巨细胞病毒感染在肠神经节发育异常中作用机制的初步研究

发布时间：2018-06-20 10:46

本文选题：Hirschsprung’病及其同源病 + 肠神经节发育异常　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分人肠神经节发育异常与巨细胞病毒感染之间相关性分析目的：探讨人肠神经节发育异常与巨细胞病毒感染之间的相关性。方法：随机选取2012年9月-2014年9月间我科的先天性巨结肠及同源病的30例患儿血标本及60份结肠标本(结肠标本包含近端肠神经节正常段结肠和远端肠神经节异常段结肠)作为肠神经节发育异常组；选取同时间段的因肠套叠肠坏死接受肠切除肠吻合的10例患儿血标本和10份结肠标本作为对照组。运用ELISA分析两组患儿血标本CMV-IgG和CMV-IgM阳性率,运用免疫组织化学方法检测两组患儿结肠标本中CMV晚期蛋白表达,运用免疫原位杂交技术检测两组患儿结肠标本中CMV-DNA,比较两组患儿结肠CMV感染的差异,同时明确CMV感染结肠的位置。结果：肠神经节发育异常组30例血标本中CMV-IgG抗体阳性者8例(26.7%),对照组血标本无阳性病例(P0.05),CMV-IgM抗体两组均为阴性；肠神经节发育异常组结肠标本共60份,其中CMV晚期蛋白阳性13份(21.7%,其中6份为HD结肠标本,7份为HAD结肠标本,均表达在肠神经节异常段结肠),对照组结肠标本中均未检测到CMV晚期蛋白表达(P0.05)；在肠神经节发育异常组中原位杂交技术检测到的CMV-DNA阳性率与CMV晚期蛋白阳性率重叠,对照组结肠中未检测到CMV-DNA。CMV-DNA与CMV晚期蛋白的阳性表达的位置不同,CMV晚期蛋白阳性表达主要集中于肠神经节发育异常段结肠黏膜层、黏膜下层以及小部分的肠肌层的血管内皮细胞,在近端正常的肠神经节内无表达,CMV-DNA在近端正常结肠段和远端异常段结肠的黏膜层、黏膜下层、肌层以及神经节均呈棕黄色阳性显色,尤其在肠神经节细胞中呈强阳性显色。结论：HCMV感染与肠道神经节细胞发育异常密切相关,可能是其发病的又重要因素。第二部分先天性MCMV感染致新生小鼠肠神经节发育异常模型的建立目的：建立先天性MCMV感染致新生鼠肠神经节细胞发育异常的模型。方法：将无MCMV感染6周龄的Balb/c小鼠按雌雄2：1合笼,根据阴栓判断孕鼠的受孕时间,选取小鼠胚胎后肠肠神经发育的起始时间点对孕鼠进行接种MCMV,在孕鼠受孕lld时行腹腔接种1ml不同浓度的MCMV病毒悬液,每种浓度5只孕鼠,观察不同浓度病毒剂量对孕鼠及胎鼠的影响,确定造模病毒浓度；确定造模病毒浓度后,选取造模所需的病毒浓度1ml,孕11d时接种造模浓度MCMV孕鼠作为模型组,含10只孕鼠,同样孕龄接种不含MCMV的细胞培养基1ml孕鼠作为对照组,含5只孕鼠,观察两组小鼠胚胎的存活率,新生小鼠的体重,同时运用PCR检测胎鼠结肠CMV-DNA,免疫组化了解新生鼠结肠MCMV蛋白的表达,原位杂交技术检测新生小鼠结肠CMV特异性核酸序列定位,观察胎鼠结肠神经嵴干细胞和新生小鼠肠神经节细胞的变化,来判断肠神经节细胞发育异常模型发生。结果：1×102PFU/ul浓度接种孕鼠腹腔后胎鼠肠道中检测不到MCMV,对孕鼠、胎鼠及新生鼠生长发育无明显影响：1×104PFU/ul浓度接种孕鼠后,在接种后36h内孕鼠全部死亡；1×103PFU/ul浓度的行孕鼠腹腔内注射后可导致孕鼠的产量降低,同时可造成存活新生小鼠肠道巨细胞病毒感染和肠神经节细胞发育异常的发生,确定造模MCMV浓度为1×103PFU/ul。造模浓度1×103PFU/ul接种的孕鼠为实验组,与对照组相比,实验组孕鼠在接种MCMV24小时内出现反应迟缓,食欲下降,活动减少,后逐渐恢复正常活动,同时观察实验组新生小鼠的体重明显低于对照组(P0.05),实验组胎鼠及新生鼠肠管CMV晚期蛋白可检测到阳性表达,CMV-DNA原位杂交也呈阳性,孕鼠腹腔接种可造成胎鼠的先天性CMV感染；实验组孕鼠中检测到新生小鼠发生肠神经节发育异常的比例约14%,肠神经节发育异常模型新生小鼠与对照组新生小鼠相比,模型新生鼠腹胀明显,解剖发现结肠远端出现狭窄及近端扩张,病理特征主要表现为结肠远端肠神经节细胞数量减少(P0.05)。结论：先天性CMV感染可造成肠神经节发育异常模型的发生,为进一步的CMV感染造成肠神经系统发育异常的机制研究奠定基础。第三部分先天性MCMV感染后细胞免疫在肠神经节发育异常中作用机制的初步探讨目的：初步探讨先天性MCMV感染后细胞免疫在小鼠肠神经节发育异常中的作用机制。方法：实验分先天性MCMV感染组和对照组,对两组胎鼠肠管进行解剖行离体培养,观察比较两组肠管的蠕动及存活时间情况,运用流式细胞学技术检测两组新生1d小鼠外周血效应性DC和CD8+CTL的差异,Western Blot方法检测两组胎鼠结肠组织特异性杀伤作用的细胞因子穿孔素和颗粒酶的表达水平；同时对小鼠胚胎GNCSCs进行分离、培养和鉴定,然后与MCMV共培养,运用Western Blot共培养后48h后GNCSCs的分子MHC-Ⅰ、Fas、TNFR1表达水平变化。结果：与对照组相比,先天性MCMV感染的实验组胎鼠肠体外培养的蠕动明显减弱,平均生长时间较正常对照组缩短(P0.05),MCMV感染模型组1d小鼠的外周血CD8+CTL和效应DC的比例增高(P0.05),模型小鼠胚胎结肠组织穿孔素和颗粒酶的表达量明显上升(P0.05)；GNCSCs与MCMV共培养后48h,与对照组比较,其形态及生长并无明显改变,但其表面分子MHC-Ⅰ、Fas和TNFR1表达量上调(P0.05)。结论：先天性CMV感染胚胎后,诱导效应DC和CD8+CTL增殖,胚胎肠道的GNCSCs的MHC-Ⅰ、Fas和TNFR1分子表达上调,可促使GNCSCs被CD8+CTL特异性识别结合诱导其凋亡,同时也可能通过CTL的细胞毒性颗粒穿孔素和颗粒酶在经典穿孔途径下造成GNCSCs坏死,造成GNCSCs发育障碍,从而导致肠神经节发育异常。
[Abstract]:Part one: correlation analysis between human intestinal ganglion dysplasia and cytomegalovirus infection
Objective: To explore the correlation between human intestinal ganglion dysplasia and cytomegalovirus infection.
Methods: 30 cases of congenital megacolon and 30 cases of homologous disease and 60 colonic specimens (colon specimens including normal colon and distal intestinal ganglion in the proximal intestinal ganglia of the proximal intestinal ganglia Duan Jiechang) were selected randomly as the intestinal ganglion dysplasia in September -2014. The blood specimens and 10 colonic specimens of 10 cases of intestinal anastomosis were used as control group. The positive rates of CMV-IgG and CMV-IgM in blood specimens of two groups of children were analyzed by ELISA, and the expression of late CMV protein in the colon specimens of the two groups of children was detected by immunohistochemistry, and CMV-DN in the colon specimens of two groups of children was detected by the immuno in situ hybridization technique. A, compare the difference of CMV infection between the two groups, and confirm the location of CMV infection colon.
Results: there were 8 cases (26.7%) of CMV-IgG positive CMV-IgG antibody in the intestinal ganglionic dysplasia group, and no positive cases in the control group (P0.05), and the two groups of CMV-IgM antibody were negative, and 60 colonic specimens from the intestinal ganglionic dysplasia group, of which 13 were CMV late protein positive (21.7%, 6 of them were HD colon specimens, 7 were HAD colonic specimens. " The expression of late CMV protein expression (P0.05) was not detected in the colon specimens of the control group. In the group of intestinal ganglia dysplasia, the positive rate of CMV-DNA was overlapped with the positive rate of late CMV protein in the group of intestinal ganglion dysplasia, and the positive rate of CMV-DNA.CMV-DNA and CMV in the colon was not detected in the control group. The positivity of the expression was different. The positive expression of CMV advanced protein mainly concentrated on the colonic mucosa of the abnormal segments of the intestinal ganglia, the submucosa and the small part of the intestinal myocutaneous vascular endothelial cells, which were not expressed in the normal intestinal ganglia of the proximal end. The CMV-DNA was in the mucosa of the proximal normal colon and the distal abnormal segment of the colon, the submucosa, and the myometrium. And the ganglion showed a positive yellow coloration, especially in intestinal ganglion cells.
Conclusion: HCMV infection is closely related to dysplasia of intestinal ganglion cells, and may be an important factor in its pathogenesis.
The second part is the establishment of neonatal rat intestinal ganglia dysplasia model induced by congenital MCMV infection.
Objective: to establish a model of intestinal ganglion cell dysplasia induced by congenital MCMV infection in neonatal rats.
Methods: the 6 weeks old Balb/c mice without MCMV infection were caged by male and male 2:1, and the pregnancy time of pregnant rats was judged according to the suppository. The starting point of the development of the hindgut nerve of the mice was selected to inoculate the pregnant mice with MCMV. The MCMV virus suspension of different concentrations of 1ml was inoculated in the pregnant rats when the pregnant rats were pregnant LLD, and each concentration was different. The effect of viral dose on pregnant rats and fetal rats was determined. After determining the concentration of the model virus, the virus concentration required by the mold making was selected 1ml. The pregnant 11d was inoculated with the model concentration MCMV pregnant rats as model group, 10 pregnant mice, and the same gestational age inoculated with 1ml pregnant mice without MCMV, including 5 pregnant mice. The survival rate of the two groups of mice, the weight of the newborn mice, the detection of CMV-DNA in the colon of fetal mice by PCR, the expression of MCMV protein in the colon of the newborn rats by immunohistochemistry, in situ hybridization technique to detect the specific nucleotide sequence of the colon CMV specific nucleic acid in the newborn mice, and to observe the changes of the colon neural crest stem cells and the intestinal ganglion cells of the newborn mice. A model of abnormal ganglion cell development was established.
Results: the growth and development of pregnant rats, fetal rats and newborn rats were not significantly affected by 1 x 102PFU/ul concentration inoculated in the intestines of pregnant rats. The growth and development of pregnant rats, fetal rats and newborn rats were not obvious. After inoculation of 1 x 104PFU/ul, all pregnant rats died in 36h after inoculation, and the yield of pregnant rats after intraperitoneal injection of 1 x 103PFU/ul concentration could lead to reduced yield of pregnant rats. At the same time, the yield of pregnant rats could be reduced. The infection of the enteric giant cytomegalovirus and the abnormal development of intestinal ganglion cells in the newborn mice were caused. The pregnant rats with the concentration of 1 x 103PFU/ul. with the concentration of 1 x 103PFU/ul. and the inoculated mice were determined to be the experimental group. Compared with the control group, the mice in the experimental group had a slow reaction, a decline in appetite, a decrease in activity, and a gradual recovery after the inoculation for MCMV24 hours. The body weight of the newborn mice in the experimental group was significantly lower than that of the control group (P0.05). The positive expression of the late CMV protein in the fetal and neonatal rats in the experimental group was detected, and the CMV-DNA in situ hybridization was also positive. The intraperitoneal inoculation of pregnant mice could cause congenital CMV infection of the fetal mice; in the experimental group, the newborn mice were detected in the intestines of the newborn mice. The proportion of abnormal ganglion development was about 14%. The newborn mice with intestinal ganglia dysplasia model had obvious abdominal distention compared with those of the control group, and the dissection found the narrowing and proximal dilatation of the distal colon, and the main pathological features were the decrease of the number of intestinal ganglion cells in the distal colon (P0.05).
Conclusion: congenital CMV infection can cause an abnormal model of intestinal ganglion development, which lays the foundation for further study of the mechanism of the abnormal development of the enteric nervous system caused by CMV infection.
The third part is the preliminary study on the mechanism of cellular immunity after congenital MCMV infection in the development of intestinal ganglia.
Objective: To explore the mechanism of cellular immunity after congenital MCMV infection in the development of intestinal ganglia in mice.
Methods: the congenital MCMV infection group and the control group were divided into two groups of fetal rat intestines in vitro. The peristalsis and survival time of the two groups of intestinal tubes were observed and compared. The difference between the peripheral blood effector DC and CD8+CTL in the two new 1D mice was detected by flow cytometry. The colonic tissue of the two groups of fetal rats was detected by Western method. The expression level of perforin and granzyme of specific cytotoxic cytokine; at the same time, the mouse embryo GNCSCs was isolated, cultured and identified, then co cultured with MCMV, and the GNCSCs molecule MHC- I, Fas, and TNFR1 expression levels were changed after co culture of Western Blot.
Results: compared with the control group, the peristaltic peristalsis in the intestinal culture of fetal rats with congenital MCMV infection was significantly reduced, the average growth time was shorter than that of the normal control group (P0.05). The proportion of CD8+CTL and effect DC in the 1D mice of MCMV infection model group increased (P0.05), and the expression of perforin and granzyme in the colon tissue of the model mice was clear. Significant rise (P0.05); GNCSCs and MCMV were co cultured with 48h. Compared with the control group, there was no obvious change in morphology and growth, but the expression of MHC- I, Fas and TNFR1 was up to up (P0.05).
Conclusion: after congenital CMV infection, the induced effect of DC and CD8+CTL proliferation, the up-regulated expression of MHC- I, Fas and TNFR1 molecules in the GNCSCs of the fetal intestinal tract can induce GNCSCs to be induced by CD8+CTL specific recognition and induce its apoptosis. Meanwhile, the cytotoxic granulin and granzyme of CTL may cause GNCSCs damage under the classic perforation pathway. Death causes dysplasia of GNCSCs, resulting in dysplasia of the intestinal ganglia.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R725.7

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