜视野,计算阳性细胞个数,最终取平均值,通过SPSS13.0软件进行统计学处理,结果用均数±标准差s表示。对所得数据行组间两两比较,P<0.05为差异有统计学意义。
实验结果：
1.神经干细胞通过Nestin染色显示,细胞核通过DAPI染色,神经元细胞通过MAP2染色。
2.神经干细胞分化为神经元细胞过程中,加入Ghrelin组(B组)分化为神经元细胞的数量明显多于普通培养组(A组)、加Ghrelin特异性受体拮抗剂[D-Lys3]-GHRP-6的组(C组)及加Ghrelin和特异性受体拮抗剂[D-Lys3]-GHRP-6组(D组)；普通培养组(A组)、加入拮抗剂组(C组)及加Ghrelin和特异性受体拮抗剂组(D组)神经干细胞分化为神经元细胞的数量相似,无明显差异。
实验结论：
Ghrelin蛋白可以明显改善神经干细胞在分化过程中的内环境,减少神经干细胞的凋亡,使神经干细胞分化为神经元细胞的数量增加。
Hirschsprung's disease (HD) is a congenital disorder that occurs in1:3000-1:5000live births. Among congenital digestive track malformations, Hirschsprung's disease (HD) remains the most frequent cause of child intestinal obstruction. Aganglionosis is attributed to a disorder of the enteric nervous system (ENS) whereby ganglion cells fail to innervate the lower gastrointestinal tract during embryonic development. It is characterized by an absence of enteric neurons along a variable region of the gastrointestinal tract, leading to functional obstruction and colonic dilatation proximal to the affected segment. Recently, several genes controlling morphogenesis and differentiation of the ENS have been identified, including RET, EDNRB, PHOX2B, ED3, GDNF and Bcl-2. At the same time, the defects of internal environment also effect on the development of embryonic intestinal nerve, including extracellular matrix, cell adhesion molecules, hypoxia, toxin and inflammation. In a word, the pathogen of Hirschsprung's disease (HD) includes many kinds of genetic factor and environmental factors.
Neurexin are prepsynaptic neuro protein and Neuroligin are postsynapdc neuro protein. The Interactions of Neurexin and Neuroligin play an important role in the occurrence and development of nerve synapse. As we know, a number of report show that the interactions of Neurexin and Neuroligin can promote the occurrence and development of nerve synapse in Central Nervous System (CNS). Intestinal tract has a complex and independent enteric nervous system (ENS), including gastrointestinal dynamic system and sensory effect system. The Structure and Function of enteric nervous system (ENS) were similitude with Central Nervous System (CNS). The functions of enteric nervous system (ENS) also depend on the perfect nerve synapse in enteric nervous system (ENS). Aganglionosis is attributed to a disorder of the enteric nervous system (ENS) in Hirschsprung's disease (HD). Neurexin and Neuroligin protein are correlated with the development and type of nerve synapse, both of them can be used for further study.
Ghrelin was first reported by Kojmia in1999, which identified as an endogenous ligand for the GH secretagogue receptor (GHS-R). Ghrelin is a novel28-amino-acid peptide that is principally released from X/A-like cells in the oxyntic mucosa of the stomach. There was report that Ghrelin could be found in all human gastrointestinal tract. Except the gastrointestinal tract, Ghrelin could be found in various organs and tissue, including pancreas, kidney, liver and hypothalamus and the tumor tissues etc.
As we have known, a number of report show that apart from its effect on energy balance, ghrelin has numerous biological actions, such as modulation of cell proliferation and survival and stimulates motility (in vitro). Jeffery reports that Ghrelin can stimulate the proliferation and differentiation of tumor cells. Currently, as we know, the function of Ghrelin include:1.promote the release of many hormone (Growth Hormone, prolactin and epinephrine, etc.).2. Whet appetite; promote food intake and gastrointestinal propulsive motivity.3. Stimulate the proliferation and differentiation of tumor cells, and inhibited apoptosis.4. Stimulate the proliferation and differentiation of many kinds of cells (immune cells and osteoblast. etc.)
Hirschsprung's disease (HD) is characterized by an absence of enteric neurons along a variable region of the gastrointestinal tract. Aganglionosis is attributed to a disorder of the enteric nervous system (ENS) whereby ganglion cells fail to innervate the lower gastrointestinal tract during embryonic development. Up to now, the pathogen of Hirschsprung's disease (HD) includes many kinds of genetic factor and environmental factors. Why the genetic factor or environmental factors happen, but the mechanism of which is unknown.
In our prophase research, we have found that Neurexin and Neuroligin are expressed in the ENS of the humane and the animal (mice), and Neurexin are presynaptic neurons protein and Neuroligin are postsynaptic neurons protein. The next experiment, we detect the expression levels of Nurexin and Neuroligin protein in different intestinal segments of patients with Hirschsprung's disease, and investigate the clinical significance of the expression.
Kawamura report that the mRNA of Ghrelin haven happened in the embryonic period, and runs through the whole life of the embryonic period. As we have known, Ghrelin can stimulate the proliferation and differentiation of many kinds of cells (immune cells and osteoblast. etc.). We can hypothesis that Ghrelin may affect the proliferation and differentiation of neural stem cell in colon during embryonic period by affect the many kinds of genetic factor (including Nurexin and Neuroligin, etc.) and environmental factors. So, we design the experiments to confirm our hypothesis.
Part I The expression and clinical significance of Nurexin and Neuroligin protein in paitients with Hirschsprung's disease
Objective:
To detect the expression levels of Nurexin and Neuroligin protein in different intestinal segments of patients with Hirschsprung's disease, and investigate the clinical significance of the expression.
Methods:
1. Twenty paitients with Hirschsprung's disease that successfully underwent standardized chemotherapy (Soave's trans-anal endorectal pull-through for Hirschsprung's disease) from October2009to June2012in Qilu Hospital, Shandong University were enrolled in our study. The study group consisted of12males and8females, ranging in age from3months to30months. Part of normal intestinal segments were resected and ganglion cells were found in resection margin by intraoperative biopsies.
2. Approximately1cm aganglionic, transitional and normal segments were harvested from the colon of the children patients. All the tissues were placed in Phosphate Buffer Solution (PBS), and were cut in the EP tube mixed with cell lysis buffer for Western-blot test.
Results:
There was no expression of Nurexin and Neuroligin protein in aganglionic segments; the expression levels in transitional segments were lower than that of normal segments.
Conclusion:
The expression levels of Nurexin and Neuroligin protein in different intestinal segments was consistent with the distribution of ganglion cells. These findings suggest the interaction of Nurexin and Neuroligin protein in ganglion cells, which may participate in the development and function of enteric nervous system.
Part II The expression and clinical significance of Ghrelin(GHRL) in patients with Hirschsprung's disease
I The expression of Ghrelin(GHRL) in plasma of paitients with Hirschsprung's disease
Objective:
To detect the expression levels of Ghrelin in normal children and patients with Hirschsprung's disease, and evaluate the relevance of Ghrelin expression to Hirschsprung's disease.
Methods:
1. Forty patients with Hirschsprung's disease from October2009to June2012in Qilu Hospital, Shandong University were enrolled in our study as study group, consisted of22males and18females. Forty normal children, consisted of21males and19females, were enrolled as control group.
2.3ml of fasting blood was drawn before and after operation in study group and control group respectively. The specimen were centrifugation10minutes at the speed of3000r.p.m. Remove supernatant (serum) and store in refrigerator at-70℃.
3. The Ghrelin serum levels were determined using ELISA by Alisei enzyme micro-plate reader. The ELISA kit was provided by Phoenix Company, American (SZK15ghrelin).
4. All ststistical analyses were performed with SPSS13.0statistical software. The t-test and variance analysis were performed to examine the differerces between study and control group. Statistical significance was assessed by comparing mean values (Means±SD). The significance level was set at P<0.05.
Results:
1. There was no dramatically difference in ages between study group(7.40+1.28months) and control group(7.45±0.98months),(P>0.05). The body weight in study group (7.25±0.38kg) was less than that in control group (8.61±0.39kg) with dramatically difference (P<0.05). The length of children in study group(63.72±1.97cm) was less than that in control group(70.79±2.07cm) dramatically (P<0.05). It might suggest that patients with Hirschsprung's disease were thinner and shorter than normal children.
2. Before the operation, the Ghrelin serum levels in study group (359.5±20.71ng/l) was higher than that in control group (300.3±15.04ng/l) with dramatically difference (P<0.05). After the operation, the Ghrelin serum levels in study group (355.9±16.97ng/l) was higher than that in control group (298.8±15.34ng/l) dramatically (P<0.05).
3. In the study group, the Ghrelin serum levels before the operation was359.5±20.71ng/l, with no dramatically difference to that after operation(355.9±16.97ng/),(P>0.05). In the control group, there was no dramatically difference in Ghrelin serum levels before and after operation (P>0.05)
Conclusion:
1. Patients with Hirschsprung's disease were thinner and shorter than normal children of same age, which may be influenced by difficult defecation or limited absorption of nutrients.
2. the Ghrelin serum levels in study group was higher than that in control group before and after operation, which suggest the correlation beween Ghrelin and incidence of Hirschsprung's disease.
3. Both in study and control group, there was no dramatically difference in Ghrelin serum levels before and after operation. Ghrelin in colon may not influent the children's growth and development in the way of endocrine into blood, but play its role by autocrine or paracrine.
Ⅱ Altered expressions of Ghrelin protein in infantile Hirschsprung's disease
Objective:
To examine the expression levels of Ghrelin (GHRL) in infantile Hirschsprung's disease, and evaluate the clinical significance of the expression.
Methods:
1. We collected40children who suffer Hirschsprung's disease between October2009and June2012, including male22cases and female18cases. The pathological files of all patients were full. The range of age was3month to12month. All children patients were treated by standardized chemotherapy (SOAVE'S trans-anal endorectal pull-through for Hirschsprung's disease). The postoperative pathological report certified that gangliocytes were detected in normal segments, no gangliocyte in narrow segments, and decreasing gangliocytes in transitional segments.
2. The aganglionic, transitional and normal segments of colon were immersed into4%paraformaldehyde dissolved in0.1M PB for24hours. These samples were then cut into4μm-5μm coronal sections after dehydration using graded ethanol and paraffin imbedding, and Immunohistochemistry staining was operated after dewaxing and hydration.
3. Immunohistochemistry Staining was observed by microscope. Six visions were collected randomly in every section under microscope (40*10magnifications). The digital images were analyzed by mage-proplus5.0, determining the integral optical density (IOD) of reactive positive positions.
4. Approximately1cm aganglionic, transitional and normal segments were harvested from the colon of the children patients. All the tissues were placed in Phosphate Buffer Solution (PBS), and were cut in the EP tube mixed with cell lysis buffer for Western-blot test.
5. Data analyses were performed using SPSS statistical program version13.0. Data was shown as mean±SD. Analysis of variance and compared between groups were employed. P<0.05was considered statistically significant.
Results:
1. Ghrelin protein was expressed on the mucous layer, stained sepia (200magnifications). In the aganglionic colonic segment Ghrelin protein expressed rarely. It was expressed moderately in the transition zone. Ghrelin protein was conspicuously expressed in the ganglionic colonic segment in patients with Hirschsprung's disease.
2. The IOD of aganglionic colonic segment was0.188±0.009, transitional segment was0.253±0.101and normal segment was0.473±0.008. There was statistically significant difference between groups.
Conclusion:
Ghrelin protein was expressed in gland epithelium or among gland epithelium on the mucous layer. The expression was different in different position of intestinal tract. The different distribution of Ghrelin protein in Hirschsprung's discase may change the local internal environment, leading to the different function of intestinal segment. The development and differentiation of gangliocyte was disordered since embryonic stage because of changed internal environment, resulting in Hirschsprung's disease.
Ⅲ Effects of Ghrelin Protein on the Differentiation of Neural Stem Cells
Objective:
To explore the effects of Ghrelin protein on the differentiation of neural stem cells (NSCs) and the clinical significance.
Methods:
1. Cell culture of neural stem cells:Pregnant SD mice provided by Laboratory Animal Centre of Shandong University were fixed after anesthesia. The embryos (12-14days) were removed and cerebral cortex tissues were collected to prepare monoplast suspension. The tissues were inoculated in no packed cell culture bottle, and then serum-free culture medium was joined. After the neural stem cell globes were cultured successfully, isolation and determination of neural stem cells were operated after p3passage.
2. Determination of neural stem cells:single neural stem cells were collected and planted on slide coated by polylysine of5ml culture dish. Immunofluorescence was detected after the cells were blocked and antibodies were added.
3. Effects of Ghrelin protein on the differentiation of neural stem cells:the sells suspension (5x105/ml) was planted on slide of5ml culture dish which was coated with polylysine. The planted dishes were divided into4groups (A, B, C, and D). The serum-free culture medium was joined into every dish, but10%fetal bovine serum was added in group A;10%fetal bovine serum and Ghrelin (10-5mol/L) were added in group B;10%fetal bovine serum and specific receptor antagonist of Ghrelin ([D-Lys3]-GHRP-6)(10-5mol/L) were added in group C;10%fetal bovine serum, Ghrelin (10-5mol/L) and specific receptor antagonist of Ghrelin ([D-Lys3]-GHRP-6)(10-5mol/L) were added in group D.
4. After culturing for48hours, differentiation of the neural stem cells in these four groups was observed separately and immunofluorescence of neuron cell was detected.
5. After stained, the number of positive cells in5random views at high magnification was calculated, the mean was adopted finally. Data analyses were performed using SPSS statistical program version13.0. Data was shown as mean±SD. Analysis of variance and compared between groups were employed. P<0.05was considered statistically significant.
Results:
1. The neural stem cells can be identified with Nestin stain, nucleus can be identified with DAPI stain, and neuron cell can be identified with MAP stain.
2. The number of neuron cells differentiated from neural stem cells in group B was more than that in group A, group C and group D. There was statistically significant difference. The number of neuron cells differentiated from neural stem cells in group D was similar with group A and C. There was no statistically difference between these three groups.
Conclusion:
Ghrelin protein improves the internal environment obviously in the process of neural stem cells differentiation, decreases the apoptosis of neural stem cells, and increases the number of neuron cells differentiated from neural stem cells.
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