Objective To clone hpaA gene of H.pylori and construct its eukaryotic expression plasmid. Methods Gene hpaA amplificated from genome of H.pylori 11637 strain by PCR was subcloned into pMD18-T vector. Then hpaA cut down from the vector with restriction enzyme was inserted to pTCAE, and the product confirmed by restriction enzyme digestion and sequence was transformed to E.coli DH5?. pT-hpaA was transfected into CHO cell by electroporation, and HpaA protein was detected by Western blot. Results hpaA gene was inserted into pTCAE and the immunological reaction band with anti-HpaA serum at MW 30 000 was detected by Western blot. Conclusion Eukaryotic expression plasmid of hpaA of H.pylori is successfully constructed. Western blot analysis demonstrates that the expression of HpaA protein can be detected in culture supernatants of transfeced CHO cells, which lays the foundation for the further study.

Objective To evaluate mid-term clinical result of drug-eluting stent (DES) implantation for patients with multivessel chronic total coronary occlusion (CTO) lesions. Methods We analyzed 89 patients who underwent interventional revascularization in their multivessel CTO lesions with DES from November 2002 to June 2005 (DES group). A control group was composed of 70 consecutive patients with multivessel CTO lesions treated with bare metal stents (BMS) from November 2000 to June 2003 (BMS group). Results There were no significant differences in basic clinical manifestations and success rate of intervention procedures between DES and BMS group. At 6-month follow-up, the cumulative rate of major adverse cardiac events (MACE) was 14.6% in the DES group and 28.6% in the BMS group (P=0.031). The incidence of restenosis was 16.9% in the DES group and 34.3% in the BMS group (P=0.011). The need for target vessel revascularization and target lesion revascularization in the DES group was significantly lower (9.0% vs 21.4%, P=0.003, and 7.9% vs 18.6%, P=0.043, respectively) compared with BMS group. Conclusion It is possible to obtain an ideal mid-term clinical outcome in patients with multivessel CTO lesions by DES implantation.

Objective To evaluate the 6-month result of implantation of drug eluted stent (DES) in diabetic mellitus (DM) patients with multivessel coronary artery disease (MVD) to compare with that of bare metal stent (BMS). Methods From November 2002 to June 2005, 443 consecutive T2DM patients with MVD without previous myocardial revascularization underwent successful percutaneous coronary intervention (PCI). There were 226 patients in BMS group, 91patients in Cypher stent group, and 126 patients in TAXUS stent group. The results during hospitalization and 6-month clinical follow-up were analyzed among the 3 groups. Results There were no difference in clinical characteristics, PCI success rate, and incidence of in-hospital major adverse cardiac events (MACE) among 3 groups. However, concerning the characteristics of coronary lesions, the rate of bifurcation lesions and lesion length in Cypher stent group and the rates of bifurcation lesions and triple coronary lesions in TAXUS stent group were significantly higher than that in BMS group, respectively (P

Objectives To compare the in-hospital outcomes of elderly patients with acute myocardial infarction (AMI)treated by interventional or conservative protocols. Patients and Methods One handred and seventy-six consecutive patients hospitalized for AMI were involved, including 95 patients underwent emergent percutaneous coronary intervention (PCI) within 24 h after the onset of AMI and 81 patients received conservative non-invasive therapies.Clinical characteristics and in-hospital cardiac events of these two divisions were analyzed. Results In the PCI group, success rate of procedure and lesions was 98.9% and 98.5%, respectively. Procedure related complication were occurred in 6 cases(6.3%) and no patient died during operative procedures. PCI group had a lower in-hospital mortality (11.6% vs 24.7%, P＜0.05) and overall cardiac events rate (2A.2%vs56.8%, P＜0.01) compared with conservative group. Patients complicated by pump failure at admission in PCI group had a lower mortality compared with their counterpart in conservative group(27.3% vs 60.9%, P＜0.05). The average hospital duration between the two groups was no significant differences. The coronary care unit (CCU) duration of the PCI group was less than that of conservative group (4±5d vs 8±5d, P＜0.05). Conclusions In elderly patients with AMI, interventional treatment can significantly decrease the in-hospital mortality and cardiac events rate compared with conservative treatment, thus gains a better short-term outcome.

To assess the variability of adhesin gene hpaA in differene H pyloristrains with PCR restriction fragment length polymorphism (RFLP) A 710 bp gene hpaA , obtained from 9 different H pylori strains, were digested by Hha Ⅰand Hae Ⅲ individually and analyzed by agarose gel electrophoresis Four different polymorphic types were found in hpaA digested with Hae III and five types with Hha I Clinical isolates of H pylori from Chongqing showed difference among them and remarkably distinguished from foreign standard strains Mongolia gerbil adapted H pylori strain,which were obtained from Mongolia gerbil infected with clinical isolate, also showed inconsistence in hpaA RFLP The hpaA gene from different H pylori strains revealed 1 variability, and this might provide an effective method for developing molecular epidemiology of H pylori

Objective To isolate and purify VacA protein secreted by Helicobacter pylori or recombinant VacA , and to investigate the effect of VacA-induced cell vacuolar change and apoptosis .Methods VacA proteins were separated and pu-rified from the culture supernatant of H.pylori ( ATCC26695 ) or from the split products of genetically engineered bacteria (pQE30-VacA-E.coli M15) expressing recombinant VacA.The VacA protein obtained was acidified and then incubated with AGS cells for 24 h at different final concentrations of 5 and 10 ng/ml before the vacuolar change and apoptosis of AGS cells were detected via microscopy and flow cytometry assay , respectively .Results H.pylori-secreted VacA and recombi-nant VacA were successfully separated and purified .The H.pylori-secreted VacA significantly induced the vacuolar change and apoptosis of AGS cells (P<0.01) while the recombinant VacA did not.Conclusion H.pylori-secreted VacA protein can effectively induce cell vacuolar change and apoptosis, but recombinant VacA can not, suggesting that the purified VacA protein secreted by H.pylori can be used to explore VacA-induced pathogenesis.

Objective To evaluate the early and mid-term results of endovascular repair for acute and chronic type B aortic dissection, and to compare the clinical outcomes between the 2 groups. Methods From May 2002 to December 2006, 50 patients with type B aortic dissection were treated by endovascular stent-graft. There were 23 patients in the acute aortic dissection (AAD) group and 27 patients in the chronic aortic dissection (CAD) group. All patients were followed up from 1 to 54 months (average, 17±16 months).The immediate and follow-up clinical outcomes were documented and compared between the 2 groups. Results Placement of endovascular stent-grafts across the primary entry tears was technically successful in all 50 patients. Compared to the CAD group, the AAD group had a higher percentage of pleural effusion (17.4% vs. 0%,P=0.04) and visceral /leg ischemia (26.1% vs 3.7%, P=0.04). Procedure related complications, including endoleak and post-implantation syndrome, occurred more frequently in the AAD group than in the CAD group (21.7% vs 3.7% and 30.4% vs 11.1%, respectively; P=0.08 and P=0.04). Kaplan-Meier analysis showed no difference in the survival rate at 4 years between the 2 groups (86.4% vs 92.3%, P=0.42 by log-rank test). However, the event-free survival rate was higher in patients with chronic dissection than in patients with acute aortic dissection (96.2% vs 73.9%; P=0.02 by log-rank test). Conclusions Endovascular repair with stent-graft was safe and effective for the treatment of both acute and chronic type B aortic dissection. However, both immediate and long term major complications occurred more frequently in patients with acute dissection than in those with chronic dissection.

Objective To investigate the efficacy and safety of 6-month and 12-month oral dual-antiplatelet therapy This work was supported by the National Key Technology Research and Development Program in the Twelfth Five-year Plan of China (2011BAIl1B07) and the Military Clinical Key Technology and Development Program (2010gxjs001)(DAPT) on patients implanted with biodegradable polymer-coated and drug-eluted long stents (BP-DES).Methods In the I-LOVE-IT 2 trial,574 patients implanted with biodegradable polymer-coated and sirolimus-eluted long stent (BP-SES) (total stent length ≥50mm) were randomized to accepting either 6-month (n=270) or 12-month (n=304) DAPT.The primary endpoint of present study was 12-month target lesion failure (TLF),including cardiac death,target vessel myocardial infarction and clinically indicated target lesion revascularization (CI-TLR).The major secondary endpoint was 12-month net adverse clinical events (NACE),including all-causes of death,myocardial infarction,stroke,all revascularization (CI-TLR plus clinically indicated nontarget lesion revascularization) and bleeding.Results For the patients implanted with BP-SES of total stent length≥ 50mm,the total stent length was 73.0 ± 22.5mm and 69.8 ± 19.4mm in the 6-month DAPT group and 12-month group,respectively (P=0.07).No significant difference existed in the incidence of 12-month TLF between 6-month DAPT group and 12-month DAPT group (11.1％ vs.9.2％,P=0.47).The incidence of NACE was similar between the 2 groups (21.9％ vs.19.7％,P=0.57).The incidence of revascularization was lower in 12-month DAPT group (5.6％) than in 6-month DAPT group (11.1％,P=0.01).Furthermore,6-month landmark analysis showed that 12-month DAPT was associated with significantly lower risk of TLF (2.6％ vs.6.3％,P=0.03) at a cost of slightly increased risk of all bleeding events (1.6％ vs.0.7％,Log-rank P=0.32) between 6 and 12-months compared to 6-month DAPT.Conclusions In patients treated with BP-SES of total stent length ≥ 50mm,12-month DAPT have similar impacts on 12-month clinical outcomes except for all revascularization.However,12 months DAPT decreased the incidence of TLF and total revascularization between 6 months to 12 months after PCI.

Objective To analyze the combination characteristics between Tir-IBD( intimin binding domain) and its ligand intimin or mutant intiminN916Y of EHEC O157 ∶H7.Methods The gene of TirIBD (tir-ibd) from EHEC O157 ∶H7 chromosome was cloned into pMD18-T vector.Thereafter,the amplified gene was cloned into prokaryotic expression plasmid pET-21 a (+).The recombinant pasmid pET-21 a( +)-tir-ibd was transformed into E.coli BL21 (DE3).After inducement,the protein Tir-IBD was successfully expressed and analyzed with SDS-PAGE and Western blot.It was purified by affinity chromatography and ionexchange chromatography and was coupled on the Ni-NTA chip of BIACore 3000.Then the ligand intimin and mutant intiminN916Y were flow through the chip and their combination characteristics were detected.Results In the present study,the gene of Tir-IBD(tir-ibd) was successfully cloned into pET-21a(+).The results of SDS-PAGE and Western blot assay showed that the protein was successfully expressed,which accounts for 15％ of total expression products,and its molecular weight was about 10×103.The purity was above 95％ after purification.After coupled on the Ni-NTA chip of BIACore 3000,their combination characteristics with ligand intimin and mutant intiminN916Y were successfully detected.The equilibrium binding constants Ka was obtained by fitting the data with the BIACore evaluation program ( Version 4.1 ).The result showed that the combination characteristics between Tir-IBD and intimin have some difference compared with that of mutant intiminN916Y and the difference is temperature dependent.Conclusion Tir-IBD of EHEC O157 ∶H7 was successfully constructed and purified.The method to analyze the combination characteristics between Tir-IBD and its ligand intimin or mutant was established.The combination characteristics between Tir-IBD and intimin or mutant intiminN916Y have some temperature dependent difference and the mutated amino acids residue is crucial for their receptor-ligand binding.

Biotechnological Pharmaceutics is a compulsory course for biotechnology undergraduates in our school. We designed and implemented an elective course named Structural Biology to help students master technological principles through practice. This elective course included in-classroom lectures and experiments; during which we encouraged students to work together, and design, prepare, implement, and complete projects; examination score of Biotechnological Pharmaceutics was used to assess learning outcomes. The results showed that students who took this course gained higher score in the examination, indicating that the elective course is effective to improve the learning effect of Biotechnological Pharmaceutics for biotechnology undergraduates.