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Sphincter of Oddi Dysfunction Model Building in Rabbits and Significance of Changes of NOS and VIP in These Organizations

Author: ZhangQiang
Tutor: WuShuoDong
School: China Medical University
Course: Surgery
Keywords: Cholecystectomy Truncal vagotomy SOM SOBP SOCA SOCF SOCD NOS(nitric oxide synthase) VIP(vasoactive intestinal polyeptide) IOD(integrated optical density)
CLC: R-332
Type: PhD thesis
Year: 2008
Downloads: 86
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Abstract


PrefaceThe musculus sphincter which encloses the bile common duct bulb and chole-pancreatic duct extreme is collectively called the sphincter of Oddi(SO).Under the normal circumstance,SO is very important in maintaining the normal bile duct pressure,promoting gallbladder to excrete and preventing from reflux.Nowaday,the exterior and interior schlors of our country have realized the sphincter of Oddi abnormality includes two parts,one is the dysfunction and another is anatomic paramorphia,the former may not combine with the latter,and the latter may not appear the sphincter of Oddi dysfunction(SOD)on the basis of anatomic paramorphia. However,sphincter of Oddi manometry(SOM)is the gold standard of detecting SOD. We deployed many kinds of methods to build SOD models and surveyed the pressure indexes of rabbit models by means of SOM,simultaneously compared them with those of control group in order to judge the results of model buiding.In addition,we investigated the level changing of Vasoactive intestinal polypeptide(VIP)and Nitric oxide synthase(NOS)by means of immunohistochemical technique(SABC method)in SO and duodenal papilla of all rabbits,in order to investigate the effect of VIP and NO in the pathogenesy of SOD.Test oneMaterial1.Groups:A total of 77 rabbits were randomized into eight groups Control group 8、Cholecystectomy group 12、runcal vagotomy group 8、Common bile duct partial ligation group 11、SO partial ligation group 9、Cholecyst partial ligation group 9、Bile duct infused pancreatic enzyme group 12、SO injected alcohol group 8,male 40,female 37.2.Apparatus and drugs1)A triple-lumen catheter with diamenter of 1.7mm,lengh of 200cm.2)Polygram pressure recorder.3)Low-compliance hydraulic infusion system.4)20%ethyl urethane,95%alcohol,animal zymine(powder)Methods1.Operative procedureethyl carbamate(1.0g/kg)i.v.,anaesthesia.All rabbits of groups were opened the abdomens through medioventral line,stoped bleeding thoroughly and closed the abdomens.(1)Control group:only open and close the abdomen;(2)Cholecystectomy group:ligate the duct of gallbladder and cystic artery,removal the cholecyst retrogradely;(3)Truncal vagotomy group:in the two sides of the esophago of pars abdominalis,dissociate left and right vaguses,hang them with suture silk,removal vagurs trunks about 1 cm respectively;(4)Common bile duct partial ligation group:in the opposite side of duodenal papilla’s debouch,cutdown the duodenum about 1 cm, retrogradely insert the asepsis epidural pipe into the common bile duct and bluntly dissect the tissue around the common bile duct,pass-by the duct with 4~# suture silk, ligate the duct onto the epidural pipe,then pull out the pipe,suture the nick of duodenum;(5)SO partial ligation group:in the opposite side of duodenal papilla’s debouch,cutdown the duodenum about 1 cm,suture the debouch of papilla lateropulsionly,observe still small cholerrhagia and suture the duodenal nick; (6)Cholecyst partial ligation group:reveal the cholecyst,the suture needle with suture silk pass-by the middle part of cholecyst,ligate and close 50%cross sectional area; (7)Bile duct infused pancreatic enzyme group:cutdown the duodenum about 1 cm, retrogradely insert the asepsis epidural pipe into the common bile duct,close the duodenal nick and fix the pipe onto the duodenum,suture the abdominal membrane and muscle,bluntly dissect a hypodermic tunnel to rabbit’s collar part,pipe proceeds through the tunnel and goes out the nick of skin,fix the pipe again,close the abdominal nick.Insert the prepared 10%animal pancreatic enzyme about 2ml into the common bile duct,after 1 hr and 2 hr,insert 2ml pancreatic enzyme again,close the pipe 10min every time after inserting drug;(8)SO injected alcohol group:cutdown the duodenum about 1 cm,in 2mm of duodenal papilla uniformly inject 95%alcohol 0.1ml into 4 parts of duodenal papilla,suture the duodenal nick.The rabbits of all groups were dieted and not refrain from water before the operation and the day of operation.Except the Bile duct infused pancreatic enzyme group which was measured the pressure 24hr after inserting drug,the rest groups were measured the pressures 1 week after operation.2.Measurementafter anaesthesia,open the abdomens through origin nick,cutdown the duodenum about 1 cm,reveal the papilla,link a triple-lumen catheter with polygram pressure recorder,retrogradely insert catheter into the common bile duct,stabilize for 30 sec and measure the pressure of CBD and SO for 3 to 5min,record the curve of pressure.3.Parameterspressure of common bile duct;basal pressure of SO;SO contractive amplitude; SO contractive frequency;SO contractive duration4.Satistical analysisThe experiment data is measurement data.Measurement data is expressed with mean±standard deviation((?)±s).Making use of the statistical analysis software of SPSS11.5,we adopted the method of One-Way ANOVA and compared every experimental group with control group.P<0.05 is significant difference,P<0.01 is extremely significant difference. Results1.SO metergasis of Cholecystectomy groupOn basal sphincter of Oddi pressure,there was a significant increase,the average was 36.61±8.60mmHg(p<0.01).2.SO metergasis of Truncal vagotomy groupOn basal sphincter of Oddi pressure,there was a significant increase,the average was 28.54±7.66mmHg(p<0.05).3.SO metergasis of Common bile duct partial ligation groupOn basal sphincter of Oddi pressure,there was a significant increase,the average was 28.45±7.02mmHg(p<0.05).4.On all the pressure parameters of the rest groups compared with them of the control group,there was no significant change.ConclusionCholecystectomy、Truncal vagotomy or common bile duct partial ligation can respectively cause the change of SO motor function in rabbits.However,the results were changes of some parameters,not all changes.Test twoMaterial1.GroupsThe method of dividing groups waw same to test one.after measuring the pressuresof above-mentioned rabbits,inject 5-10ml air into the ear-edge vein of the rabbits and kill them,fastly take out of the duodenal papilla and SO,put into 4%paraform and fix them.2.Apparatus and drugs1)First antibody:VIP antibody,NOS antibody.Second antibody:goat anti-rabbit IgG.2)5%BSA confining liquid,SABC,compound digestive juice,DAB color reagent kit.3)C-7070/BX 41 collect image system4)MetaMorph micrograph analysis systemMethods(1)Fix fresh tissue with 4%paraform for 24hr,paraffiln imbedding in routine,sect serial sections by 5μm,bake them for 25min at 65℃;(2)Deparaffinage to water in routine;(3)30%H2O2 inactivate endogenous activating enzyme,after soaking them for 10min,wash with distilled water;(4)Heat repair antigen:immerge section into 0.01M citrate balanced solution(PH6.0),microwave oven heat them to boiling,after 10min interval,repeat twice.After cooling,wash twice with PBS;(5)ropwise 5%BSA confining liquid,room temperatlure 20min.discard unnecessary liquid,not wash; (6)Dropwise prper diluted one anti-rabbit IgG,4℃staying overnight,PBS wash; (7)Dropwise two antibiotin goat anti-rabbit IgG,37℃incubation 20min,PBS wash; (8)0.05%DAB colouration,control reaction time under the Microscope,lotic water wash and stop reaction;(9)Hematoxylin lightly afterstain,dehydration,transparence, mounting with resin.(10)PBS replaced the first antibody and the rest procedures were same to above-mentioned operation,which was regarded as the negative control. Results judgementWhen nucleus is blue and cytoplasm is buffy in smooth muscke cell of SO、cellula columnoepithelialis of papilla mucosa and phorocyte of submucosa on NOS and VIP,we can regard it positive.At first,we selected 5 eyeshots in every slice and collected image by 40×objective lens.On every image,we utilized the analytical system of micrograph which is called MetaMorph/ C-7070/BX41,and measure the Integrated OD of positive area.Results were recorded in the table and prepared for the statistical analysis.Statistical analysisThe Integrated OD is measurement data.Measurement data is expressed with mean±standard deviation((?)±s).Making use of the statistical analysis software of SPSS11.5,we adopted the method of One-Way ANOVA and compared every experimental group with control group.P<0.05 is significant difference,P<0.01 is extremely significant difference.Results1.Changes of Integrated OD of NOS and VIP in SO1)Truncal vagotomy group:on the Integrated OD of NOS and VIP,there was a significant increase,the mean was 34.04±27.24(P<0.01)and 45.82±36.35(P<0.01) respectively.2)Common bile duct partial ligation group:on the Integrated OD of NOS and VIP,there was a significant increase,the mean was29.40±15.99(P<0.05)and 53.06±61.24(P<0.01)respectively.3)Cholecyst partial ligation group:on the Integrated OD of NOS and VIP,there was a significant increase,the mean was 53.72±47.74(P<0.01)and 71.95±62.34(P<0.01)respectively.4)Bile duct infused pancreatic enzyme group:on the Integrated OD of NOS,there was a significant increase,the mean was 27.36±24.85(P<0.05).5)On all the Integrated OD of the rest groups compared with them of the control group,there was no significant change.2.Changes of Integrated OD of NOS and V1P in mucous membranes of papilla Cholecyst partial ligation group compared with the control group,there was a significant increase,on the Integrated OD of NOS,the mean was 29.78±13.08(P<0.05).The rest groups compared with the control group,there was no significant change on the Integrated OD of NOS and VIP.3.Changes of Integrated OD Average of NOS and VIP in muscularis mucosae and submucosa of papillaIn Cholecystectomy group and Bile duct infused pancreatic enzyme group,on the Integrated OD of NOS,there was a significant increase,the mean was 81.28±47.39(P<0.01)and 75.47±73.11(P<0.05)respectively.In Common bile duct partial ligation group,on the Integrated OD of VIP,there was a significant increase,the mean was 129.09±182.35(P<0.01).The rest groups compared with the control group,there was no significant change on the Integrated OD of NOS and VIP.ConclusionsTruncal vagotomy、common bile duct partial ligation、cholecyst partial ligation or bile duct infused pancreatic enzyme can respectively cause the change of quantity of NOS and(or)VIP in smooth muscle of SO、papilla mucosa、submucosa or muscular layer of rabbits,and the tendency of change is increasing.

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