Purpose To indicate the expression of nattokinase (NK) in Pichia pastoris , an emulsion was prepared with the purified NK to prepare polyclonal antibody. In order to establish sandwich enzyme-linked immunosorbent assay (ELISA) to assay NK in organism, furthermore to lay the foundation for researching in vivo metabolism and function of NK. Methods The NK gene was cloned into a Pichia pastoris expression vector pHBM905A to construct the recombinant plasmid pPRONK.The recipient cell of Pichia pastoris GS115 was transformed with pPRONK which had been cut by restriction enzyme Sal I , under the induction of methanol. The expressed production is purified by salting out and ultrafiltration membrane. An emulsion was prepared with the purified NK and injected into rabbits to prepare polyclonal antibody. Results NK was expressed and identified by SDS-PAGE.The molecular mass of expressed production is about 27 kD.The fibrin plate assay indicated that the NK protein can cleavage fibrin effectively. ELISA analysis indicated that the polyclonal antibody titer is about 1:8 000. Western blot demonstrated that there was a special strap nearby 27 kD. Conclusion NK was successfully expressed in Pichia pastoris , the production can cleavage fibrin effectively and it had great immunogenicity.

Objective:To explore relationship among severity of cerebral infarction (CI) ,arteriosclerosis and serum level of cystatin C (CysC) in young and middle‐aged patients with atherosclerotic cerebral infarction (ACI) .Methods:A total of 82 young and middle‐aged ACI patients treated in our hospital from Feb 2013 to Sep 2015 were enrolled .According to CI volume ,they were divided into small infarction group (n=36 ) , medium infarction group (n=22 ) and large infarction group (n=24);according to CI severity ,they were divided into mild CI group (n=54) and severe CI group (n=28);ac‐cording to atherosclerotic plaque nature ,they were divided into stable plaque group (n=45) and unstable plaque group (n=37) .Another 46 healthy people were regarded as healthy control group .Serum CysC level during emergency period and recovery period and carotid intima‐media thickness (IMT) were measured and compared among all groups .Results:Com‐pared with emergency period ,there was significant reduction in serum CysC level in all subgroups of ACI during recovery period , P<0. 01 all;compared with healthy control group ,there were significant rise in serum CysC level [recovery peri‐od:(0.81 ± 0.24) mg/L vs .(1.03 ± 0.13) mg/L vs .(1.09 ± 0.19) mg/L vs .(1.18 ± 0.10) mg/L] during emergency period and recovery period in small ,medium and large infarction group ,and that of large infarction group was significantly higher than those of small and medium infarction group (P<0.01 all) .Compared with healthy control group ,there was significant rise in serum CysC level [recovery period:(0.81 ± 0.24)mg/L vs .(1.07 ± 0.15)mg/L vs .(1.19 ± 0.16)mg/L] during emergency period and recovery period in mild and severe infarction group ,and that of severe infarction group was significantly higher than that of mild infarction group ,P<0.01 all .Compared with healthy control group ,there were sig‐nificant rise in serum CysC level[(0.81 ± 0.24)mg/L vs .(1.18 ± 0.15)mg/L vs .(1.39 ± 0.27)mg/L]during emergency pe‐riod and IMT [(0.72 ± 0.10) mm vs .(1.24 ± 0.17) mm vs .(1.30 ± 0.14) mm]in stable plaque group and unstable plaque group ,and those of unstable plaque group were significantly higher than those of stable plaque group ,P<0. 01 all .Conclu‐sion:The serum CysC level significantly rises in ACI patients ,it can be used as an index for ACI prevention and treatment .

Objective To investigate the expression and clinical significance of connective tissue growth factor(CTGF) in colorectal cancer.Methods The expressions of CTGF in 62 patients' colorectal cancer tissues and their corresponding adjacent tissues were detected by SP immunohistochemical method.The results were statistically analyzed.Results The positive rates of CTGF in colorectal cancer and adjacent tissues were 61.3% and 19.4% respectively,and the difference between them was significant(P0.05).Conclusion CTGF may play an important role in the occurrence of colorectal cancer,which contributes a lot to guide clinical treatment and prognosis.

BACKGROUND: Previous studies have confirmed that nano-hydroxyapatite/collagen (nHAC) and mesenchymal stem cells for repair of bone defect have the ability of bone formation in vivo. OBJECTIVE: To observe the effects of vascular endothelial growth factor (VEGF) and bone marrow mesenchymal stem cells (BMSCs), nHAC composite in the repair of femoral defect in rats. METHODS: Sprague-Dawley rat models of middle part of the femur defect were established and randomly assigned to two groups. Control group was implanted with BMSCs/nHAC composite. Experimental group was implanted with VEGF/BMSCs/nHAC composite. At 2, 4 and 8 weeks postoperation, imaging and histology observation of femoral samples were performed. At 8 weeks postoperation, scanning electron microscopy was performed in new bony callus environment. RESULTS AND CONCLUSION: nHAC composite implantation in the rats did not show rejection or inflammatory reaction. Moreover, bone formed rapidly using VEGF and BMSCs, nHAC composite, which exhibited better bone regeneration capacity compared with BMSCs/nHAC composite. The way of ossification mainly was endochondral ossification. It is presumed that VEGF promoted the formation of local microvessels, differentiation and proliferation of osteoblasts, speeded up the speed of endochondral ossification, shortened bone repair time, and elevated the quality and velocity of osteanagenesis.

Objective To set the criteria of mild spinal tuberculosis and investigate the ettect of standard chemotherapy regimen for further establishing the clinical classification of spinal tuberculosis and standardizing management.Methods According to the criterion,a total of 89 patients with mild spinal tuberculosis were enrolled for outpatient conservative management and follow-up.Ambulant treatments were carried out in all patients,including nutrition support and standard chemotherapy regimen.The regimen was consisting of four first-line antituberculosis drugs (rifampicin,isoniazid,ethambutol and pyrazinamide).All patients were followed up one month later,then every 3 months for the following 12 months,and subsequently at intervals of half a year.The clinical manifestations,kyphosis progression,neurological status,erythrocyte sedimentation rate and liver function were analyzed.Results A mean of 30.62± 13.20 (range,18-46) months' follow-up was achieved in 85 patients,whose tuberculosis lesions were cured completely.Another 4 patients,who were diagnosed with drug-resistant tuberculosis later,had underwent surgery for progressive bone destruction and no response to chemotherapy.The mean visual analogue scale score and Cobb's angle was 5.6± 1.6 and 6.25°±3.11° before chemotherapy,and 2.1 ± 1.1 and 12.36° ±6.31 °at the last follow-up time,respectively.Signals of vertebral body and intervertebral disc returned to normal in 6 patients,while solid bony fusion of adjacent segment was achieved in 79 patients.Asymptomatic mild kyphosis was observed in 69 patients.2 patients with sinus before treatment all healed.No neurological deficit was found.Conculusion For patients early diagnosed with mild spinal tuberuclosis,standard chemotherapy regimen could work safely and effectively for healing the tuberculous lesion,avoiding surgery as well as preventing kyphosis,vertebral instability and neurological deficit.Mild spinal tuberculosis that was early diagnosed could be considered as a subtype of spinal tuberculosis.

Objective To investigate the feasibility and safety of designed perioperative surgical treatment for advanced gastric cancer.Method From March 2006 to Sep.2009,24 patients with advanced gastric cancer were enrolled for this study,of which 14 cases were graded as Ⅳ M0,10 as Ⅳ M1,including liver metastasis in 5 cases,peritoneal seeding in 4 and lung metastasis in 1.All patients received 2-4 cycles of chemotherapy of PCF or ECF regimen,followed by intended surgical resection and postoperative 2-3 cycles of adjuvant chemotherapy of the same regimen as used during the preoperative course.Result Seven Cases abandoned surgical therapy,a total of 17 cases completed preoperative chemotherapy,surgical resection and postoperative chemotherapy.R0 resection was achieved in 16 cases(94%).There was no perioperative mortality,and total clinical response rate was 75%,overall pathological response rate was 82%.In 59% surgical cases (10 cases) serum CEA and CA199 has returned to normal;In 71%patients tumor pathological stage degraded at the cost of Ⅲ-Ⅳ grade of neutropenia,thrombocytopenia,anemia,severe nausea and vomiting in 79%,8%,13%,and 54% cases respectively.Fever was encountered in 2 cases with grade Ⅲ-Ⅳ neutropenia.Conclusion Designed perioperative chemotherapy regimen renders advanced gastric carcinoma patients operable at the cost of acceptable complications.

Objective To introduce the application of double liver hanging maneuver in anatomical right hemihepatectomy and share our own experience. Methods Twenty-four patients underwent right hemihepatectomy using double liver hanging maneuver, and the data were collected prospectively after operation. Another 49 patients underwent right hemihepatectomy using the traditional methods,serving as the control group. Results Retrohepatic tunnel was constructed in 27 patients, double liver hanging maneuver was successfully performed in 24 anatomical right hemihepatomy and failed in 3 patients due to the near proximity between the tumor and the middle line. Compared with the control group, blood loss was much less (t=3. 191 ,P＜0.05), ALT and liver function recovered more quickly postoperatively and the difference in operative duration between the 2 groups was not significant (t=-1. 695,P＞0. 05). There was about 1-2 cm wide space located between retrohepatic IVC and dorsal liver when the 2 tapes were tracted, and no injury in hepatic short veins and retrohepatic IVC occurred during the operation. Conclusion The double liver hanging maneuver can make anatomical right hemihepatectomy more accessible and safe.

Objective:To explore the correlation among serum levels of resistin ,oxidized low density lipoprotein (ox‐LDL) ,high sensitive C reactive protein (hsCRP) and severity of acute ischemic cerebrovascular diseases .Methods :A total of 92 patients with acute ischemic cerebrovascular diseases ,who were treated in our hospital from Nov 2013 to Nov 2014 ,were selected ,including 36 cases with transient ischemic attack (TIA ,TIA group) ,29 cases with re‐versible ischemic brain damage (RIBD ,RIBD group) and 27 cases with focal cerebral infarction (FCI ,FCI group) . Another 70 healthy volunteers were regarded as normal control group .Serum levels of resistin ,ox‐LDL and hsCRP were measured and compared among all groups .Pearson correlation analysis was used to analyze the correlation a‐mong serum levels of resistin , ox‐LDL , hsCRP and severity of acute ischemic cerebrovascular diseases . Results:Compared with normal control group ,there were significant rise in serum levels of resistin [ (0.26 ± 0.42)μg/L vs . (0.63 ± 0.38)μg/L vs .(0.91 ± 0.45)μg/L ,(0.89 ± 0.42)μg/L] ,ox‐LDL [ (334.3 ± 142.5) mg/L vs .(451.7 ± 15.8) mg/L vs .(518.3 ± 205.7) mg/L ,(520.7 ± 198.9) mg/L] and hsCRP [ (5.8 ± 4.9) mg/L vs .(8.7 ± 7.6) mg/L vs .(13.5± 9.1) mg/L ,(13.6 ± 7.4) mg/L] in TIA group ,RIBD group and FCI group (P< 0.01 all);those of RIBD group and FCI group were higher than that of TIA group (P<0.01 all) ,and there were no signifi‐cant difference in above indexes between RIBD group and FCI group (P>0.05 all) .Pearson correlation analysis in‐dicated that serum levels of resistin , ox‐LDL and hsCRP were significant positively correlated with severity of acute ischemic cerebovascular disease (r=0.473~0.902 , P<0.01 all) .Conclusion:Serum levels of resistin ,ox‐LDL and hsCRP can reflect severity of acute ischemic cerebrovascular diseases ,which is worth extending .

Background and purpose:Hepatic epithelioid hemangioendothelioma (HEHE) is an extremely rare, vascularly original tumor, and would be misdiagnosed easily in the clinical and imaging characteristics. This study aimed to investigate the imaging features of HEHE and our experience in clinical diagnosis and treatment, and to provide important reference for the diagnosis and treatment of HEHE in the future.Methods:This study retrospectively analyzed clinical manifestations, imaging features, pathological characteristics and treatment effects of 5 HEHE cases confirmed by pathology at Department of Integrative Cancer, Fudan University Shanghai Cancer Center.Results:The ratio of male to female patients was 2 to 3. HEHE predominantly occurred in middle-aged female patients whose ages range from 26 to 65 (mean=45.6). Imaging features of HEHE included multifocal hepatic disease (n=2), systemic multi-center multi-tissue occurrence (n=3). The ultrasound images showed isoechoic or hypoechoic lesions with no obvious blood lfow signal in the lesions. CT plain scan showed isointensity or hypointensity, while MR unenhanced with hypointense T1 signal and hyperintense T2 signal. The density or signal was uneven. Contrast-enhanced CT and MR images showed “slow in slow out”, obvious and variable degrees of peripheral rim enhancement. High FDG uptake showed delayed imaging characteristics (PET/CT). DSA angiography showed the tumor blood vessels were slim. After transcatheter arterial chemoembolization (TACE) surgery, lipiodol deposition within the lesion was not ideal. Under the microscope, tumor cells showed epithelial differentiation; angiogenesis was also visible. Immunohistochemistry staining showed CD31 and CD34 positive in all the 5 cases. Two cases treated with TACE combined with high intensity focused ultrasound (HIFU) and/or radiofrequency ablation (RFA) had good result.Conclusion:The clinical and radio-logical characteristics of HEHE are distinctive. Currently, for patients with systemic multi-organizational multi-center lesions, TACE combined with HIFU and/or RFA might be the most effective treatment method.

Objective To investigate the causes of spinal tuberculosis retreatment and its surgical treatment strategy.Methods Between May 2010 and May 2014,96 patients with spinal tuberculosis who had been operated before were retreated.The dates of them were reviewed.There were 51 males and 45 females with mean age of 39.7 years.Deciding upon the revision surgical procedure should be determined by last operation approach,the direction of compression of spinal cord,the position of sinus and extent of foci.The retreated patients were compared with 481 unretreated patients in the following index including sex,age,duration of disease,focus range,nutrition,drug resistant tuberculosis,debridement,stability of instrumentation,postoperative regular chemotherapy,associated tuberculosis.Results The surgery duration time was 160-280 min,average 210 min,and the blood loss was 400-1500 ml,average 600 ml.The VAS score before the operation was 6-9 (average 7.5) and 1-3 (average 1.5) at the last follow-up,the difference was statistically significant.Neurological deficits in 21 patients clinically improved at least one grade according to the ASIA grading system at last follow-up.Kyphosis and scoliosis degrees were corrected significantly postoperatively and the correction was 9.5°±3.6° at the final follow-up.The average angle loss was 3.5°±1.1°.There was significant difference between the kyphosis angles preoperatively,postoperatively and final follow-up.Kirkaldy-Willis function score showed that the total fine rate was 88％.There were 35 patients whose tuberculosis bacterial culture and drug sensitive experiments suggested drug resistance.Wound healing delayed in 7 patients.24 cases had sinus formation,13 cases in which were healed after wound dressing,and 11 cases undertook operation again.Conclusion The causes of spinal tuberculosis retreatment include uncompleted debridement,drug resistant strains of tuberculosis,irregular postoperative anti-tubercular treatment,poor preoperative nutritional status and failure of spinal stability reconstruction.The key of successful revision surgery includes radical debridement,strut grafting with autologous iliac bone block,proper reconstruction of spinal stability,individualized chemotherapy according to the drug-resistance,and the appropriate use of irrigation and drainage postoperatively.