Objective:To clone the fused gene of hVEGF_(165) and hirudin,construct a eukaryotic expression vector hVEGF_(165)-FH/pcDNA3.0 and express the vector in human endothelial cell strain. Methods: hVEGF_(165) cDNA fragment was amplified by PCR and fused hirudin peptide was synthesized by primer annealing. The fused hVEGF_(165 ) and hirudin gene was cloned into vector pcDNA3.0 to construct a new vector hVEGF_(165)-FH/pcDNA3.0 and the recombinant plasmid was confirmed by restriction enzyme and auto-sequencing. The correctly cloned plasmids were subjected to rapid in vitro transcription and translation identification. Liposome mediated, recombinant plasmids were used to transfect human endothelial cell strain and the products were identified. Results: hVEGF_(165)-FH was successfully fused and formed a 711 bp target fragment. The recombinant vector hVEGF_(165)-FH/pcDNA3.0 was successfully transformed into human endothelial cell strain which expressed the fusion protein. Conclusion: The recombinant vector hVEGF_(165)-FH/pcDNA3.0 can express fused protein in human endothelial cell strain, which paves a way for further study on the activity of the fusion protein and gene therapy.

Objective To detect the effects of glucocorticoids on the expression of toll-like receptor 4 (TLR4) on CD14+ monocytes in peripheral blood from patients with chronic obstructive pulmonary disease (COPD) and explore the relationship between inherent immunity deficiency and the pathogenesis of COPD.Methods A total of 30 hospitalized acute exacerbations of COPD (AECOPD) patients from 2012 January to March were divided them into 2 groups of mild-moderate (A,n =15) and severe-critical (B,n =15) according to pulmonary functions.The same basic treatment (anti-infection,oxygen inhalation,bronchial smooth muscle relaxing and cough relief) was administered.And glucocorticoids were added to Group B.Two weeks later,AECOPD achieved a clinical remission and it was designated as a stable COPD group.Another 30 healthy cases over the same period were selected as control group.Flow cytometry was used to measure the relative mean fluorescence intensity (rmfi) and relative positive cell percent (rpcp) of TLR4 onr CD14 positive peripheral blood monocytes.Conventional pulmonary function test (FEV1 percentage of predicted value,FEV1/FVC value) was performed.Results The value of rmfi for TLR4 of AECOPD group was 5.14 ±0.26 and rpcp (4.24 ±0.17)％ and they were similar to those of COPD group [4.46 ±0.24 and (3.49 ± 0.24) ％ respectively,all P ＞ 0.05].AECOPD and COPD groups were both significantly lower than normal control group [13.75 ±0.28 and (10.25 ±0.13)％ respectively,all P ＜0.01].The rmfi of TLR4 of Group B was 3.12 ±0.15 and rpcp (3.17 ±0.10)％.And both were significantly lower than those of Group A [5.48 ±0.23 and (5.12 ±0.20)％ respectively,all P ＜0.01].The value of rmfi for TLR4 of Group B before glucocorticoid treatment was 3.12 ±0.15 and rpcp (3.17 ±0.10)％.And both were significantly lower than those of later glucocorticoid treatment [5.24 ± 0.25 and (5.44 ± 0.19) ％ respectively,all P ＜ 0.01].Both values were significantly lower than those of normal control group [13.75 ± 0.28 and (10.25 ± 0.13) ％ respectively,all P ＜ 0.01].The expression of TLR4 on CD14 + monocytes in peripheral blood of AECOPD patients was positively correlated with FEV1 percentage of predicted value(r1 =0.824,P＜0.001) and FEV1/FVC (r2 =0.756,P＜0.001).Conclusions Innate immunodeficiency plays an important role in the pathogenesis of COPD.And the expression of TLR4 is an important indicator for disease severity.Glucocorticoids treat COPD through inhibiting airway inflammation response and enhancing defensive function of innate immune system.

Objective To investigate laparoscopic treatment of hepatic cysts in the right posterior lobe.Methods A four-port laparoscopic fenestration and drainage was performed in 35 patients with symptomatic hepatic cysts in the right posterior lobe(the lesion was located at the segment Ⅳ in 21 patients and at the segment Ⅶ in 14 patients) from January 1998 to December 2005.For cysts located at the superior segment of the right posterior lobe,the operation was performed through the suprahepatic approach. The liver was pressed along the anterior and inferior direction by using a pair of traction tongs for the exposure of hepatic cysts.Then the cyst was fenestrated and filled with pedicled greater omentum.For cysts located at the inferior segment of the right posterior lobe,the operation was conducted through the infrahepatic approach.The hepatocolic ligament,right triangular ligament,and connective tissues between the liver and the kidney were opened by using a harmonic scalpel.The liver was lifted with the traction tongs,and the cyst was fenestrated and filled with greater omentum.Results Laparoscopic fenestration and drainage was accomplished in all the 35 patients,without conversions to open surgery.The suprahepatic approach was adopted in 15 patients and the infrahepatic approach,in 20 patients.The operation time was 30～95 min(mean,46 min).No surgery related complications occurred.Pathological examinations in the 35 patients showed congenital cysts of liver.After operation,patients' symptoms all disappeared.The postoperative hospitalization time was 2～5 days (mean,3.8 days).All the 35 patients were followed for 6～36 months(mean,34 months).The cyst did not completely disappeared but significantly subsided in 2 patients(suprahepatic approach),without remarkable symptoms.The cysts were not obviously enlarged within 6 months.Conclusions Laparoscopic fenestration and drainage via suprahepatic or infrahepatic approach is an effective treatment for hepatic cysts in the right posterior lobe.

Objective To develop an RP-HPLC method for the content determination of heseridin in Hezhong Lipi Pills.Methods The Agilent HC-C18 column (250 mm×4.6 mm, 5μm) was used with methanol-0.1% phosphoric acid (35∶65) as the mobile phase at the flow rate of 1.0 mL/min. The detection wavelength was 283 nm, and the column temperarute was 40℃.Results The linear range of hesperidin was 0.15-3.64μg,r=1.000 (n=6). The average recovery was 98.97%, RSD=0.90% (n=6).Conclusion The method is accurate, simple, sensitive and with good repeatability, which can be applied to the quality control ofHezhong Lipi Pills.

Objective To investigate the diagnosis and treatment of latent perforation of peptic ulcer (LPPU) during laparoscopic cholecystectomy (LC). Methods A retrospective analysis was made on clinical data of 13 cases of LPPU diagnosed during LC from January 1994 to November 2005. Results All the 13 cases were misdiagnosed before the LC and clarified as having LPPU during the LC. Three cases of anterior duodenal bulbar perforation (0.4~0.5 cm in size) were laparoscopically repaired, whereas conversions to open repair were performed in 5 cases of duodenal perforation (0.5~1 cm in size) and 5 cases of gastric perforation (0.6~1 cm in size) because of severe adhesion or difficult performance. No surgical complications occurred. Twelve cases were followed for 6~36 months (mean, 27 months). The symptoms disappeared and gastroscopy showed healed ulcer. No recurrence of perforation was found. Conclusions Underestimation of LPPU leads to misdiagnosis, and preoperative gastrosopy can prevent it. For small perforation, laparoscopic repair and abdominal drainage can be employed. But in most cases, a conversion to open surgery is required. It is necessary to give regular internal medicine for peptic ulcer after operation.

OBJECTIVE:To optimize the extraction technology of Qingjie atomized liquid. METHODS:The extraction technol-ogy of Qingjie atomized liquid was optimized by orthogonal test with hyperoside content as index,using the amount of added wa-ter,extraction time and extraction times as factors,and the verification test was conducted. RESULTS:The optimal extraction tech-nology was as follows as 12-folds water,extracting for 2 times and 1 hour each time. The content of hyperoside was 4.129 μg/g in 3 validation tests(RSD=1.81%,n=3). CONCLUSIONS:The optimal extraction technology is stable and practical,and can pro-vide reference for the formulation of technology and quality standard for Qingjie atomized liquid.

Objective To investigate the awareness of amputees to his appearance and their social activities. Methods 45 amputees were investigated with Body Shape Measuring for Amputee self-designed and Social Activities Questionnaire. Results For body shape measuring, the top 3 awareness amputees always feeling were: wearing loose clothing to cover the prosthesis (55.56%); only able-bodied can be attracted (48.89% ); losing of limbs damage the body and affect their daily living (40%). For the social activities, the total score was more in lower limb amputees than in upper limb amputees (P<0.05). For the items, there was no difference in amusement and sport, pastime, community participation, and join in workshop (school) (P>0.05) between the patient with upper and lower limbs losing, but there was in sociality (P<0.05). Conclusion Amputees fall in social withdrawal for limb losing and inferiority, which may associate with what limb lost.

Objective: To investigate the effect of human vascular endothelial growth factor on restenosis after angioplasty. Methods: A rabbit model of injured carotid artery was established using percutaneous transluminal angioplasty. The pcDNA3/hVEGF165（500 μg,n=12） and pcDNA3 （500 μg,n=12） were separately transfected into injured arterial wall with 30 min incubation. The carotid artery was imaged by arotic angiography at the end of week 2 and week 4. Pathology analysis and Northern blot analysis were performed for harvested injured artery segment. Results: Arotic angiography showed carotid artery diameter narrowness were obviously lessened at week 2 and week 4 in experimental group than that in control group; H-E stains showed lumina narrow ratio were obviously reduced at week 2 and week 4 in experimental group than that in control group［（9.58±1.35）% vs (31.72±1.72)%；(18.09±2.93)% vs (44.05±3.28)%， P<0.01 ］; By Northern blot analysis, the expression of hVEGF165mRNA in experimental group were upregulated than in contol group. Conclusion: pcDNA3/hVEGF165 can be transfected into smooth muscle cell and continue to secret bioactivity protein at least for 4 weeks; it can accelerate reendothelialization and prevent restenosis.

Objective:To evaluate the influence of prophylactic intra-coronary injection nitroglycerin with diltiazem in patients of acute ST segment elevation myocardial infarction(STEMI) Methods:This study was conducted in 212 patients with acute STEMI who underwent primary percutaneous coronary inter- vention(PCI)in our center from January 2001 to December 2006.The patients were randomly assigned to receive intra-coronary nitroglycerin with dihiazem(n = 87)or none of them(125)before intervention.The clinical data,coronary angiography data after intervention,ST-segment resolution,area under the curve of CK-MB,ejection fraction(EF)and left ventricular end-diastolic di- mension several days alter operation and the major adverse cardiac events(MACE)were analyzed systemically. Results:Ten(11.5%)patients in study group and 28(22.4%)in control group had MACE(P = 0.042).The difference of re-hospitalization was also significant(19.5% vs 37.6%,P = 0.008).No/slow reflow occurred less in study group than in con- trol group,but the difference was not significant.Corrected TIMI frame count was lower(34.4?11.3 vs 38.9?17.2,P = 0.034) and myocardial blush grade was higher(2.65?0.6 vs 2.41?0.7,P =0.018)in study group.ST-segment resolution,area under the curve of CK-MB in 5 days after operation,EF and left ventricular end-diastolic dimension in study group were more significant than those in control group(P