Objective To construct a bait plasmid in bacterial two-hybrid system.Methods A cDNA fragment encoding for rat AMP-activated protein kinase ?2(AMPK?2) was amplified by PCR and inserted into bacterial expression vector pBT.After confirmation with restricted endonuclease digestion and sequence analysis,bacterial reporter strain XL-1 Blue MRF' was transformed with pBT-AMPK?2 plasmid and the expression of the recombinant bait fusion protein was detected.To test whether the bait fusion protein had the capability of self-activation,the XL-1 Blue MRF' cells were cotransformed with the pBT-AMPK?2 plasmid and empty pTRG vector,and screened on 3-amino-1,2,4-triazole(3-AT) Selective Screening Medium plates.Results Restriction digestion and sequence analysis revealed that the AMPK?2 code sequence was correctly inserted into pBT with a right reading frame.pBT-AMPK?2 expressed ?cI/AMPK?2 fusion protein.Colonies were obtained on no 3-AT Nonselective Screening Medium plates when XL-1 Blue MRF' cells were cotransformed with recombinant pBT-AMPK?2 and empty pTRG vector,while none grew on 3-AT plates,indicating that the recombinant plasmid pBT-AMPK?2 expressed AMPK?2/?cI fusion protein correctly,and was incapable of activation of the reporter cassette in the absence of an interaction partner.Conclusion The recombinant plasmid pBT-AMPK?2 could be used as "bait plasmid" to screen cDNA library.

With the advent of post-genomic era, identification of protein-protein interaction has become another hot spot of protein research and promoted the invention, development and complement of relative techniques. Major large-scale high-throughput methods, such as two-hybrid system, bacteriophage display, mass spectrometry, protein microchips and bioinformatics have provided a global view of protein-protein interaction, and are hopeful to play an important role in proteomics. Each method may have its specific strengths and weaknesses as well as differences in coverage, to some extent, the data based on these methods can complete each other. Here, recent progress of these large-scale high-throughput methods and their applications for studying protein-protein interaction are reviewed.

Objective [WT5”BZ] To evaluate the preliminary clinical results of the endovascular grafts for the treatment of abdominal aortic aneurysms (AAA).[WT5”HZ]Methods [WT5”BZ] Fourteen patients with infrarenal abdominal aortic aneurysms underwent transluminal endovascular graft placement for the exclusion of AAA.[WT5”HZ]Results [WT5”BZ] Two patients received tubular and 12 patients received bifurcated endograft, the placement was successful in all cases. Aortography carried out immediatly after the procedure showed the AAA were completely excluded by endografts, and no endoleaks both on the proximal or distal connections. Myocardial infarction developing in a patient postoperatively was successfully treated by thrombolysis. Perioperative death occurred in 2 cases. Technical success at 30 days was 85 7%. 24 month follow up in 12 cases found migration of the graft in none and endoleaks in 2 cases.[WT5”HZ] Conclusion [WT5”BZ] Based on our initial results and a rather short follow up period of 24 months, the endovascular treatment of AAA with stent graft is safe and effective. Long term follow up is needed to evaluate the feasibity of this procedure.

Objective To evaluate the mid-term outcomes of endovascular repair of descending thoracic aortic aneurysm(DTAA) in high-risk patients.Methods Twenty four patients with DTAA received stent-grafts implantation between January 2001 and July 2007 and their clinical data were retrospectively analyzed.All patients were considered high-risk for open surgical repair due to the location or rupture of the aneurysm,high age and severe comorbidities.Spiral computed tomography angiography and 3-dimentional reconstructions were performed at 1,3,6 and 12 months postoperatively and thereafter every year.Results The primary operational success rate was 100%.There were 2 deaths(8.3%) during perioperative period due to multiorgan failure and acute myocardial infarction respectively.Two(8.3%) patients had severe post-operational complications(1 stroke and 1 acute renal insufficiency).Eleven cases of type Ⅰ(46%) endoleak were detected immediately after the operation and among them,the endoleak disappeared after balloon dilatation in 3 cases while the other 8 patients were treated conservatively.Follow-up of 1 to 60 months(mean 18.6?4.2 months) was completed in 19 patients(79.2%).Stent displacement with type Ⅰ endoleake was found in 1 patient at 4 years after the operation and delayed type Ⅲ endoleak occured in another patient at 2 years post-operation.Both patients received endovacular repair again and the endoleaks were closed successfully.One patient died of colon cancer during the follow up.Complete thrombosis of the thoracic aneurysm sac and no stent migration or endoleak was found on the followup CT at 3 months after the operation in all the patients.The decrease in maximal aneurysm diameter was 0-18 mm(mean 6.3?3.1 mm).The prosthetic vascular grafts implanted in 4 patients with preliminary carotid subclavian bypass surgery were patent during the follow-up period.Conclusion Treatment of descending thoracic aortic aneurysm in high-risk patients with endovascular approach showed acceptable early mortality and morbidity and may be considered as a treatment alternative for carefully selected patients.

Objective To explore the application of near infrared fluorescent dye C-dots in cell labeling.Methods B16 melanoma cells were cultured with C-dots of various diameters.The distribution of C-dots in cells was observed by laser confocal fluorescent microscope.The proliferation of both C-dots-labeled and unlabeled cells were measured by MTT colorimetry.The viability of labeled cells was checked by trypan blue staining.Results C-dots with diameter of 5 or 10 nanometers can be used to label cells.The near infrared fluorescence of C-dots was taken into the cells and observed on the cellular membrane.No difference of cellular viability was found between the labeled and unlabeled cells.Conclusion C-dots can be used as an intracellular marker for the research on trace and proliferation of cells.

Objective To screen the proteins interacting with AMP-activated protein kinase ?2(AMPK?2)in rat brain cDNA library by bacterial two-hybrid system,and to investigate the biological role and the regulatory mechanism of AMPK?2 in brain.Methods The recombinant pBT-AMPK?2 was used as the bait to screen a rat fetal brain cDNA library by bacterial two-hybrid system.The plasmids of positive colonies were extracted and analyzed by DNA sequencing and BLAST search in GenBank.Results Seven proteins binding to AMPK?2 were screened out,including polyubiquitin,small heat shock protein 8,phosphofructokinase,cytochrome C oxidase subunit Ⅰ(COXⅠ),HLA-B-associated transcript 3(BAT3)isoform 1,protein tyrosine phosphatase receptor type D(Ptprd)and islet-brain 1(IB1).Conclusion Polyubiquitin,HSP8,phosphofructokinase,COXⅠ,BAT3 isoform 1,Ptprd and IB1 can interact with AMPK?2 in brain.

Objective To study the feasibility and efficacy of endovascular thoracic aortic repair supported by preliminary arterial bypass. Methods From June 2003 to Jan. 2005, eight patients underwent endovascular repair (EVR) combined with supportive arterial reconstruction in the thoracic aorta. Five patients had Debakey type Ⅲ aortic dissection aneurysm (ADA), and the remaining 3 had descending thoracic aortic aneurysm (DTAA), aortic arch aneurysm and aortic arch pseudoaneurysm respectively. The proximal landing zone was measured less than 15 mm in all instances by contrast-enhanced CT scan and digital subtraction angiography. The preliminary right-left axillary bypass was performed in an ADA case in whom the distance from the primary entry site to the origin of the left common carotid artery was longer than 15mm,and the right- left carotid and left carotid-subclavian bypass in the remaining 7 cases. EVR was conducted 1 week after the bypass. Results All procedures were performed successfully. However, the DTAA patient died of the hemispheric cerebral infarction and subsequent multiple system organ failure, albeit the uneventful recovery from the prior cervical reconstruction. Type Ⅱ endoleak occurred at the completion of EVR in the patient receiving the axillary bypass, but disappeared on 3-month CT. No neurological deficits or limb ischemia developed perioperatively or during the follow-up raging from 3 to 22 months, and complete thrombosis of the thoracic aortic false lumen or of the aneurysm/pseudoaneurysm was revealed on CT at 3 months in the 7 patients. Conclusion The adjunctive surgical bypass appeared to be feasible and effective in creating extra proximal landing zone for endovascular thoracic aortic repair, and thus broaden its application.

BACKGROUND:The endothelial dysfunction is the pathogenesis of arteriosclerotic disease, the quantity and function of endothelial progenitor cells are decreased within the cycle, leading to a poor capacity of neovascularizatio, the efficacy of stem celltransplantation alone is unclear, the combination of cytokines and gene-modified stem cells is the hotspot.
OBJECTIVE:To observe the effect of stromal cel-derived factor-1 on the neovascularization after endothelial progenitor cells transplantation.
METHODS:Unilateral hindlimb ischemia model was established in 20 athymic nude mice, and the mice were randomly divided into four groups:combined group (intravenous endothelial progenitor cells+intramuscular stromal cel-derived factor-1), endothelial progenitor cells group (intravenous injection of endothelial progenitor cells), stromal cel-derived factor-1 group (intramuscular injection of stromal cel-derived factor-1), and blank control group (intramuscular M199). The skin temperature of ischemic hindlimbs and survival of animals after transplantation were observed. The ratio of capil ary/skeletal muscle fiber was counted. The expression of CD31 and endothelial nitric oxide synthase were detected.
RESULTS AND CONCLUSION:The fluorescence-labeled endothelial cells were embedded in ischemic hindlimb muscles after celltransplantation. Of the 20 nude mice, two mice died. The rate of ischemic hindlimb reserving was respectively 80%, 75%, 20%and 0 in combined group, endothelial progenitor cells group, stromal cel-derived factor-1 group, and blank control group. The capil ary/muscle fiber ratio in combined group and endothelial progenitor cells group was higher than that of blank control group (P<0.01). The combined group was greater than endothelial progenitor cells group, and endothelial progenitor cells group was greater than stromal cel-derived factor-1 group (P<0.05). The capil ary density in combined group and endothelial progenitor cells group were higher than that in blank control group (P<0.01), and stromal cel-derived factor-1 group was also more than blank control group (P<0.05). The combined group was greater than endothelial progenitor cells group, and endothelial progenitor cells group was greater than stromal cel-derived factor-1 group (P<0.05). The positive rate of endothelial nitric oxide synthase was 73.33%and 53.33%in combined group and endothelial progenitor cells group respectively (P>0.05). Endothelial progenitor cells can migrate to ischemic tissues, endothelial progenitor cells transplantation can promote neovascularization, and stromal cel-derived factor-1 augments the neovascularization after celltransplantation, in which endothelial nitric oxide synthase is involved.

Objective To study the expression of polycystin1 and polycystin2 in dissection aorta specimens by Real time PCR and immunohistochemisty. Methods Twelve descending dissection aorta specimens from patients of acute Stanford B dissection were taken during surgery; 12 normal descending aorta specimens were taken from multi-organ donors. The G APDH gene was used as control, gene expression of polycystinl and polycystin2 were compared in these two groups with Real time PCR. The expression of polycystinl and polycystin2 protein were showed with immunohistochemisty in the specimen of these two groups. Three high magnify fields were randomly chosen to count the expression of polycystin1 and polycystin2 protein. The counting of polycystin1 and polycystin2 protein were compared between these two groups. Results The gene expression of polycystinl in AD group is 0. 32 fold as that in the control group (P ＜ 0.01 ) and polyeystin2 in AD group is 0.34 fold as that in the control group (P ＜0.01 ); The protein expression of polyeystinl was 0.47 folds as that in the control group ( P ＜ 0.05 ) and polycystin2 in AD group is 0.35 folds as that in the control group ( P ＜ 0.01 ). Conclusions The expression of polycystin1 and polycystin2 on gene and protein level were down regulated in AD group. The down regulation of polycystinl and polycystin2 may play a pivotal role in the development of AD.

Objective To study operative morbidity and mortality of infrarenal abdominal aortic aneurysm (IAAA) and analyze the correlation between the preoperative or intraoperative risk factors and the morbidity or mortality. Methods Between Jan 1993 and Dec 2001, 120 IAAA cases undergoing surgery were analyzed. Preoperative cardiac, pulmonary, hepatic and renal condition, time of intraoperative aortic clamping as well as emergent operation were taken into account to evaluate the independent determinants of operative morbidity and mortality. Results The operative morbidity was 56.7% and mortality was 10.0%, with the mortality of elective surgery of 5.4% and emergent surgery of 66.7%. Conclusion Preoperative coronary heart disease, hypertention, renal dysfunction and massive blood transfution intra- or postoperatively adversely influenced the postoperative outcome significantly.