| Objective:Hirschsprung’s disease(HSCR)is a deformity of the infantile digestive system characterized by the lack of intestinal ganglion cells in some or all of the colon.The continuous spasm and stenosis of affected intestinal lead to increased peristalsis of proximal colon and compensatory hypertrophy,eventually leading to the difficulty in feeding,serious constipation,growth disorders and bloating,the severe patients can have blood stool,fever and diarrhea(Hirschsprung associated enterocolitis,HAEC),and even neonatal intestinal obstruction or intestinal perforation,endangering the patient’s life,which can lead to death.HSCR is the second one of gastrointestinal malformation,second only to anorectal malformation,in which the incidence of newborns is about 1:5000,and the incidence of men and women is about 4:1.At present,there are many theories about its etiology.It is thought that the pathogenesis is that when the precursor cells derived from the vagal nerve crest form the enteric nervous system(ENS),the migration,colonization,differentiation and development process are abnormal due to various reasons and form the intestinal canals with aganglionosis,but the specific molecular mechanism is not clear.At present,the gold standard of HSCR diagnosis is rectal suction biopsy,but due to the atypical disease symptoms,many normal children also have the symptoms of abdominal distension,constipation and vomiting,and the data analysis involved in the genetic testing is too complicated,as well as domestic specific national conditions and other reasons,now the domestic diagnosis depends mainly on clinical manifestations and laboratory examinations,so the preoperative diagnosis is quite difficult.Meanwhile,there are also some children have poor surgical results in the process of treatment,and there has a lack of clear follow-up indexes,so the etiology and clinical study of Hirschsprung’s disease has always been one of the hot spots in pediatric surgery.At present,there are two common theories,that is,the theory of "genetic abnormalities"and the theory of "micro-environment anomalies".According to the theory of "genetic abnormalities",about 20%of children patient have definite family history and their parents or siblings have a high risk of relatives’ pathogenesis.Genotyping can detect specific gene abnormalities or deletions,and the common disease-related genes are oncogene tyrosine protein kinase(RET),endothelin B receptor(EDNRB),transcription factor SOX 10 or glial cell line-derived neurotrophic factor gene(GDNE)and so on.However,most of the cases are also found to be sporadic in the study.Therefore,this theory cannot fully explain the pathogenesis of HSCR,so some scholars have proposed the theory of "micro-environment anomalies ",which suggests that the development of embryonic stem cell requires specific space and environment.When the surrounding microenvironment is abnormal,it may also cause abnormalities of the differentiation and development of neural crest cells into ENS.The non-genetic factors are diverse,including brain-gut axis(BGA)functional imbalance,intestinal immune dysfunction,damage of intestinal mucosal barrier function,infiltration of local inflammatory factors and many other aspects,among them,there is a research shows that excessive changes in the composition of the extracellular matrix can lead to the premature colony,differentiation,adhesion and maturation of intestinal neural crest cells,and the cells cannot be properly migrated to the distal colon,resulting in the deficiency of neuron and glial cells in colon.But there also has a study which points out that pelvic nerve plexus dominated organs of children patient can also have normal-developed ganglion cells.Therefore,the theory of "micro-environmental anomalies " is also one-sided in the etiology study of HSCR.At the same time,due to the pathogenesis of the basic theory is not perfect,it leads to many problems of HSCR diagnosis and treatment in clinical work.In the study of the central nervous system,some scholars have suggested that the development of central nervous cells and neural networks is a process that has both temporal and spatial characteristics,that is,both the expression and regulation of a specific gene at a specific time and the influence of the external environment in the structural space.In view of the similarities and interrelationships between the central nervous system and the enteric nervous system,this project draws on the methodology of research on the central nervous system,proposes that genetic factors and microenvironment factors combine and interact with each other and affect the developmental abnormalities of the enteric nervous system during the embryonic period leading to HSCR.In conclusion,this research will propose gene-environment interaction theory from the aspect of ENS abnormal synaptic development and combine the characteristics of"genetic abnormalities" and "micro-environment anomalies" theory,studying the expression and interaction molecular signaling mechanism of neuroligin(Nlgn)gene which is a postsynaptic membrane protein widely studied in the central nervous system(CNS)and fibronectin(FN)which is the main component of the extracellular matrix in children with HSCR.Further research on the secreted FN in the serum of HSCR children reveals the relationship between the expression of Nlgn as well as FN and the pathogenesis of HSCR from the perspective of abnormal ENS development.This study looks for the specific and economical serum diagnostic indicators,also provides a theoretical basis for the pathogenesis of HSCR,and a practical direction for the clinical diagnosis and treatment of HSCR.Methods:The intestinal tissues and blood samples collected in this study were all from inpatients between 2013 and 2015 in the Department of Pediatric Surgery of Qilu Hospital.Among them,there were 57 patients who were pathologically diagnosed with HSCR(1 month-5 years old,37 males and 20 females,33 cases of short-segment type and 24 cases of long-segment type),57 blood samples in children patient with indirect inguinal hernia(IIH)diagnosed at the same time which were used as the control group.Colon samples were resected into three parts:stenosis segment(no ganglion area),migration and expansion segment(with ganglion cell area)and the margin segment(normal area).The cut part of fresh intestine tissue was promptly stored in liquid nitrogen and used for subsequent Western blot and qRT-PCR detection.The expression of Nlgn,FN and JAK/STAT3 signal pathway in each group was observed;The other part of tissue was fixed in paraformaldehyde for immunohistochemistry and immunofluorescence staining.In the first blood test,blood samples of patients with HSCR and IIH were collected,then after centrifugation,the upper serum was taken and stored in a refrigerator at-80 ℃,for detecting FN in serum of each group by enzyme-linked immunosorbent assay(Elisa)technique.The differences between HSCR children as well as non-HSCR children and between children with long-segment Hirschsprung disease as well as short-segment Hirschsprung children were compared.The significance of serum FN for the diagnosis and clinical classification of HSCR was evaluated.Take Wistar male and female rats paired randomly and mated separately,after pregnancy,40 pregnant rats were cultured solely to gestational age of 16,18,20 days,then kill the pregnant rats as well as 24-day-old neonatal rat and collect the area of colon tissue.The expressions of Nlgn,FN and Stat3 were detected by the above methods to study the correlation in embryonic development.Culturing adrenal pheochromocytoma cell line PC 12(poorly differentiated)routinely(this cell line has the same source with enteric neural crest cells and is mostly used in the nervous system research).Producing the FN-Knockdown-PC12 cell line(KD)with low expression of FN by virus transfection,and Western blot was used to detect the expression of Nlgn,FN and JAK/STAT3 signaling pathway.Cell proliferation,migration and differentiation were also detected.The experimental data were expressed as "Mean ± SD",unpaired t-test was used for comparison between the two groups,and one-way analysis of variance(ANOVA)was used for the comparison among the three groups.SPSS 10.5 and GraphPad Prism 5.0 software were used for the statistical analysis,and P values less than 0.05 are considered to be statistically significant.Results:(I)Through the detection of colon tissues of fetal mice with different gestational age,we found that the expression of FN decreased and the expression of the Nlgn increased with the gradually mature of embryo,and the JAK/STAT3 signaling pathway was activated,which can promote the development and improvement of ENS.(2)Immunohistochemical staining results of tissue samples in different segment of HSCR children showed that the expression of FN decreased from aganglionic segment,ganglionic segment to normal segment,while the expression of Nlgn increased and JAK/STAT3 signaling was activated.Immunofluorescence staining showed that FN protein and Nlgn protein mainly expressed in myenteric plexus,and the two may have interaction.(3)The results of Elisa test showed that the concentration of FN in serum may be related to the pathogenesis of HSCR.Serum FN concentrations in HSCR children were significantly increased,but there was no significant difference between long-term HSCR and short-term HSCR groups.(4)qRT-PCR and Western blot confirmed that the PC 12 cells in FN-KD group(experimental group)had lower expression of FN than that in negative control group,while the expression of Nlgn protein in experimental group increased,and the level of phosphorylated Stat3 increased.(5)Interleukin-6(IL6)activated Stat3 as an activator of JAK/STAT3 signaling pathway.With the increase of time and concentration,the expression of Stat3 and phosphorylated Stat3 increased,as well as Nlgn.(6)The results of MTT assay showed that the cell proliferation of FN-KD-PC12 group was inhibited compared with that of the control group.The results of wound healing test showed that the migration rate of FN-KD-PC12 increased at 24H,but decreased at 48 H.Cell cloning experiments showed that the formation of FN-KD-PC12 clone decreased.All above differences were statistically significant.(7)Morphologically,after removal of horse serum and increasing of fetal bovine serum in cell culture,the PC 12 cells in two groups both had differentiation from low differentiation to high differentiation,and the differentiation of FN-KD-PC12 cells was faster,of which the neuron structure was more obvious.Conclusion:In summary,according to the above results,we can draw the following conclusions in this study:(1)During the process of embryonic neural crest cells gradually evolving into ENS,as the embryos become more and more mature,there is a negative correlation between FN and Nlgn,indicating that FN may be related to the formation and synaptic maturation of ENS,and this process involves in JAK/STAT3 signaling pathway.(2)FN and Nlgn are expressed in the colon tissue of myenteric plexus,and related to the severity of the colon lesions.Such changes may result in the myenteric plexus ENS abnormalities,and synaptic function abnormal affect the transmission of nerve signals in the ENS,and this process is also related to the abnormal activation of JAK/STAT3 signaling pathways.(3)The difference of the serum FN concentration between each group suggests that FN may have an important role in the preoperative diagnosis of HSCR,but which is not related to the clinical classification and the severity of the disease.(4)The vitro experiments confirmed that FN can regulate the expression of Nlgn via JAK/STAT3 signaling pathway and affect the development of ENS.The changes of FN can inhibit the cell proliferation,colonization and migration.The dynamic process of neural crest cell microenvironment during embryonic development should be treated consistently. |
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