| ObjectiveBy detecting the gene polymorphism of IL-1 p-511 and TNF-a-308 and its transcriptome in the gastirc mucosa of patients who have Helicobacter pylori correlated diseases(HPCD)and in different syndromes of Chinese Medicine,controlled with the normal people,the mechanism of HPCD and its development have been discussed in the view of syndromes of Chinese Medicine,which may predict the occurrence and its development of diseases and promote the new-age research of HPCD transited malignance from health.Method1.Source of casesThe Chronic Gastritis(CG)and Peptic Ulcer(PU)pathological specimens of mucosa were gathered from patients who visited the endoscopy department of affiliated hospital of GuangZhou University of Chinese Medicine,from Sep 2012 to Oct 2014 by our team,in which 186 cases of CG conforming with the inclusion criteria were included,73 cases of Chronic Superficial Gastritis(CSG),87 cases of CSG,20 cases of Chronic Atrophic Gastritis(CAG),6 cases of CAG and erosion,61 cases of patients with PU.24 cases of patients with Gastric Cancer(GC)were collected from May 2014 to Oct 2014 in gastrointestinal surgery department of affiliated hospital of GuangZhou University of Chinese Medicine,including 2 cases of patients with gastric lymphoma.According to diagnose criteria of syndromes of Chinese Medicine and syndromes element differentiation,all the case were classified into such groups as damp-heat syndrome in spleen and stomach with 88 cases,including 75 Hp positive while 13 negative,spleen-qi deficiency syndrome with 78 cases,including 59 cases Hp positive while 19 negative,dishamony syndrome between liver and stomach with 81 cases,including 59 cases Hp positive while 22 cases negative,stagnation of blood stasis syndrome with 24 cases,including 20 cases Hp positive while 4 negative,20 healthy people recruited from GuangZhou University of Chinese Medicine were classified as the normal group.All the general information of subjests was signed the informed documents.2.Collection of pathologic specimensAll the subjects in different groups were examined by electronic gastroscopy,4 specimens collected in the great gastric curvature of lesser curvature of stomach antrum of every subject,at a distance of 2 or 3 centimeters from pylorus by biopsy forceps.The 10 GC spcimens were collected in tumor resected or near the tumor at a distance of 2 or 3 centimeters with 3 pieces respectively or its distal part with 4 pieces,one of which was detected Hp infection by rapid urease tests,which one of which was fixed in 4%paraformaldehyde for histopathological detection,the other two specimens were put into separated freezeing tubes with DNA or RNA Storaging Solution and cryopreserved in the liquid nitrogen and then transferred to-80 ? low temperature refrigerator for generation sequencing and next generation sequencing.3.Observation and detection of indicatorsObserv e the Hp infection and its extent of infections for the gastric mucosa by the rapid urease tests and methylene blue staining and the inflammation of the gastric mucosa,the active degree,or the pathological progression of different stages with microscope by the method of HE.4.Detection of gene polymorphismThe extracted gastric mucosa DNA of every subjects were amplified and identified,then detected gene polynmorphism of IL-1 ?-511 and TNF-?-308 of every specimen by using double DNA chain termination direct sequencing menthod.5.The qualitative analysis of transcriptomeThe extracted gastric mucosa RNA of every subjects after reverse transcription were amplified and identified and then analysised the difference of product of different syndrome in Chinese Medicine in qualitative way by next generation sequencing.Results1.General information comparisonThe average age of damp-heat syndrome in spleen and stomach group,spleen-qi deficiency syndrome group,disharmony syndrome between liver and stomach group,and stagnation of blood stasis group were all higher than the normal gourp(P<0.05);the incidence of disharmony syndrome between liver and stomach group and damp-heat syndrome group of male were higher significantly than that spleen-qi deficierey syndrome(P<0.05),while the age,clinical course,sex and distribution of HPCD in rest four syndromes have no significant difference(P>0.05).2.The syndrome distribution of HPCDThe frequency of CG in disharmony syndrome between liver and stomach was higher than damp-heat syndrome in spleen and stomach and spleen-qi deficiency syndrome that was higher than stagnation of blood stasis syndrome;The frequency of peptic ulcer in damp-heat syndrome in spleen and stomach was higher than spleen-qi deficiency syndrome,which was higer than disharmony syndrome between liver and stomach and stagnation of blood stasis syndrome;The frequency of GC in damp-heat syndrome was highest,then were stagnation of blood stasis syndrome,spleen-qi deficiency syndrome and disharmony syndrome between liver and stomach,but is no significant difference(P>0.05).3.The different diseases entity and syndrome of HPCD,the infections and the extent of infections of HpThe infection and extent of Hp in GC patients and PU patients were significant higher than CG(P<0.05);the infection and extent of Hp in GC was higer than PU,but has no significant difference(P>0.05).In terms of syndrome,the tendency that Hp infection of damp-heat syndrome in spleen and stomach was higher than that stagnation of blood stasis syndrome that was higher than spleen-qi deficiency syndrome that was higher than disharmony syndrome between liver and stomach group exists;The tendency that the extent of infections of Hp of stagnation of blood stasis syndrome was higher than that damp-heat syndrome in spleen and stomach that was higher than spleen-qi deficiency syndrome group that was higher than disharmony syndrome between liver and stomach group,while there are no significant difference among the groups(P>0.05).4.The comparison of different syndrome and pathological histology of HPCDThe Hp infections of gastric mucosa was highly close to the degree of chronic inflammation,inflammation activity,atrophy and dysplasia(P<0.05),while there was no significant difference with intestinal metaplasia(P>0.05).The tendency that the degree of inflammation,atrophy,intestinal metaplasia and dysplasia of gastric mucosa in GC was higher than PU,which was higher than CG;while the tendency that the degree of inflammation activity in PU was higher than GC that was higher than CG(P<0.05).In terms of syndrome,the tendency that the inflammation and atrophy degree of gastric mucosa in disharmony syndrome between liver and stomach was the highest;the tendency that the inflammation activity degree of gastric mucosa in spleen-qi deficiency syndrome was the highest;the tendency that the dysplasia degree of gastric mucosa in stagnation of blood stasis syndrome was the highest,but has no significant difference(P>0.05).While the tendency that the intestinal metaplasia degree of gastric mucosa in damp-heat syndrome in spleen and stomach was higher than stagnation of blood stasis syndrome,which was higher than disharmony syndrome between liver and stomach and spleen-qi deficiency syndrome(P<0.05).5.The analysis of different diseases and syndrome of HPCDHPCD was positive correlate with stomachache,gastric distension,coated tongue(P<0.05),so does between the different syndrome of HPCD and stomachache,gastric distension,heavy limbs and coated tongue in TCM while was negative correlate with tongue picture,(P<0.05).however,there is no significant difference between HPCD and the such clinical syndrome as abdominal distension,appetite,belching,sour regurgitation,pulse,sodoes the different syndrome of HPCD(P>0.05).6.The relationship of gene polymorphism of IL-1?-511,TNF-?-308 and Hp infectionThe IL-1(3-511 genetype and distribution of Hp infections in HPGD patients'gastric mucosa showed that CT genetype was higher than that TT genetype that was higher than that CC genetype,in aspect of TNF-a-308,its genetype showed that GG was higher than that AG.Besides,the frequency of AG gengtype in Hp positive was higher thanthat in Hp negative.(P>0.05).There was no significant difference between the distribution of IL-1?-511 and TNF-a-308.The tendency that frequency of T allelomorph of IL-1?-511 in Hp positive were higher than that Hp negative exists,while the frequency of G allele of TNF-a-308 in Hp positive were higher than that Hp negative.There was no significant difference between the distribution of genetype and allele of IL-1?-511,TNF-?-308 and the degree of Hp infections(P>0.05).7.The relationship between the gene polymorphism of IL-1?-511,TNF-?-308 and HPCDThe tendency that CSG was higher than PU,which was higher than CAG and CG in terms of frequency of CC,CT,TT genetype of IL-1?-511 exists.In the aspect of allele frequencyC allelomorph was higher than T in CSG,but T allelomorph was higher in PU,CAG and CG,however,there is no significant difference(P>0.05).The difference diseases of HPCD exist in the form of TNF-?-308 GG genetype,but there is no significant difference in the distribution of genetype and allelomorph(P>0.05).8.The relationship between syndrome of HPCD and IL-1?-511,TNF-a-308 gene polymorphismThe tendency that frequency of IL-1(3-511 TT genetype of damp-heat syndrome in spleen and stomach was higher than that disharmony syndrome between liver and stomach that was higher than that spleen-qi deficiency syndrome that was higher than that stagnation syndrome of blood stasis group,but there is no significant difference(P>0.05).The tendency that frequency of TNF-a-308 GG genetype of damp-heat syndrome in spleen and stomach was higher than that spleen-qi deficiency syndromethat was higher than that disharmony syndrome between liver and stomach that was higher than that stagnation,but there is no significant difference(P>0.05).The tendency that frequency of TNF-a-308 AG genetype of disharmony syndrome between liver and stomach was higher than that spleen-qi deficiency syndrome that was higher than that damp-heat syndrome in spleen and stomach that was higher than that was higher than that stagnation syndrome of blood stasis,but there is no significant difference(P>0.05).The tendency that frequency of IL-1?-511 C allele gene of spleen-qi deficiency syndromewas higher than that disharmony syndrome between liver and stomachthat was higher than that stagnation syndrome of blood stasis that was higher than that damp-heat syndrome in spleen and stomach,but there is no significant difference(P>0.05).In terms of distribution of allele gene,the frequency of A allele gene in all groups apart from the stagnation of blood stasis group were higher than that of the control group,while G allele gene were lower than the control group,but there is no significant difference(P>0.05).9.The analysis of different gene and function of Hp infection:Compared to the Hp negative,there was 3705 obvious different gene in the Hp positive group,which of 2697 have the function of up-regulating.GAS was first and then the ribosomal protein S4,closed protein 2,CXC115 and MTL sequenced successively.And the rest of diifferent genes play such roles as protein synthesis and metabolism,signal transmission,cytoskeleton with the function of down-regulating,locating in pathways like NF-?B,MAPK,Toll receptor and Wnt.10.The analysis of different gene and function of different diseases of HPCD:In terms of expression of different gene of different diseases of HPCD,the 1759 gene of GC have the function of up-regulating while compared to the normal subjects..GAS was first and then defense cytokines-?6,CXC115,CXC118 sequenced successively.The 2208 different genes,such as TNF-?,IL-1?,pepsinogen 5.pepsinogen 3,have down-regulating function.Compared to CG,the 4264 different genes in GC,such as IL-1?,IL-8,TNF-?,have up-regulating function,while 337 like DEFB4A and HCN4 do the opposite jobs.Compared to para-carcinoma tissue,348 different genes in tumor tissue have up-regulating function,292 play opposite function,both of which participate in protein transduction activity,material metabolism,enzyme catalysis,regulating biological functions and pathways like p53?Wnt?NF-?B.11.The analysis of different gene and function of different syndrome of HPCD:After the comparsion the GC patients with different syndrome such as damp-heat syndrome in spleen and stomach,stagnation syndrome of blood stasis,spleen-qi deficiency syndrome with the normal subjects,the results showed that different genes in syndrome in TCM and deficiency and excess consititution play dominant function in protein transduction activity,material metabolism,enzyme catalysis,regulating biological functions.Conclusion:Our research detectde the gastric mucosa of HPCD by histopathological detection,generation sequencing and next generation sequencing technology,we reach the following concultions1.The inflammatory factor IL-1 beta-511-T allele and TNF-alpha-308-AG genotype might be the susceptibility genes of Hp infection,which migt also close to damp-heat syndrome in spleen and stomach and disharmony syndrome between liver and stomach.Hp could increase the level of gastric mucosa inflammatory respones with IL-1 beta-511 and TNF-alpha,caused gastric mucosal atrophy,intestinal metaplasia,and dysplasia,even cancre.The results were identical to the previous reseaches,which about mRNA and protein expression of IL-1 beta and TNF alpha in gastric mucosa.2.The different genes of HPCD participated in protein synthesis and metabolism,signal transmission and cytoskeleton etc.The HPCD getting worse might be related to cells consititution,cells abnormal proliferation and inflammatory cytokines etc,including the pathways activation and abnormal expression of genes.While the different genes of different syndrome,susceptible consititutions participated in cells migration and adhesion,fat,polysaccharide and protein synthesis and metabolism.The new sequencing method did the excellent jobs in syndrome and constitution theory in TCM from point of view of molecule,making explanation for different syndromes and susceptible diseases in different constitutions,providing solid theoretical basic for the progression of diseases caused by Hp infections. |
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