發(fā)布時間：2018-06-20 10:46

  本文選題：Hirschsprung’病及其同源病 + 腸神經(jīng)節(jié)發(fā)育異常�。� 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：第一部分人腸神經(jīng)節(jié)發(fā)育異常與巨細(xì)胞病毒感染之間相關(guān)性分析 目的：探討人腸神經(jīng)節(jié)發(fā)育異常與巨細(xì)胞病毒感染之間的相關(guān)性。 方法：隨機選取2012年9月-2014年9月間我科的先天性巨結(jié)腸及同源病的30例患兒血標(biāo)本及60份結(jié)腸標(biāo)本(結(jié)腸標(biāo)本包含近端腸神經(jīng)節(jié)正常段結(jié)腸和遠端腸神經(jīng)節(jié)異常段結(jié)腸)作為腸神經(jīng)節(jié)發(fā)育異常組；選取同時間段的因腸套疊腸壞死接受腸切除腸吻合的10例患兒血標(biāo)本和10份結(jié)腸標(biāo)本作為對照組。運用ELISA分析兩組患兒血標(biāo)本CMV-IgG和CMV-IgM陽性率,運用免疫組織化學(xué)方法檢測兩組患兒結(jié)腸標(biāo)本中CMV晚期蛋白表達,運用免疫原位雜交技術(shù)檢測兩組患兒結(jié)腸標(biāo)本中CMV-DNA,比較兩組患兒結(jié)腸CMV感染的差異,同時明確CMV感染結(jié)腸的位置。 結(jié)果：腸神經(jīng)節(jié)發(fā)育異常組30例血標(biāo)本中CMV-IgG抗體陽性者8例(26.7%),對照組血標(biāo)本無陽性病例(P0.05),CMV-IgM抗體兩組均為陰性；腸神經(jīng)節(jié)發(fā)育異常組結(jié)腸標(biāo)本共60份,其中CMV晚期蛋白陽性13份(21.7%,其中6份為HD結(jié)腸標(biāo)本,7份為HAD結(jié)腸標(biāo)本,均表達在腸神經(jīng)節(jié)異常段結(jié)腸),對照組結(jié)腸標(biāo)本中均未檢測到CMV晚期蛋白表達(P0.05)；在腸神經(jīng)節(jié)發(fā)育異常組中原位雜交技術(shù)檢測到的CMV-DNA陽性率與CMV晚期蛋白陽性率重疊,對照組結(jié)腸中未檢測到CMV-DNA。CMV-DNA與CMV晚期蛋白的陽性表達的位置不同,CMV晚期蛋白陽性表達主要集中于腸神經(jīng)節(jié)發(fā)育異常段結(jié)腸黏膜層、黏膜下層以及小部分的腸肌層的血管內(nèi)皮細(xì)胞,在近端正常的腸神經(jīng)節(jié)內(nèi)無表達,CMV-DNA在近端正常結(jié)腸段和遠端異常段結(jié)腸的黏膜層、黏膜下層、肌層以及神經(jīng)節(jié)均呈棕黃色陽性顯色,尤其在腸神經(jīng)節(jié)細(xì)胞中呈強陽性顯色。 結(jié)論：HCMV感染與腸道神經(jīng)節(jié)細(xì)胞發(fā)育異常密切相關(guān),可能是其發(fā)病的又重要因素。 第二部分先天性MCMV感染致新生小鼠腸神經(jīng)節(jié)發(fā)育異常模型的建立 目的：建立先天性MCMV感染致新生鼠腸神經(jīng)節(jié)細(xì)胞發(fā)育異常的模型。 方法：將無MCMV感染6周齡的Balb/c小鼠按雌雄2：1合籠,根據(jù)陰栓判斷孕鼠的受孕時間,選取小鼠胚胎后腸腸神經(jīng)發(fā)育的起始時間點對孕鼠進行接種MCMV,在孕鼠受孕l(wèi)ld時行腹腔接種1ml不同濃度的MCMV病毒懸液,每種濃度5只孕鼠,觀察不同濃度病毒劑量對孕鼠及胎鼠的影響,確定造模病毒濃度；確定造模病毒濃度后,選取造模所需的病毒濃度1ml,孕11d時接種造模濃度MCMV孕鼠作為模型組,含10只孕鼠,同樣孕齡接種不含MCMV的細(xì)胞培養(yǎng)基1ml孕鼠作為對照組,含5只孕鼠,觀察兩組小鼠胚胎的存活率,新生小鼠的體重,同時運用PCR檢測胎鼠結(jié)腸CMV-DNA,免疫組化了解新生鼠結(jié)腸MCMV蛋白的表達,原位雜交技術(shù)檢測新生小鼠結(jié)腸CMV特異性核酸序列定位,觀察胎鼠結(jié)腸神經(jīng)嵴干細(xì)胞和新生小鼠腸神經(jīng)節(jié)細(xì)胞的變化,來判斷腸神經(jīng)節(jié)細(xì)胞發(fā)育異常模型發(fā)生。 結(jié)果：1×102PFU/ul濃度接種孕鼠腹腔后胎鼠腸道中檢測不到MCMV,對孕鼠、胎鼠及新生鼠生長發(fā)育無明顯影響：1×104PFU/ul濃度接種孕鼠后,在接種后36h內(nèi)孕鼠全部死亡；1×103PFU/ul濃度的行孕鼠腹腔內(nèi)注射后可導(dǎo)致孕鼠的產(chǎn)量降低,同時可造成存活新生小鼠腸道巨細(xì)胞病毒感染和腸神經(jīng)節(jié)細(xì)胞發(fā)育異常的發(fā)生,確定造模MCMV濃度為1×103PFU/ul。造模濃度1×103PFU/ul接種的孕鼠為實驗組,與對照組相比,實驗組孕鼠在接種MCMV24小時內(nèi)出現(xiàn)反應(yīng)遲緩,食欲下降,活動減少,后逐漸恢復(fù)正�；顒�,同時觀察實驗組新生小鼠的體重明顯低于對照組(P0.05),實驗組胎鼠及新生鼠腸管CMV晚期蛋白可檢測到陽性表達,CMV-DNA原位雜交也呈陽性,孕鼠腹腔接種可造成胎鼠的先天性CMV感染；實驗組孕鼠中檢測到新生小鼠發(fā)生腸神經(jīng)節(jié)發(fā)育異常的比例約14%,腸神經(jīng)節(jié)發(fā)育異常模型新生小鼠與對照組新生小鼠相比,模型新生鼠腹脹明顯,解剖發(fā)現(xiàn)結(jié)腸遠端出現(xiàn)狹窄及近端擴張,病理特征主要表現(xiàn)為結(jié)腸遠端腸神經(jīng)節(jié)細(xì)胞數(shù)量減少(P0.05)。 結(jié)論：先天性CMV感染可造成腸神經(jīng)節(jié)發(fā)育異常模型的發(fā)生,為進一步的CMV感染造成腸神經(jīng)系統(tǒng)發(fā)育異常的機制研究奠定基礎(chǔ)。 第三部分先天性MCMV感染后細(xì)胞免疫在腸神經(jīng)節(jié)發(fā)育異常中作用機制的初步探討 目的：初步探討先天性MCMV感染后細(xì)胞免疫在小鼠腸神經(jīng)節(jié)發(fā)育異常中的作用機制。 方法：實驗分先天性MCMV感染組和對照組,對兩組胎鼠腸管進行解剖行離體培養(yǎng),觀察比較兩組腸管的蠕動及存活時間情況,運用流式細(xì)胞學(xué)技術(shù)檢測兩組新生1d小鼠外周血效應(yīng)性DC和CD8+CTL的差異,Western Blot方法檢測兩組胎鼠結(jié)腸組織特異性殺傷作用的細(xì)胞因子穿孔素和顆粒酶的表達水平；同時對小鼠胚胎GNCSCs進行分離、培養(yǎng)和鑒定,然后與MCMV共培養(yǎng),運用Western Blot共培養(yǎng)后48h后GNCSCs的分子MHC-Ⅰ、Fas、TNFR1表達水平變化。 結(jié)果：與對照組相比,先天性MCMV感染的實驗組胎鼠腸體外培養(yǎng)的蠕動明顯減弱,平均生長時間較正常對照組縮短(P0.05),MCMV感染模型組1d小鼠的外周血CD8+CTL和效應(yīng)DC的比例增高(P0.05),模型小鼠胚胎結(jié)腸組織穿孔素和顆粒酶的表達量明顯上升(P0.05)；GNCSCs與MCMV共培養(yǎng)后48h,與對照組比較,其形態(tài)及生長并無明顯改變,但其表面分子MHC-Ⅰ、Fas和TNFR1表達量上調(diào)(P0.05)。 結(jié)論：先天性CMV感染胚胎后,誘導(dǎo)效應(yīng)DC和CD8+CTL增殖,胚胎腸道的GNCSCs的MHC-Ⅰ、Fas和TNFR1分子表達上調(diào),可促使GNCSCs被CD8+CTL特異性識別結(jié)合誘導(dǎo)其凋亡,同時也可能通過CTL的細(xì)胞毒性顆粒穿孔素和顆粒酶在經(jīng)典穿孔途徑下造成GNCSCs壞死,造成GNCSCs發(fā)育障礙,從而導(dǎo)致腸神經(jīng)節(jié)發(fā)育異常。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R725.7

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