The Preliminary Study Of The Biological Functions Of Plasticity Gene JHP947

Objectives: To investigate the relationship between JHP947 gene of Hp and gastroduodenal diseases, and its relationship with JHP940,JHP947,JHP949, cagA, and to study the functions of JHP947 gene by its derivative mutant strains.Methods:1. Hp strains were isolated form gastric biopsy samples of patients with gastritis, peptic ulcer (PU) and gastric carcinoma (GC) . PCR-based methods was used for detecting JHP940, JHP947, JHP949, cagA. Experimental data were analyzed by theχ~2 test and partitions ofχ~2. The expression of JHP947 in HpJ99 was confirmed by Western blot hybridization. 2. Gene mutagenesis was performed by gene-replacement based on a PCR technique. To create the JHP947 knockout strain, PCR fragments containing JHP946-JHP947-JHP948 were cloned into the plasmid. Inverse PCR amplification excluding the JHP947 was performed using primers flanking the JHP947 gene. A km resistance gene cassette was inserted to replace the deleted gene resulting in pT△JHP947::km recombinant plasmid, which was transformed into E. coli DH5a for multiplication. the purified piasmid DNAs were introduced into HpJ99 to to knockout chromosomal JHP947 gene by electrotransformation. Inactivation of the genes was validated by PCR and Western blotting. 3. cDNA microarray technology was employed to investigate the effects of JHP947 of Helicobacter pylori on the gene expression profile of AGS cells. AGS cel1s were incubated with JHP947-wildtype strain J99 and the JHP947-knockout strain J99-947△Hp respectively.Total RNAs of cells treated with J99 and J99-947△were extracted for gene expression profile assay by cDNA microarrays. The identified expression profile was further verified by RT-PCR. 4. The in vivo effects of JHP947 on the expression levels of ESE-3A, NDRG1, MATP in gastric epithelial cells and on the inflammation levels of gastric mucosa were investigated by animal studies. Twenty-seven special pathogen free (SPF) C57BL/6 mice were randomly divided equally into 3 groups, which were challenged with J99, J99-947△and PBS respectively, at a dose of 109CFU at 0, 2, 4 days. Mice were sacrificed 4 weeks after the last challenge, and went through the following experimental assays, rapid urease test, culture of Hp, histological examination and immunofluorescent histochemistry of gastric mucosa.Results: 1. Positive rates of JHP940,JHP947,JHP949,cagA that in gastritis and peptic ulcer and gastric carcinoma were JHP940-30.6%,22.6%,40.0% ,JHP947-3.2%,32.2%,33.3%,JHP949-0,3.2%,13.3%,cagA -74.2%,93.5%,86.7%,respectively. Positive rates of JHP947 that in gastric carcinoma and peptic ulcer was higher in gastritis( P<0.00227,χ2 =12.04; P<0.00417,χ2 =8.64). Positive rates of JHP949 that in gastric carcinoma was higher in gastritis( P<0.00417,χ2 = 8.48). Positive rates of JHP947 with positive and negative JHP949 were 66.7%(2/3) and 14.3 %(15/105) ,respectively(P<0.05). Positive rates of JHP947 with positive and negative JHP940 were 25%(8/32) and 14.3%(9/67) ,respectively(P>0.05). Western blot hybridization using anti-JHP947 antibodies showed that JHP947-specific protein was expressed in HpJ99 and clinical strains(JHP947+), but not in Hp26695(JHP947-). 2.PCR amplification and Western blot hybridization showed that contrary to its parental strain, JHP947 knockout strains had neither JHP947 gene sequence and nor JHP947-specific protein. 3. The microarray assays showed that the expression of 18 genes was significantly up-regulated and108 genes was significantly down-regulated in AGS cells treated with J99. Included genes are those involved in tumorigenesis, cell cycle,signal transducer activity,transcription regulator activity,transporter activity,enzyme regulator activity,catalytic activity.The results of RT-PCR were consistent with those of gene expression pattern.4. The result of rapid urease test and culture of Hp indicated that the positive rates in J99 and J99-947△group were both 100% while 0% in PBS group. The result of histology examination indicated that garstirc mucosa is all normal in PBS group; in J99 group, 33.3%(3/9) had slightly anabrosis, 66.7%(6/9) had seriously anabrosis; in J99-947△group, 22.2%(2/9) is normal, 77.8%(7/9) had slightly anabrosis.The degree of anabrosis seems to be more severe in J99-947△than in J99. The result of immunofluorescent histochemistry of gastric mucosa indicated that the expression level of ESE-3A, NDRG1, MATP is significantly higher in J99-947△than in J99(P<0.05).Conclusions:1. The JHP947 gene is closly associated with peptic ulcer and gastric carcinoma.2. The microarray assays showed that the expression of 18 genes was significantly up-regulated and 108 genes was significantly down-regulated in AGS cells treated with J99. Included genes are those involved in tumorigenesis, cell cycle,signal transducer activity,transcription regulator activity,transporter activity.3. The degree of anabrosis seems to be more severe in Hp with JHP947gene than in Hp without JHP947gene.4. In vivo, JHP947 may induce tumorigenesis by inhibiting anti-oncogene(MTAP,NDRG1).