| Part OneThe distribution and activation of macrophages and phenotype change of Interstitial cells of Cajal in Human Hirschsprung's diseaseObjective:Detect the distribution and activation of macrophages and phenotype changes of ICCs in human HD specimens,and to explore the relationship between the activation of macrophages and phenotype change of ICCs,and its role in the development of HAEC.Methods:Collect the specimens of human HD and divide into proximal colon and distal colon,HE staining was used and evaluated the degree of bowel inflammatory injury,CD68 and iNOS double immunoflourescence staining was used to evaluate the classically activated macrophages,CD68 and Argl double immunoflourescence staining was used to evaluate the alternatively activated macrophages,c-kit immunohistochemistry staining was used to test the phenotype changes of ICCs,Western Blot was used to detect the protein expression of iNOS,TNF-a,IL-1?,Arg1,CD34,c-kit.RT-PCR was used to detect the mRNA expression of those cytokines.Results:HE staining revealed that there exist amount of immune cells in the mucous layer and submucosa in the proximal dilated colon of HAEC group,the colon histopathological score suggested that the inflammatory injury score in the proximal dilated colon of HAEC group was evaluate compared with other groups.CD68 and iNOS double immunoflourescence staining revealed that the M1 macrophages were increased in proximal dilated colon of HAEC group,but there were no M1 macrophages distribute in the distal colon of HAEC group and HD group.CD68 and Argl double immunoflourescence staining revealed that the M2 macrophages were increased in distal colon of HAEC group and HD group,but there were no M2 macrophages distribute in the proximal colon of HAEC group and HD group.c-kit immunohistochemistry staining revealed that the ICCs were decreased in the proximal dilated colon of HAEC group compared with HD group,and there were no ICCs in the distal colon of both HAEC and HD group.Western Blot revealed that the protein expression of iNOS,TNF-a,IL-1? were increased in the dilated colon of HAEC group compared with HD group,Arg1 expression were elevated in the distal colon of both HAEC and HD group,c-kit expression was decreased in the dilated colon of HAEC,CD34 showed no obviously change between two groups.RT-PCR showed that mRNA expression of iNOS,TNF-a and IL-1? were increased in the dilated colon of HAEC group compared with HD group,Arg1 expression were elevated in the distal colon of both HAEC and HD group,c-kit expression was decreased in the dilated colon of HAEC.Conclusions:The M1 macrophages and inflammatory cytokines include TNF-a,IL-1?were obviously increased in the dilated colon of HAEC,meanwhile,the c-kit positive ICCs were decreased.Part TwoEffect of Macrophages activation on the phenotype changes of ICCs and colon slow waves in Ednrb null mouse model of HDObjective:Detect the activation of macrophages and phenotype changes of ICCs and colon slow waves in different period of Ednrb null mouse model of HD,and to explore the effect of the activation of macrophages on phenotype change of ICCs and slow waves in HAEC.Methods:Collect the specimens of 1w,2w and 3w old HD mice and wild-type mice,HE staining was used and evaluated the degree of bowel inflammatory injury,CD68 and iNOS double immunoflourescence staining was used to evaluate the classically activated macrophages,CD68 and Argl double immunoflourescence staining was used to evaluate the alternatively activated macrophages,c-kit immunohistochemistry staining was used to test the phenotype changes of ICCs,Western Blot was used to detect the protein expression of iNOS,TNF-a,IL-1(3,Argl,CD34,c-kit.RT-PCR was used to detect the mRNA expression of those cytokines.Electrophysiological recorder was used to record the slow waves,liposomal clodronate were used to delete the macrphages and then observe the macrophages activation,phenotype changes of ICCs and colon slow waves.Results:HE staining revealed that there exist amount of immune cells in the mucous layer and submucosa in the proximal colon of 3w old mice of HD group,the colon histopathological score suggested that the inflammatory injury score in the proximal dilated colon of 3w old mice of HD group was evaluate compared with 1w and 2w old mice groups.CD68 and iNOS double immunoflourescence staining revealed that the M1 macrophages were increased in proximal dilated colon of 3w old mice of HD group,but there were no M1 macrophages distribute in the distal colon of all HD groups.CD68 and Arg1 double immunoflourescence staining revealed that the M2 macrophages were increased in distal colon of all HD group,but there were no M2 macrophages distribute in the proximal colon of all HD groups,c-kit immunohistochemistry staining revealed that the ICCs were decreased in the proximal dilated colon of 3w old mice of HD group compared with 1w and 2w old HD group,and there were no ICCs in the distal colon of all HD groups.Western Blot revealed that the protein expression of iNOS,TNF-a and IL-1? were increased in the dilated colon of 3w old mice of HD group compared with 1w and 2w HD group,Argl expression were elevated in the distal colon of all HD groups,c-kit expression was decreased in the dilated colon of 3w old mice of HD group,CD34 showed no obviously change between two groups.RT-PCR showed that mRNA expression of iNOS,TNF-a and IL-1? were increased in the dilated colon of 3w old mice of HD group compared with lw and 2w HD group,Argl expression were elevated in the distal colon of all HD group,c-kit expression was decreased in the dilated colon of 3w old mice of HD group.The rhythm and frequency of colon slow waves in the 3w old mice of HD group were destroyed.After treatment with liposomal clodronate,the macrophages were significantly decreased in the dilated colon of 3w old mice of HD group,and c-kit expression was evaluated,simultaneously,rhythm and frequency of slow waves were recovered.Conclusions:Classically activated macrophages and secretion of inflammatory cytokines in the dilated colon of HD mice damage the phenotype of ICCs and inhibit colon slow waves.Part ThreeThe effect of TNF-? on the phenotype changes and pacemaker currents of ICCs.Objective:Isolate ICCs from mouse colon,stimulate with TNF-a and detect the phenotype changes and pacemaker currents of ICCs.Methods:The ICCs were isolated from the 3 old week mouse colon by enzyme digestion,after 3 days culture,the experiment group were added lOng/ml TNF-a,the control group were added equivalent NS,then culture for 24h,CD34 and c-kit double immunoflourescense staining were used to detect the phenotype changes of ICCs,RT-PCR were used to detect the mRNA expression of CD34,c-kit and pacemaker currents related gene including Ryr3,Itpr3,Calml,TRPC4,Ano1,DIG2,DIG4.Patch clamp technique was used to record the pacemaker currents of ICCs.Results:The numbers of synapse of ICCs in single cell and networks were decreased in experiment group under optical microscope scanning,immunoflourescense staining revealed that the c-kit expression was decreased in experiment group,but CD34 expression had no change.RT-PCR showed that mRNA expression of c-kit was significantly decreased in experiment group,CD34 expression had no change,Ryr3,Itpr3,TRPC4,Ano1 expression were decreased,but Calm 1?DIG2.DIG4 expression had no changes.The pacemaker currents of ICCs in experiment was significantly decreased.Conclusions:TNF-a could significantly inhibit the expression of c-kit and pacemaker related gene in ICCs,furthermore inhibit the pacemaker currents of ICCs.Part FourThe phenotype changes of ICCs and its mechanism research on LPS-induced Zebrafish bowel inflammationObjective:To explore the expression of miR221,phenotype changes of ICCs and intestinal peristalsis on LPS-induced Zebrafish bowel inflammation.Methods:The control group zebrafish were breed with egg water,the experiment group zebrafish were breed with 1,10,50?g/ml LPS from 48hpf to 11dpf.Collect the zebrafish specimen at 3dpf,5dpf,7dpf,lldpf.HE staining was used to evaluate the inflammatory injury.miR221 expression was detected by RT-PCR.The protein level of TNF-a and kita were tested by Western Blot.Wholemount preparation of zebrafish tissue and observe the c-kit expression under laser confocal scanning microscope,flourescent tracer were used to detect the zebrafish intestinal peristalsis.Microinject the miR221-MO and Mismatch-MO into the zygote within 0.5-1h,and breed the zebrafish larval according to the above methods,collect the zebrafish tissues and detect the c-kit expression and intestinal peristalsis.Results:HE staining revealed that the inflammatory injury was serious under the LPS stimulation in 3dpf,5dpf,7dpf,1 ldpf,moreover with the increased concentration of LPS,the inflammatory injury was more significant.Western Blot showed that the TNF-a expression was gradually elevated according to the increased concentration of LPS,at the same time,the kita expression was decreased.TNF-a and kita mRNA expression showed the same tendency with their protein levels.Whole-mount and kita immunoflourescense staining showed no expression of kita in any groups of 3dpf,5dpf,and kita expression was gradually decreased according to the increased concentration of LPS in 7dpf,11dpf.Flourescent tracer revealed the intestinal peristalsis was gradually decreased according to the increased concentration of LPS in 7dpf,11dpf.Targetscan predict that there exist a complementary sequence between the miR221 and kita,and RT-PCR confirmed that miR221 expression was gradually increased according to the increased concentration of LPS,while kita expression was decreased.After microinject Mismatch-Mo and miR221-MO,the miR221 expression in miR221-MO group in 3dpf,5dpf,7dpf was significant decreased upon LPS stimulation,and kita expression was increased as detected by RT-PCR and Western Blot,intestinal peristalsis was also recovered to some extend.However,due to the fade effect of miR221-MO,the expression of miR221 was elevated and kita was decreased in 1 1dpf.Conclusions:LPS induced TNF-a promoted over-expression of miR221 inhibit the expression of kita,as well as restrain the intestinal peristalsis. |
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