| Background:Interstitial cells of Cajal(ICC)in the gastrointestinal(GI)tract which is considered to be pacemaker cells can generate and propagate of independent slow wave rhythmic electrical activity,and regulate the gallbladder smooth muscle relaxation or contraction,and play a very important role in the process of gastrointestinal motility disorder disease.Recent studies found that ICCs also exist in the biliary system,such as the gallbladder,bile duct and sphincter of Oddi.It can also generate and propagate of independent slow wave rhythmic electrical activity,and regulate the gallbladder smooth muscle relaxation or contraction.Acute cholecystitis is a common disease in gastroenterology clinic,caused by obstruction of the cystic duct,chemical irritation and bacterial infection.Recent studies found that inflammatory reaction will lead to the number of interstitial cells of Cajal,function,morphology and ultrastructure changes,ultimately affect the dynamic function of the gastrointestinal tract.Therefore,this study research the morphology,distribution and function of gallbladder interstitial cells of Cajal in acute cholecystitis,and observe the relationship between interstitial cells of Cajal and biliary motility system.It can broaden the understanding of the interstitial cells of Cajal function,and help to elucidate the mechanism of interstitial cells of Cajal in biliary motility system disorder disease,to provide a new method for the clinical diagnosis and treatment in the future.Aim:1.By common bile duct ligation method to construct the animal model of acute cholecystitis,to observe the pathological changes of gallbladder tissue in inflammation.2.To observe the morphology and distribution of interstitial cells of Cajal in different periods of inflammation.3.In the animal model of acute cholecystitis and in different periods of inflammation,to compare the difference of interstitial cells of Cajal in morphology and distribution with the normal gallbladder interstitial cells of Cajal,and the difference in distribution of the bottom of gallbladder to the neck.4.To study the effect of neutrophils on the expression of SCF/c-kit and apoptosis in gallbladder interstitial cells of Cajal.5.To investigate the mechanism of interstitial cells of Cajal in biliary motility system disorder disease.Materials and Method:1.Thirty adult guinea pigs were randomly divided into 3 groups,including normal group,bile duct ligation for 24 hours group and bile duct ligation for 48 hours group.Guinea pigs within acute cholecystisis model were induced by common bile duct ligation.2.To extract gallbladder interstitial cells of Caja from 10-to 14day-old guinea pigs,and co-culture with neutrophils in normal group,bile duct ligation for 24 hours group and bile duct ligation for 48 hours group,and divided into four groups,respectively,single-culture group,co-culture 1 group,co-culture 2 group and co-culture 3 group.3.HE staining and immunofluorescence were used to observe the degree of gallbladder inflammation and the distribution of interstitial cells of Cajal.4.By the technology of Western Blotting,Rt-PCR and TUNEL,to observe the effect of neutrophils on the expression of SCF/c-kit and apoptosis in gallbladder interstitial cells of Cajal.Results:1.Gallbladder spicemen with HE staining in each group,normal group were no obvious inflammation.Compared with normal group,bile duct ligation for 24 hours group were in mild inflammation,bile duct ligation for 48 hours group were in significant inflammation.2.Gallbladder ICCs did not reveal significant morphological differences between the single-culture group and the co-culture 1 group.In contrast,in both the co-culture 2 group and the co-culture 3 group,gallbladder ICCs synapses was shortened,with fewer connections among ICCs,and the ICC network structure was partly broken.The morphological changes were more pronounced in co-culture 3 group than in co-culture 2 group.3.The numbers of interstitial cells of Cajal were decreased during the gallbladder within acute inflammation,and in normal group,bile duct ligation for 24 hours group and bile duct ligation for 48 hours group were respectively 61.6667±2.08167、54.0000±1.73205、38.3333± 1.52753(P<0.001).The number of interstitial cells of Cajal cells in each groups gallbladder tissue were increased from the bottom of gallbladder to the neck,but with the development of the change of inflammation the number of interstitial cells of Cajal were all reduced.In the normal group,the numbers of interstitial cells of Cajal cells of the gallbladder neck,the gallbladder body and the gallbladder bottom were respectively 70.0000±2.00000,60.6667±3.78594,53.6667±4.04145.In the bile duct ligation for 24 hours group,the numbers of interstitial cells of Cajal cells were 60.3333± 2.51661,55.0000±1.00000,46.6667±3.51188,respectively.In the bile duct ligation for 48 hours group,the number of interstitial cells of Cajal cells were 50.3333±1.52753,39.0000±1.00000,25.6667±3.05505,respectively(all P<0.05).4.The protein levels of both c-kit and SCF were significantly lower in the co-culture groups than in the single culture group.Both SCF and c-kit protein levels were the lowest in co-culture 3 group.And the ratios of c-kit and β-actin were 0.5943± 0.07095,0.5503 ± 0.03109,0.3363 ± 0.04712,and 0.2327 ± 0.04974,respectively(F= 33.360,P = 0.000).5.SCF and c-kit mRNA levels were much lower in co-culture 3 group than in the other groups.Compared with the single-culture group,in co-culture 1 group,co-culture 2 group,and co-culture 3 group,SCF mRNA levels were 1.0000 ± 0.00000 vs.0.9673 ± 0.02887 vs.0.6630 ± 0.07119 vs.0.4350 ± 0.14584,respectively,and c-kit mRNA levels were 1.0000 ± 0.0000 vs.0.9570 ± 0.02961 vs.0.7130 ± 0.05839 vs.0.4287 ± 0.10130,respectively(F-values were 31.721 and 57.066,respectively;all P=0.000).6.The density of ICC that underwent apoptosis was significantly higher in the co-culture groups than in the single-culture group,with co-culture 1 group showing a higher density than the single-culture group,and co-culture 3 group showing a higher density than co-culture 2 group,too(1.0000 ± 0.00000 vs.1.6667 ± 0.57735 vs.5.3333± 0.57735 vs.10.6667 ± 0.57735,F = 192.889,P = 0.000).Conclusion:1.The changes of the number,distribution and morphological in gallbladder interstitial cells of Cajal will play a role in the pathogenesis of acute cholecystitis.2.Gallbladder interstitial cells of Cajal play a very important role in regulating mechanism of gallbladder motility function,and may be involved in the pathogenesis of biliary motility disorders. |
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