| Investigated background and objective It was knowned that H pylori infection was a definited carcinogenic agent and the first impulse factor of homo-sapiens gastric carcinogenesis,especially typical intestine type in the process of chronic superficial gastris (CSG)- chronic atrophic gastris(CAG)- intestinal melaplasia(IM)- atypical hyperplasia(AH)- gastric carcinoma(GC) , which had been prefered by Correa.GC was a species of genopathy and its carcinogenesis was the gradually accumulated change of multiple genes those possibly made roles in diaparate phases. It was associated with not only proto- oncogene activation and anti-oncogene mutation ,but also cell apoptosis genes, which intimately participate in the gastric carcinogenesis.Possibly apoptosis related genes make critical roles in the process of gastric cancerization with H pylori infection. At present , it is not yet clarity that the sure effect and the mechanism of cell apoptosis regulation with H pylori infection,also the correlation with them ,and the apoptosis inducing and inhibiting genes maybe make different roles in different phases.So it is necessary to explore the pro- apoptotic and antiapoptotic machanism of them in the process of gastric cancerization with H pylori infection. Fas-associated factor 1 (FAF1)was a protein recently discoved and had been demonstrated to be a component of the death inducing signaling complex (DISC) in Fas-mediated apoptosis, whose N-termial region specificly combinated with Fas to enhance the Fas-mediated apoptosis. FAF1 was down-regulated in gastric cancer tissue specificly. Now it is known little about the role of FAF1,and our early study had tentatively confirmed the prior conclusion,furthermore,no report about the correlation and mechanism of action between H pylori infection and FAF1 as a proapoptotic factor in gastric cancerization. B-cell lymphoma/Leukemia-2(Bcl-2) was a anti- apoptotic gene which was discovered earliestly and investigated deeply and extensively up to now.It was known that Bcl-2 was a most important factor to inhibit the apoptosis of tumor cell. Survivin was a member of inhibitors of apoptosis proteins(IAP) family and a most strongest antiapoptotic factor,which affected on the assemble point of all apoptosis pathway.Survivin expression was negative or low in the normal ripe human tissue,but positive and high in human majority tumor tissue, which to hint it important in carcinogenesis and deve- lopment .The machanism of action yet know little between H pylori infection and Bcl-2 as antiapoptotic factors in gastric cancerization,also together with FAF1 as a proapoptotic factor. In conclusion,in this study we aim to clarity the correlation and possible machanism of action between H pylori infection and those genes related with cell apoptosis regulation, furthermore, combining with clinical correlated information, to explore the effect of those genes on the early diagosis , infiltration ,metastasis and prognosis of gastric cancer .Which to offer some referred molecular biological parameters for the early diagosis and treatment of gastric cancer. Methods To explore the correlation between H pylori infection and FAF1 ,Bcl-2 and Survivin expression in gastric carcinogenesis,also cell apoptosis,H pyloriinfection was evaluated by Rapid Urease Test ,Methylene blue and Warthin-Starry staining method,and the expression of FAF1 ,Bcl-2 and Survivin were detected by immunohistochemical (IHC) staining ,while the level of cell apoptosis were detected by terminal deoxynucleosides mediated nick end labeling (TUNEL)staining,including 62 cases of chronic superficial gastris (CSG), 55 cases chronic atrophic gastris(CAG),52 cases intestinal melaplasia (IM) ,46 cases atypical hyperplasia(AH),65 cases gastric carcinoma(GC). Furthermore,to explore the correlation between H pylori infection and genes transcription,the expressin level of FAF1 mRNA,Bcl-2 mRNA and Survivin mRNA were detected by Fluorescent Qanlity Real-time polymerase chain reaction.Additionaly,combinating with the pathematology and follow-up data of clinical cases , to explore the effect of those genes on the early diagosis , infiltration ,metastasis and prognosis of gastric cancer.ResultsChapter I1. The positive rate of H pylori infection during the course of gastric cancer progression. The positive rate of H pylori infection was 53.6% with three methods combined,and increased with the degree of carcinogenesis of gastric mucosa .The positive rate was 30.6%,49.1%,55.8%,65.2%,69.2%,respectively. The infection rate of H pylori in CAG,IM,AH,GC were higher than that in CSC respectively(p<0.05),and GC were higher than CAG(p<0.05).2. The correlation between the expression of FAF1,Bcl-2 and Survivin protein and cell apoptosis. The positive rate of FAF1 protein expression decreased with the degree of carcinogenesis of gastric mucosa. The positive rate was 72.6%,65.5%,63.8%,54.3%,36.8% respectively. The positive rate of FAF1 protein expression in AH were lower than that in CSG (p<0.01),and GC were lower than CSG,CAG,IM respectively(p<0.01).While, the positive rate of Bcl-2 and Survivin protein expression increased with the degree of carcino- genesis of gastric mucosa. The positive rate of Bcl-2 expression was 16.1%, 21.8%,32.7%,50.0%,61.5% respectively. The positive rate of Bcl-2 expression in IM was higher than in CSG (p<0.05),and AH and GC were higher than CSG and CAG,respectively(p<0.01).The expression of Survivin was negative in CSG,and weak in non-carcinoma pathological tissue,but strong in gastric cancer tissue. The positive rate of Survivin expression was 0,9.1%,17.3%,45.7%,63.1% respectively. The positive rate of Survivin expression in AH and GC were significant higher than in CSG ,CAG and IM(p<0.01).The AI increased in the phase of CSG and CAG, while decreased in the phase of AH and GC gradually. The AI in CAG and IM were significant higher than in CSG (p<0.01), while AH and GC were lower than CSG,CAG and IM respectively(p<0.01).During the course of gastric cancer progression ,the AI with FAF1 positive expression were higher than that with FAF1 negative among all groups(p<0.05, p < 0.01),howerver, the AI of Bcl-2 positive expression were lower than negative ,especially in IM,AH and GC(p<0.05,p<0.01),and the AI of Survivin positive expression were lower than negative in CAG, IM,AH and GC( p<0.05).During the course of gastric cancer progression, there was negative correlation between FAF1 positive and the degree of carcinogenesis of gastric mucosa(r=-0.251,p<0.05), but there were positive correlation between Bcl-2 and Survivin positive and the degree of carcinogenesis of gastric mucosa (r=0.361, 0.535 respectively, p all<0.01).There were negative correlation between FAF1 positive rate and Bcl-2 and Survivin positive rate(r=-0.958, -0.971 respectively,p all < 0.05),howerver positive colleration was found between Bcl-2 and Survivin postive rate(r=0.995,p<0.01). There was only positive correlation tendency between FAF1 positive rate and AI(r=0.738,p>0.05),while negative correlation tendency between Bcl-2 and Survivin positive rate and AI(r=-0.748,-0.782, p all >0.05).3. The correlation between H pylori infection and cell apoptosis. We found the AI of H pylori positive was higher than that of H pylori negative in CSG (t' =2.055,p<0.05),while lower in GC (t' =2.817,p<0.05).There were no difference in other groups(p>0.05).4 . The correlation between H pylori infection and the expression of FAF1,Bcl-2 and Survivin protein. We found the positive rate of FAF1 protein expression with H pylori positive was lower than that of H pylori negative in AH and GC, and there was significant difference in GC(p<0.05). There were no difference in other groups, though the positive rate of FAF1 protein expression with H pylori positive was a little high than that with H pylori negative(p>0.05).The positive rate of Bcl-2 protein expression with H pylori positive was higher than that of H pylori negative in all group,but only there was significant difference in GC(p<0.05). The positive rate of Survivin protein expression with H pylori positive was higher than that of H pylori negative in all group,but only there was significant difference in AH and GC(p<0.05).Chapter II1. The expression of FAF1,Bcl-2 and Survivin mRNA during the course of gastric cancer progression. During the course of gastric cancer progression (CSG→CAG→IM→AH→GC),FAF1mRNA expression was detected in different gastric mucosa lesion groups,and the relative quantity in GC was lower. The Bcl-2 mRNA positive rate was 22.6%(14/62),30.9(17/55),40.4%(21/52), 58.7%(27/46),69.2%(45/65) respectively. The Survivin mRNA positive rate was 0,12.7%(7/55),25.0%(13/52),54.3%(25/46),75.4%(49/65)respectively.The level of FAF1mRNA expression in different groups were0.136±0.008, 0.131±0.009,0.128±0.010,0.094±0.032,0.006±0.003 respectively. Bcl-2 mRNA were 0.095±0.015,0.115±0.012,0.173±0.027,0.224±0.042,0.368±0.036 respectively. SurvivinmRNA were 0, 0.068±0.007, 0.347±0.060, 0.553±0.080, 1.646±0.356.We can conclude FAF1 mRNA decreased gradually with the degree of carcinogenesis of gastric mucosa ,but Bcl-2 mRNA and Survivin increased in the same course. The level of FAF1 mRNA expression were lower in IM,AH and GC than that in CSG(p<0.05,p<0.01),and AH was lower than IM(p<0.01),including GC was lower than IM and AH(p<0.01). The level of Bcl-2 mRNA expression were higher in IM,AH and GC than that in CSG and CAG(p<0.01),and AH was higher than IM(p<0.01),including GC washigher than IM and AH(p<0.01). The level of Survivin mRNA expression were higher in IM,AH and GC than that in CAG(p<0.05, p<0.01),and AH was higher than IM(p<0.05),including GC was higher than IM and AH(p<0.01).The level of three genes expression showed difference in IM firstly, the most significant in GC, Following in AH.2. The correlation among the level of FAF1mRNA ,Bcl-2 mRNA and Survivin mRNA during the course of gastric cancer progression. During the course of gastric cancer progression, there was negative correlation between the level of FAF1 mRNA expression and the degree of carcinogenesis of gastric mucosa (r=-0.827,p<0.01), but there were positive correlation between the level of Bcl-2 and Survivin mRNA expression and the degree of carcinogenesis of gastric mucosa(r=0.945, 0.918 respectively, p all < 0.01) with Spearman Correlations test .There were negative correlation between the level of FAF1 mRNA expression and the level of Bcl-2 and Survivin mRNA expression (r=-0.911, -0.890 respectively,p all<0.01),howerver positive colleration was found between Bcl-2 mRNA and Survivin mRNA (r=0.906,p<0.01) with Bivariate Correlations analysis.3. The correlation between mRNA and protein of FAF1,Bcl-2 and Survivin during the course of gastric cancer progression. During the course of gastric cancer progression ,there were positive correlation between the level of FAF1,Bcl-2 and Survivin mRNA expression and the level of Bcl-2 and Survivin protein expression (r=-0.298, 0.324, 0.403 respectively,p all<0.01),so there were consistency between them respectively.4. The correlation between H pylori infection and the expression of FAF1,Bcl-2 and Survivin mRNA during the course of gastric cancer progression We found the level of FAF1 mRNA expression with H pylori positive was little higher than that of H pylori negative in CSG and CAG , but this tendency altered in IM, howerever, the level of FAF1 mRNA expression with H pylori positive was lower than that of H pylori negative in AH and GC(p<0.05,p<0.01).The level of Bcl-2 mRNA expression with H pylori positive was higher than that of H pylori negative in IM,AH and GC respectively(p<0.05, p<0.01). The level of Survivin mRNA expression with H pylori positive was higher than that of H pylori negative in IAH and GC respectively(p<0.05). H pylori infection can gradually down-regulated the level of FAF1mRNA expression,but up-regulated the expression of Bcl-2 and SurvivinmRNA expression during the course of gastric cancer progression. Chapter III1. The correlation on FAF1,Bcl-2 and Survivin protein expression and clinical pathological characteristics in gastric cancer It was showed that there were close correlations between FAF1 expression and tumor size, Histological grade, Tumor infiltration, nodus lymphaticus metastasis, distance metastasis and TNM clinical stage( p<0.01),and between Bcl-2 expression and tumor infiltration, nodus lymphaticus metastasis, and TNM clinicalStage( p<0.01), meanwhile between Survivin expression and tumor histological grade, Tumor infiltration, and TNM clinical stage(p <0.05, p <0.01).There were negative correlation between the level of FAF1 protein expression and the level of Bcl-2 and Survivin protein expression in gastric cancer (r=-0.236,-0.293, respectively,p all<0.01),howerver positive colleration was found between Bcl-2 protein and Survivin protein (r=0.210,p<0.05) .2. The prognosis factors investigation on the correlation of FAF1,Bcl-2 and Survivin and clinical pathological characteristics in gastric cancer. Single factor COX risk model assay showed the level of FAF1,Bcl-2 and Survivin expression ,tumor size , anatomic site, tumor infiltration, nodus lymphaticus metastasis, distance metastasis and TNM clinical stage histological grade all were markely correlated with the prognosis of patients with gastric cancer by radical resection.FAF1 expression was protect factor ,and others were risk factors. Independent risk factors analysis showed FAF1,Bcl-2 and Survivin were not indepent risk factors which influenced on the prognosis of patients with gastric cancer radical resection.3. The correlation on FAF1,Bcl-2 and Survivin expression and survival in gastric cancer by radical resection.The median surviral time was prolonged and surviral rate increased with the degree of FAF1 expression increased, Howerver,with Bcl-2 and Survivin expression increased the median surviral time was shorten and surviral rate decreased. There were signifcant defferences for 1 year, 3 years and 5years surviral rate (χ~2=1.029,χ~2=2.302,χ~2=6.218,respectively, p all <0.01). There were significant differences for 3 years and 5years surviral rate(χ~2=13.102,χ~2=3.017,respectively,pall<0.01) among the Bcl-2 groups.There was significant difference among the Survivin groups (-,+,++,+++)for 1year, 3 years and 5years surviral rate (χ~2=36.463,χ~2=43.787,χ~2=8.646,respectively, pall<0.01).With Log-Rank methods,there were significant difference among the different degree three groups of FAF1.Besides Bcl-2(- and+) groups,there were significant difference in other Bcl-2 groups(p<0.01, p<0.05). Besides survivin(- and +,+and ++) groups, there were significant difference in other survivin groups(p<0.01, p<0.05).Conclusion1. In this study on the protein and cell fields ,we noticed the FAF1,Bcl-2 and Survivin related apoptosis regulation genes expression level altered obviously in the process of CSG→CAG→IM→AH→GC,with FAF1 expression increased and Bcl-2 and Survivin decreased, which showed proapoptosis factor FAF1 mediated and regulated cell apopotosis attenuated, while anti- proapoptosis factors Bcl-2 and Survivin mediated cell apoptosis to enhance , especially in AH the apoptosis process was repressed obviously ,with AI decreasing the same time, so the disbalance between cell proliferation and apopotosis to induce gastric carcinogenesis.2.In this study on the gene and protein fields ,we explored the relationship between H pylori infection and FAF1,Bcl-2 and Survivin,and found H pylori infection could down-regulated the level of FAF1 gene and portein expression, but up-regulated the expression of Bcl-2 and Survivin genes and proteins expression in the process of CSG→CAG→IM→AH→GC ,which all showed the disblance between the FAF1 and Bcl-2,Survivin possibly was a mechanism of gastric carcinogenesis induced by H pylori infection.3. In our clinical study, we found FAF1,Bcl-2 and Survivin all were intimately correlated with the biological behaviour of gastric cancer and clinical pathology characteristic, also were factors which influenced on the surviral prognosis of patients with gastric cancer radical resection. he higer FAF1 protein level was correlated with good prognosis,and higer Bcl-2 and Survivin protein level were correlated with poor prognosis.
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