Study On Gene Regulation Networks Of Etiology And Pathogenesis Of Hirschsprung’s Disease

PART ONE EXPRESSIONAL BRIEF SUMMARY OF GANGLIONIC MARKERS IN HIRSCHSPRUNG’ S DISEASEObjective: To study the Expressing characteristics and significance of these neural markers(include BMP2, BMP4, Ret, CXCR4, CR, GDNF, NSE, S-100, CAD, Bcl-2 etc) in Hirschsprung’ Disease, and then discuss the etiological correlation and clinical diagnosis significance between these neural markers and Hirschsprung’ Disease.Methods :immunohistochemistry was used to detect the expression of these several neural markers(include BMP2, BMP4, Ret, CXCR4, CR, GDNF, NSE, S-100, CAD, Bcl-2 etc) in the colonic wall of 45 patients with HSCR( include the normal section, expansion section and spasm segment of HSCR group), 10 cases of Hirschsprung allied disease(HAD) and 10 cases of control group and the results were compared with routine HE staining.Results: The ganglion cells can be viewed in the nerve plexus of dilated bowel segment wall when HE staining, but deletion of the ganglion cells in the spasm segment. The diagnosis results of all markers betweenimmunohistochemistry staining and HE staining is accordance. 50% to 70% cases expression of BMP2, BMP4, Ret,CXCR4 and GDNF positive, above 90% cases expression of CR, NSE, S-100, CAD positive.Bcl-2 was expressed positive in the neonatal HSCR and the Hirschsprung allied disease, but not in the normal control group and well developed HSCR cases.Conclusion: These markers(include GDNF, BMP2, BMP 4, Ret and CXCR4)was involved in the enteric nervous system development by activating distinct signaling pathways and related to the etiology of part of HSCR.. Bcl-2 can be used for the identification of HSCR and HAD because it is only expressed positive in the immature neural crest cell. The superior clinical diagnosis value of HSCR combined with CR, S-100 and CAD immunohistochemistry inspection, the combined use of these indicators, the ganglion cells and nerve fibers were complementary positive color, can increase the accuracy of diagnosis.PART TWO DETECTION OF THE HUMAN GENE EXPRESSION PROFILE AND RELATED GENETIC SCREENING FOR CHILDREN WITH HIRSCHSPRUNG’S DISEASEBackground: We investigated the genome microarray of colorectal lesions(spasm segment) in children with Hirschsprung’s disease(HSCR) and analyzed the results. Additionally, we preliminarily screened for differential expression genes in children with HSCR.Methods: Microarray technology was used to test the human gene expression profiles for the colorectal lesions(spasm segment) in six children with HSCR and three normal colon tissues. The results were analyzed by the P-values and absolute fold changes et al. The meaningful differential expression genes were preliminary screened in children with HSCR, and the target gene will be analyzed in the next verification and analytical investigation.Results: In the more than 20,000 detected human genes, preliminary screenings showed 3850 genes are differential genes with up-regulated expressions whose P-values were < 0.05 and whose absolute fold changes were > 2-fold. There are 645 differential genes with down-regulated expressions whose P-values were < 0.05 and whose absolute fold changes were > 2-fold. Of the up-regulated genes, 118 were involved in classic signal pathways, and 11 down-regulated genes were involved in classic signal pathways, with a P-value of < 0.001 and an absolute fold change > 2-fold.Conclusion: HSCR etiology is very complex and often involves multiple gene changes. Microarray technology can produce large amounts of gene expression data simultaneously. Analyzing this data using varioustechniques may provide a fast and efficient method for researching new targets and directions for research on HSCR pathogenesis.PART THREE EXPRESSIONAL ANALYSIS OF WNT5 A, GLI-1 AND BMP7 IN COLON TISSUE OF CHILDREN WITH HIRSCHSPRUNG’S DISEASEObjective: To study the expression and distribution characteristics of the WNT5 a, GLI-1and BMP7 in normal section(anastomotic stoma), dilated section, and spastic section of children with Hirschsprung’s disease. To verify the result of the genome microarray, and compare with the result of normal colonic tissue. To investigate the etiological relevance of Hirschsprung’s disease with them and it’s clinical significance of molecular biology.Methods: 75 cases of children’s colonic tissue samples who was definite diagnosed as Hirschsprung’s disease in our hospital and underwent the radical operation, were collected as test group and divided into normal section(anastomotic stoma), dilated section, and spastic section; 10 cases of normal colonic tissue samples who wasn’t Hirschsprung’s disease but such as trauma et al, were collected as control group. Using immunohistochemical method to detect the expression of WNT5 a, BMP7, GLI-1 in colonic walls of 75 cases of HSCR(normal section(anastomotic stoma), dilated section, and spastic section) and 10 cases of normal control group, and Contrastive analysis compared with HE staining; Westernblotting was performed to detect the expressional levels of WNT5 a, GLI-1, BMP7 protein in different parts of the colonic organization of children with Hirschsprung’s disease; Quantitative real-time PCR was used to detect the expressional levels of WNT5 am RNA, GLI-1 m RNA, BMP7 m RNA in different parts of the colonic organization of children with Hirschsprung’s disease.Results: 1) HE staining shows that the ganglion cells can be detected in the nerve plexus of normal section and dilated section of children with Hirschsprung’s disease and normal colon, while the deficiency of ganglion cells occurred in the spasm segment. 2) Immunohistochemical methods shows that WNT5 a, GLI-1, BMP7 are expressed in the colonic tissue of normal section and dilated section of children with Hirschsprung’s disease and normal colonic tissue with ganglion cells, while they were no significant expression in the spasm segment without ganglion cells. 3) Western blotting test shows that the expressional level of WNT5 a, GLI-1, BMP7 protein in the colonic tissue of spasm segment was significantly lower than it’s expressional level in dilated section and normal section of children with Hirschsprung’s disease(P﹤0.01, n=75); 4) q RT-PCR detection shows that the expressional level of WNT5 am RNA, GLI-1 m RNA, BMP7 m RNA in the in the colonic tissue of spasm segment was significantly lower than it’s expressional level in dilated section and normal section of children with Hirschsprung’s disease(P﹤0.01, n=75); But it’s expressional level was no significant statistical difference between dilated section and normal section(P﹥0.05, n = 75);Conclusion: 1) Intensive WNT5 a, GLI-1 and BMP7 staining was detected in the colonic tissue of dilated section and normal section of children with Hirschsprung’s disease which has ganglion cells; while theexpressional level decreased significantly in the spasm segment due to a lack of ganglion cells. 2) The results are in conformity with the result of genome microarray. 3) The expressional difference of the WNT5 a, GLI-1 and BMP7 in the colonic tissue may be related to they involved in morbidity of Hirschsprung’s disease as development related genes, and it’s specific molecular biology mechanism needs further study.PART FOUR STUDY ON THE ROLE OF WNT5 A PLASMID IN MICE HIRSCHSPRUNG’S DISEASE BY TRANS-PLACENTAL RNAIObjective: To structure expressional plasmid of RNA interference segments of WNT5 a gene. Down-regulated the expressional level of WNT5 a gene in pregnant Balb/C mice by certain amount of RNAi plasmid through tail vein injection in a different time window(E8.5- E14.5). Research the expressional level change characteristic and the expressional positioning of WNT5 a in embryonic intestinal tissue during the different periods(E8.5 E14.5) of Balb/C mice embryonic development. Investigate the role of WNT5 a in influence of phenotype of fetal mice gastrointestinal development and in the development of the enteric nervous system in mice, and the pathogenetic correlation of the mice with Hirschsprung’s disease.Methods: 1) To structure expressional plasmid of WNT5 a gene by directional cloning the targeted RNA interference segments of WNT5 a geneto the carrier pc DNA6.2?-GW/Em GFP-mi R(Life, the Catalog no. K4936-00) through recombinant DNA technology, and then amplification, sequencing and validation; 2) The Offspring mice of Down-regulated expressional level of WNT5 a gene were obtained by injecting the RNAi plasmid of WNT5 a into pregnant Balb/C mice through tail vein from the fetal intestinal development at E8.5, in a different time window with different concentration, injection volume and injection speed( injection time). 3) To execute the mice of postnatal two days and the embryon of injection of plasmid after 48 h, take out the whole digestive tract(from the stomach to the rectum) and then divide into foregut, midgut and hindgut to prepare frozen section. The expression of WNT5 a and its downstream gene and the developmental situation of the enteric nervous system are detected by immunohistochemistry; fluorescence microscope to observe the expression of green fluorescent protein; The expressional level of WNT5 a and its downstream gene m RNA was detected by QRT-PCR; The expressional level of WNT5 a and its downstream gene protein was detected by Western blot.Results: 1) The shuttle plasmid pc DNA6.2?-WNT5 a 1, 2, 3, 4 and the negative control plasmid pc DNA6.2 ?-scrambled were built successfully, and it is consistent between the designed oligomeric single-strand DNA sequences and the recombinant clone sequencing by sequencing and validation; 2) The expression of WNT5 a genes in the intestinal tissue can be down-regulated effectively due to the WNT5 a plasmid can transit through the placental barrier after tail vein injection of pregnant Balb/C mice; The effect of transfection is influenced by plasmid concentration, injection volume, injection speed(injection time) and weight of pregnant mice; 3) Immunohistochemical detection showed that the hindgut of offspring mice is dysplasia and lack of ganglion cells after interference; The q RT-PCR resultsshowed that the expression level of WNT5 a m RNA in the foregut and midgut is increased significantly than the hindgut. The Western-blot results showed that the expression of WNT5 a protein was consistent with the results of q RT-PCR. 4) The manifestation of Hirschsprung’s disease can be find after down-regulated the expansion of WNT5 a in fetal mice, it included that the hindgut of fetal mice was dysplasia, the rectum was dysontogenesis and the colon dilated.Conclusions: 1) The expressional plasmid of RNA interference segments of WNT5 a gene was constructed successfully, and finished the research of Quantified Trans-placental RNAi prosperously, and perfected the RNAi technology platform; 2)The study confirms that the WNT5 a play an important role during the development of the enteric nervous system; The Migration and colonization of intestinal neural crest stem cells in the gut may be inhibited and lead to the Hirschsprung’s disease after down-regulated the expression of WNT5 a.