Objective:To express Helicobacter pylori oipA gene with different fragments and determine the protein and its peptides with finest antigenicity.Methods:oipA gene was obtained from international standard Hp NCTC 11637 and cloned into vector pGEM-T then was identified by PCR and sequencing.Six different fragments of oipA gene were amplified by PCR with the template pGEM-T/oipA.The gene fragments and expressing vector pET-42a were ligated.The recombinant vector were transformed into BL-21.The correct recombinants were identified by PCR,enzyme digestion and sequencing.Recombination protein and the peptides were detected by Western Breeze chemiluminescent after induced by IPTG.Optimization of the time and IPTG concentration for induction were conducted.The expressed products were affinity purified by Ni-NTA of His?Bind.The antigenicity of the recombination protein was determined with Western blot indicated by goat anti-Hp polyclonal antibody,and the recombination protein with finest antigenicity was selected with indirect ELISA.Results:The six oipA gene fragments could all expressed and the products were recognized by goat-anti Hp polyclonal antibody.The finest peptide with antigenicity was the smallest peptide.Conclusion:OipA gene fragments could be expressed in prokaryotic system and the smallest peptide had the finest antigenicity.It may be used as the reagent to detect corresponding antibody in serum.

Objective To investigate MR findings and analysis of misdiagnosis of atypical invasive pituitary adenoma.Methods The MR findings of twenty cases of atypical invasive pituity adenomas confirmed by pathology were reviewed ,which were misdiag-nosed as chordomas or meningiomas.Results All the twenty cases showed iso-or slightly hypo-signal on T1 WI,iso-or slightly hy-per-signal on T2 WI;Enhanced scan displayed heterogeneous enhancement.The dynamic enhancement curve showed rapid enhance-ment phase.The bilateral cavernous sinuswere infringed in nine cases,in which the pituity and pituity stalk were not well seen. Among the twenty cases,twelve cases with suprasellar and anterior cranial fossa extension were misdiagnosed as meningiomas;eight cases with clival destruction were misdiagnosed as chordomas.Conclusion The MR features of atypical invasive pituity adenomas are various.In order to avoid misdiagnosis,a comprehensive analysis should be based on a variety of signs.

Objective To investigate the value of spiral CT for the diagnosis and treatment of congenital anorectal malformations (CARM). Methods Thirty children with CARM underwent lateral upside-down plain film in abdomen and spiral CT scanning in pelvic cavity before surgical operation. The distance between rectal blind end and anal fossa in two imaging modalities was measured. The correlation between the measurement results in two imaging modalities was compared with operational results. Sacral deformation was diagnosed according to 3D reconstruction of CT images. Results The measurement results obtained from CT were correlated positively with those of operation (r=0.99, P<0.01). CARM combined with curved sacrum was diagnosed in one child. No correlation was found between the measurement results of X-ray and operation (r=0.31, P>0.05). Conclusion Spiral CT scanning can accurately describe the atresic level of rectum and malformation of sacrum. It can provide reliable evidence for surgeon to choose the best surgical method and to judge prognosis.

Objective To study the effect of Hydrogen on ischemia/reperfusion(I/R)-induced cardiocyte apoptosis and apoptosis related proteins expression in diabetic rats .Methods Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) ,then feed 4 weeks before build ischemia/reperfusion model .60 SD male rats ,weight during 300-350 g ,were randomly divid-ed to six groups :non-diabetic sham-operated group (NS) ,non-diabetic I/R group(NI/R) ,non-diabetic hydrogen treated group (NH) ,diabetic sham-operated group(DS) ,diabetic I/R group(DI/R)and diabetic hydrogen treated group(DH) ,each group has 10 rats .The cardiac muscle I/R model was made by 30 min occlusion of the left anterior descending coronary artery and 2 h reperfu-sion .The rats in Hydrogen group were treated with 5 mL/kg hydrogen by intraperitoneal administration at the beginning of reper-fusion .The apoptosis index (AI) was calculated by TUNEL .The positive expressions of Bcl-2 ,Bax ,Caspase-3 in cardiomyocytes were respectively detected by immunohistochemistry .Results The apoptotic rates of cardiomyocytes and the positive expressions of Bcl-2 ,Bax ,Caspase-3 were significantly increased(P<0 .01) ,Compared with I/R group ,the apoptotic rates of cardiomyocytes in hydrogen treatment group were obviously decreased(P<0 .01) ,and the positive expressions of Bcl-2 were increased(P<0 .01) ,at the same time ,the positive expressions of Bax ,Caspase-3 were decreased(P<0 .01) .Conclusion Hydrogen inject by intraperitoneal method on myocardial ischemia-reperfusion injury of diabetic rats has a protective effect ;Its mechanism may be related to its inhibi-ting myocardial apoptosis by advanced the Bcl-2 protein expression and reduced the Bax 、Caspase-3 protein expression .

OBJECTIVE:To prepare nanostructured lipid carrier of adefovir dipivoxil(ADV-NLC),and optimize the formula-tion. METHODS:Using stearic acid and glycerin monostearate as solid lipid,oleic acid as liquid lipid,Gemini surfactant and poly-sorbate 80 as emulsifier,sodium dodecyl sulfate (SDS) as stabilizer,solvent dispersion ultrasonic method was used to prepare ADV-NLC. And using particle size,polydispersity index,Zeta potential,encapsulation efficiency as indexes,single factor test was conducted to screen Gemini surfactant-polysorbate 80 ratio,emulsifier dosage(ratio of emulsifier to water phase),drug-lipid ratio, solid-liquid lipid ratio. RESULTS:The formula was as follow as 3% emulsifier (Gemini surfactant-polysorbate 80 ratio of 1:2), 4.5% drug-lipid ratio,solid-liquid lipid ratio of 6:5. The average particle size of the prepared ADV-NLC was(48.83±2.65)nm, polydispersity index<0.3,Zeta potential was(-28.7±1.8)mV,encapsulation efficiency was(77.65±0.03)%(n=3). CONCLU-SIONS:ADV-NLC is successfully prepared,and the formulation is reasonable and feasible.

Objective To investigate the effect of B7-H3 on human hepatocellular carcinoma cell line HepG2 mediating regulation on human peripheral blood CD8+T cell activation,cell cycle and secretion of IL-17.Methods The expression of the B7-H3 on HepG2 cells was detected by RT-PCR and FCM respectively.B7-H3 was silenced by PGPU6/GFP/neo-B7-H3shRNA plasmid via cathodolyte liposome transfection method.CD8+T cells were sorted from healthy human peripheral blood with immunomagetic beads.The effect of HepG2 cells on activation,cell cycle and cytokine secretion of CD8+T cells which was stimulated by PHA or PMA respectively were analyzed by FCM.Results B7-H3 was highly expressed on HepG2 cells,and PGPU6/GFP/neo-B7-H3shRNA plasmid could effectively block down its expression.Otherwise,HepG2 cells could inhibit the expression of CD69,the early activation phenotype of T cell,blockade B7-H3 on HepG2 cells could significantly attenuate the inhibitory effects.Likewise,blockade B7-H3 on HepG2 cells apparently reversed the inhibitory effects of HepG2 cells on CD8+T cell cycle through down-regulating the cell number in G0/G1 phase and up-regulating the cell number in S phase;Moreover,HepG2 cells caused a sharp increase of IL-17 which was secreted by CD8+T cells and the level of IL-17 was further up-regulated after blocking down B7-H3.Conclusion HepG2 cells highly expressed B7-H3 that could promote the inhibitory the effect of HepG2 on expression of CD69 and cell cycle of CD8+T cells.HepG2 cells were able to up-regulate the level of IL-17 secreted by CD8+T cells,in which B7-H3 played an inhibitory role.

Paired basic amino acid cleaving enzyme 4 (PACE4),a subtilisin-like endoprotease,which is thought to play a significant role in tumor occurrence and development.Its over or low expression may lead to enhanced proliferation and invasion of tumor cells,and even increase the malignant degree.The specific regulation according to the roles of PACF4 expression in different tumors may be helpful for tumor treatment and prognosis improvement.

Objective: To optimize Helicobacter pylori(H.pylori)oipA gene and develop strain of vaccine in Attenuated Salmonella typhimurium SL7207 haxboring recombinant plasmid pVAX1-oipA and pVAX1-optioipA, then to study its protection against H.pylori in C57BL/6 mice.Methods: oipA gene was modified by codon optimization,inserting the Kozak sequence and engineering CpG motifs.The optioipA gene and oipA gene was inserted into the eukaryotic expressing vector pVAX1 respectively to develop recombinant plasmid pVAX1-optioipA and pVAX1-oipA.The AGS cells were transfected by pVAX1-oipA,pVAX1-optioipA and pVAX1 respectively.Then the OipA protein expression was confirmed by Western blot.pVAX1-oipA,and pVAX1-optioipA were converted to LB5000 for methylation and then the modified eukaryoticvector was electrotransfered to final host SL7207 respectively, SL7207/pVAX1-oipA, SL7207/pVAX1-optioipA was identified with PCR and enzyme digestion.Specific serum IgG antibody was detected by indirect ELISA after oral inoculation with vaccine strains.Mice were challenged with live Sydney strain(SS1)three times at 0,2,4 d(5 × 10~8CFU/each mouse).Fonr weeks after challenge, the mice were sacrificed.The colonization of H.pylori in gastric mucosa were detected by rapid urease test and quantitative culture of H.pylori.Results: The AGS cells transfected with pVAX1-oipA and pVAX1-optioipA had expressed corresponding production, moreover, the expression level of pVAX1-optioipA was higher than those of pVAX1-oipA,SL7207/pVAX1-oipA and SL7207/pVAX1-optioipA confirmed with PCR,enzyme digestion;Two weeks after last immunization,immunized mice.were induced to produce anti-OipA IgG antibodies, and the levels of anti-OipA IgG antibody in pVAX1-optioipA group was obviously higher than in pVAX1-oipA group(P＜0.01); After challenged with SS1, the infection rate of pVAX1-oipA and pVAX1-optioipA group was 60%(9/15)and 26.67%(4/15), which was significantly lower than 100%(10/10)in the control group(P＜0.01).Conclusion:Attenuated Salmonella typhimurium SL7207 containing pVAX1-oipA, pVAX1-optioipA is successfully constructed.It can protect mice from H pylori infection and optimizing oipA gene can enhance the protection efficiency.

Objective To understand the status of oipA gene of Helicobacter pylori (H.pylori) and to evaluate the relationship between oipA gene and digestive disease, Methods Biopsy gastric mucosa were obtained from 360 patients with digestive disease under gastroscopy. H.pylori was isolated from urease positive and some negative samples, and then identified by microscope, urease test and 16 S rRNA PCR. oipA and cagA genes of the isolates were amplified by PCR and the status of oipA signal region was analyzed after sequencing. The relationship between functional oipA gene of H. pylori and digestive disease was investigated. Results One hundred and six isolats were obtained. Seventy-two strains were positive for cagA gene and 87 were positive for oipA gene in signal region. Eighty strains were on open status in signal region called functioal oipA gene and 1 was on off status. The status of other 6 strains were uncertain. The frequency of functional oipA in ulcer and atrophic gastritis were higher than that of cagA(100% and 78.26% vs 58.33% and 39.13%).Conclusion Functional oipA gene is closely related with ulcer and atrophic gastritis.

Objective:To analyze the morbid risk factors of chronic tubulointerstitial nephropathy(CTN)with an unknown origin in Wenzhhou City.Method:810 residents in several big districts were investigated.652 subjects,which had complete data and were coincided with the inclusion standard,were analyzed by an unmatched case-control study.The case group contained 19 cases coincided with the diagnostic criteria for CTN with unknown causes.The controlled group contained 633 cases.Thirteen probable risk factors were selected to be investigated:age,sex,body height,body weight, occupation,education,allergic history,addiction to smoking or wine,family history,the respective exposure to heavy met- als,radioactive ray and poisons.A history of medications,including caulis aristolochiae manshuriensis,antipyretic analge- sic,antibiotics,etc.was also investigated.The odds ratio(OR)of CTN with an unknown origin caused by risk factors were calculated with Logistic regression analysis.The obtained data were investigated,and the risk factors were screened out. All the data were analyzed with SPSS 12.0.Result:The homogeneity test showed there was no significant difference in age, sex,occupation and education between the case and controlled groups.The exposure to heavy metals or caulis aristolochiae manshuriensis was screened as the valid risk factor of CTN with OR 17.50 and 103.594 respoetively(P