| Part One The clinical research for identifing the relationship between Helicobacter pylori and irritable bowel syndromeObjective:To identify the correlationship between the Helicobacter pylori(Hp)and the irritable bowel syndrome(IBS),including the relationship between the Hp infection and IBS subtypes(IBS-D,IBS-C and IBS-M).Methods:During Janurary 2015 to December 2015,322 outpients(Renmin Hospital of Wuhan University)who were diagnosed as IBS were selected as the IBS positive group.Meanwhile,403 healthy people who did physical examination in the same period at Renmin Hospital of Wuhan University were selected as the control group.C-13 urea breathe test(C-13 UBT)and rapid urease test(RUT,under endoscrope)were used to identify the Hp infection status of all the 725 candidates.Results:Average age of IBS positive group and control group were 46.4±7.3 and 39.4±6.1 years old.The ratio between male and female were 1.22:1 and 1.08:1 seperately.The main complaints of the IBS group were typical unspecific abdominal symptoms of IBS.The numbers of subtypes of IBS were 156,93 and 73(IBS-D,IBS-C and IBS-M).274 patients used C-13 urea breathe test and 48 patients used rapid urease test to testify the Hp infection in IBS group,while 309 candidates used C-13 urea breathe test and 94 candidates used rapid urease test to testify the Hp infection in control group.The result indicates 219 patients(219/322,68.0%)infected by Hp of IBS group comparing to 162 patients(162/403,40.2%)infected of control group(P<0.05).The IBS patients took 219 of all the Hp infected candidates,meanwhile,the IBS patients took 103 of all the Hp infected negative candidates,the OR value was 2.13.For relationship between subtypes of IBS and Hp infection,we found that IBS-D group has 127(127/156,81.4%)infected patients,IBS-C group has 47(47/93,50.5%)infected patients and IBS-M group has 45(45/73,61.6%),there is a significantly diffenence among these groups(P<0.05).Conclusion:There is a correlationship between Helicobacter pylori and irritable bowel syndrome,as well as a relationship between Helicobacter pylori infection and irritable bowel syndrome's subtypes.Part TwoSetting up the animal model for Helicobacter pylori infectionObjective:It is widely admitted that Helicobacter pylori(Hp)has an intimate relationship with a great quantity of digestive system diseases.To deeper investigate of the mechanisms of Hp,scienstists tried to set up a stable and reliable aninal model using different animal types such as different kinds of mice and rats.Due to the specific condition for culturing Hp and sucessefully implanted Hp in vivo,the previous results were not optimistic enough.This research is to set up a reliable aninal model of Hp infection.Methods:Helicobacter pylori(Hp,code number:NTCT 11637)were donated from Professor Nonghua Lv from Nanchang University.After successfully cultivation of Hp(Mueller-Hinton-Agar CM0337;mixed antibiotics of Vancomycin,Polymyxin,Trimethoprim and Amphotericin;37 0 C micro aerobic environment in constant temperature incubator;),the third genetration were observed under light microscope to testify its morphology.48 Male SD rats were divided into six groups randomly.All environment and feeding condition are the same for all the rats.The Hp infection procedure was completed by intragastric administration to the SD rats which numbered group one to group six(Hp infection groups),while group five were treated with 0.9%physiological saline as placebo group(P0)and group six treated with nothing as blank group(B0).Four weeks later,all the rats from six groups were sacrificed and testified if Hp infected through fast urease test(FUT)and gastric mucosa biopsy.Results:The morphology of Helicobacter pylori we observed under light microscope was like a short rod(some people describe it as crescent shaped),with no contamination of other bacteria or fungus.The solution of the Hp was made to a final concentration of 1×109 CFU without any particle or precipitant.Four weeks later,after sacrifice of the rats,the Hp infectiong effect through fast urease test(FUT)and gastric mucosa biopsy were observed.The Hp infection groups presented strong positive result throught FUT test comparing negative result of placebo and blank groups.The biopsy proved that the Hp infection groups had non specific appearance(as chronic gastritis)comparing with the negative Hp infection groups.Conclusion:The culture of Helicobacter pylori in vitro is proceeded successfully.The establishment of this in vivo SD rat model of Hp infection proved this is a stable and reliable way to set up an animal model for chronic gastritis for the future research.Part ThreeInfluence of Helicobacter infection on gastrointestinal hormones and colonic motility in ratsObjective:Helicobacter pylori(Hp)have been proved to be chief criminal of gastric diseases.It can even be detected outside upper digestive track like lung and kidney.Scientists found that Hp might modulate hormone secretion by influencing brain gut axis.However,the specific relationships among Hp infection,hormone secretion and colonic motility are still undefined.This study is to clarify the relationships among Hp infection,secretion of gastrointestinal hormones and contraction of colon muscles of SD rats.Methods:Hp(NTCT 11637)were cultivated to the third generation separately in a micro aerobic culture box.Male SD rats were divided into six groups randomly.Afterwards,the Hp infection procedure was completed by intragastric administration to the SD rats which numbered group one to group six.Four weeks later,all the infection rats were confirmed of Hp infection by C-13 urea breath test(C-13UBT)and gastric mucosa biopsy.Colonic motility was assessed by counting the numbers of fecal pellet output and recording the contractions of isolated colonic strips through isometric force transducer.Plasma hormones including gastrin,cholecystokinin(CCK),substance P(SP)and neuropeptide Y(NPY)were determined using Enzyme Immunoassay Kits.The colonic motility closely related proteins' expressions were tested separately by Western blot technique.Results:Hp infection significantly increased fecal pellet output compared with negative Hp infection groups at the 28th day(M group mean 20 i± 2.63 vs PO group mean 13 ± 1.34 vs BO group mean 11 ± 1.33;p<0.05).Hp infection induced increasing of fecal output number was obviously attenuated and also had a decreased dry weight of stool.The mean dry weight of stool of model groups(M1-M4)was 0.22±0.01 g,comparing the mean weight of PO group 0.41±0.03 g and BO group 0.38±0.04 g(p<0.05).However,during the Hp suspension intragastric administration treatment,there was no statistically significant difference between the PO group and BO group in either number of fecal pellet,or dry weight of stool(p>0.05).The colonic motility closely related proteins' expressions were tested separately by Western blot technique.The mean tension of longitudinal muscle(LM)strips' spontaneous contraction of Hp infection groups were significantly greater than the negative Hp infection groups(Hp infection groups,1.63±0.17 g vs PO group,0.77±0.10 g vs BO group 0.83±0.11 g;p<0.05).Meanwhile,the mean tension of circular muscle(CM)strips' spontaneous contraction of Hp infection groups were obviously higher than the negative Hp infection groups(Hp infection groups,0.75±0.10 g vs P0 group,0.36±0.04 g vs B0 group,0.27±0.04 g;p<0.05).Nevertheless,no statistically significant difference can be found between the P0 group and the B0 group in the tension and frequency of spontaneous contraction(p>0.05).Significantly differences can be found between Hp infection groups(M1,M2,M3,M4)and negative Hp infection groups(P0 and BO),concerning the concentration of gastrin,cholecystokinin(CCK)and substance P(SP)(Gastrin:mean of model groups,66.95±9.94 pg mL-1 vs P0 group 26.78±4.97 pg mL-1 and Bo group 23.51±4.37 pg mL-1,p<0.05;CCK:mean of model groups,187.49±28.41 pg mL-1 vs P0 group 74.44±11.30 pg mL-1 and Bo group 83.07±14.24 pg mL-1,p<0.05;SP:mean of model groups,151.25± 17.33 pg mL-1 vs P0 group 63.95±9.03 pg mL-1 and Bo group 53.06±8.17 pg mL-1,p<0.05),while no statistically difference can be found between P0 and B0 group(p>0.05).However,the level of neuropeptide Y(NPY)had no obvious variety in the Hp infection groups(M1-M4)compared to the negative Hp infection groups.Conclusion:Hp infection probably activate the regulation of brain-gut peptides and gastrointestinal hormones which could directly or indirectly interact with their associated receptors in smooth muscle cells,upregulate the colonic motility closely related proteins.As a result,Hp infection can induce increased count of fecal pellet output during and after Hp infection process and increased colon motility of smooth muscles. |
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