Objective To detect whether there is neurotropism following end-to-side neurorrhaphy by means of the neuron retrograde tracing technique. Methods Forty female Sprague-Dawley rats were randomly divided into 4 groups: tracing main branch of musculcutaneous nerve(MC) of end-to-side group, tracing MC main branch of normal group, tracing MC motor branch of end-to-side group and tracing MC motor branch of normal group.In two end-to-side groups, the MC was transeeted, then an 1 mm epineukral window was created on the ulnar nerve. Distal end of MC nerve was sutured to the windowed ulnar nerve by means of end-to-side neurorrhaphy.In two normal control groups, MC and ulnar nerves were just exposed. Five months post operation, by means of retrograde Fluoro-Gold neuron tracing technique,the number of C5～ T1 anterior horn motoneurons and dorsal root ganglion sensory neurons of all groups were counted. Results In two tracing MC main branch groups: the motor neuron counts in end-to-side group was 245.2 ± 93.8, the motor neuron counts in normal group was 846.7 ± 264.8, and counts of end-to-side group was 30.0% of the normal control group (P＜ 0.01). The sensory neuron counts in end-to-side was 434.7 ± 160.4, the sensory neuron counts in normal group was 1545.2 ± 287.4, and counts of end-to-side group was 28.1% of the normal control group (P ＜ 0.01). The per-centage of motor neuron in end-to-side group was 0.36 ± 0.09, there was no difference between end-to-side group and normal control group(P＞ 0.05). In two tracing MC motor branch groups: the motor neuron counts in end-to-side group was 72.3 ± 35.3, the motor neuron counts in normal group was 189.7 ± 57.0, and counts of end-to-side group was 38.1% of the normal control group (P ＜ 0.01). The sensory neuron counts in end-to-side was 110.8 ± 52.5, the sensory neuron counts in normal group was 157.9 ± 50.0, and counts of end-to-side group was 70.2% of the normal control group (P ＞ 0.05). The percentage of motor neuron in end-to-side group was 0.40 ± 0.14, the difference between end-to-side group and normal control group was signifieant(P ＜ 0.01 ). Conclusion Neurotropism in collateral spouting after end-to-side neurorrhaphy is not significant.

To produce specific antibodies against malachite green ( MG) , one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay ( icELISA ) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value ( IC50 ) of 0. 96 μg/L, a linear range ( IC20-IC80 ) of 0. 1-8. 1 μg/L and a limit of detection ( LOD, IC10 ) of 0. 05 μg/L for determination of MG. The assay showed cross-reactivity of 18. 1%, 26. 5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0 . 1%) . The average recoveries of MG from spiked fish samples were from 87. 3% to 107. 3%. Good correlation (R2=0. 999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner.

Objective To investigate the relationship and mechanism between the serum level of high mobility group box protein-1(HMGB1)and the mortality rate in patients with sepsis.Methods The serum levels of HMGB1,superoxide dismutase(SOD)and malondialdehyde(MDA)in 48 patients with sepsis were determined.The clinical outcomes in those patients were recorded and anlyzed.Results After the onset of sepsis,the serum HMGB1 levels of both death group and survival group were increased gradually and peaked at 72h after the onset of the disease.The semm HMGB1 levels of death group were much higher than those of survival group except at 24h(t=6.07,6.20,24.43,all P＜0.05).The activity of serum SOD of death group was markedly lower than that of survival group at 12h,24h,48h and 72h(t=10.24,20.61,11.67,33.33,all P＜0.05),and the level of serum MDA of death group was significantly higher than those of survival group at all time points(t=26.06,22.17,23.86,9.49,5.95,all P＜0.05).There was a significantly positive correlation between the serum HMGB1 and MDA level.Conlusioa The increase in serum HMGB1 level may be the important reasoll for the increased mortality rate in patients with sepsis;Oxidant/antioxidant imbalance may be olle reason for the increase in serum HMGB1 level.

Objective To detect the doubling time of airway smooth muscle cells of human and rat by the xCELLigence in-strument,a real time cellular analyzer.Methods The airway smooth muscle cells were separated by collagenase-pancreatin digestion from the rat airway.Then,added to the different holes of E-plate of xCELLigence instrument with the planting destiny of 3 000 cells/well.and so were the human airway smooth muscle cells.The E-plate was then placed on the xCELLi-gence instrument to monitor cell proliferation for 100 hours to calculate the doubling time by using the RTCA Software Package 2.0 software.Results The doubling time of human airway smooth muscle cell calculated by the real time cellular analyzer was 23.96±0.47 h,which was consistent with the data provided by the reference.The doubling time of rat airway smooth muscle cell was 18.62±0.15 h,and the computational process was simple,time-saving and also effective.Conclusion The xCELLigence instrument can be used to calculate doubling time of airway smooth muscle cells of human and rat, which provides experimental methods and reference data for the basic respiratory disease research.

Objective To investigate spiral CT,PET/CT and clinical manifestations in non-HIV infectious penicilliposis marneffei lung disease, to improve diagnostic level of this disease.Methods Imaging manifestations of 6 cases of non-HIV infectious penicilliposis marneffei confirmed by bronchofiberscope lung biopsy and/or pus culture were analyzed retrospectively All cases underwent chest CT,two had brain MRI,and two had PET/CT scan before treatment.Imaging appearances were observed and combined with clinical dates and literatures.Results Unilateral lung lesion was detected in 1 case,bilateral lungs lesions in 5 cases.Multiple patchy consolidation,stripe shadow in bilateral lungs or right lung were found in 3 cases, multiple nodes in 2 cases, mass with nodes in 1 case.Among 6 cases, 2 had septa interlobulare thickness,1 had tracheal fistula,2 had pericardium and pleura involvement,4 had bone destruction,1 had the brainand liver involvement.6 cases showed multiple lymphadenectasis,amalgamation and necrosis in bilateral hilar,mediastinaand the neck.2 had abdominal cavity and or retroperitoneal lymphadenectasis.On PET/CT,2 cases showed high uptake,and the range of SUV value were 1.4-13.9.Initial misdiagnosis by imaging was found in 5 cases.6 patients recovered after anti fungus treatment.Conclusion Imaging appearances of non-HIV infectious penicilliposis marneffei mainly reveals as multiple patchy consolidation,nodes and mass in bilateral lungs, all accompany with lymphadenectasis, many with bone destruction,lack of specificity, which needs lesion biopsy and pus culture to make confirmed diagnosis.

BACKGROUND:Currently, the enzymatic digestion combined with magnetic activated cel sorting for isolating microvascular endothelial cel s are cumbersome and do harm to cel s. Therefore, how to simplify the isolation and culture of human dermal microvascular endothelial cel s to obtain highly purified endothelial cel s in vitro becomes a hotspot.
OBJECTIVE:To explore a simple and effective cultivation method of microvascular endothelial cel s from diabetic patient skins in vitro, and to detect the cel growth.
METHODS:Diabetic patients with chronic foot wounds after amputation were enrol ed to col ect the limb proximal skin and topical skin around the wound superficial dermal tissue. Human dermal microvascular endothelial cel s were obtained using adherent method and trypsin method, fol oewd by purified utilizing trypsin digestion and repeated attachment method when passage culture.
RESULTS AND CONCLUSION:Human dermal microvascular endothelial cells were obtained successfully, Primary cultured endothelial cells completely adhered to the wall at 24 hours, entered the logarithmic phase at the 10th day, and the cell concentration reached 80%at the 12th-13th day. While the passage cells grew more actively than primary cells, and fully covered the bottom in a“cobblestone”arrangement after 5-7 days of culture. Immunohistochemical staining showed that cultured cells were positive for FVIII and CD31-associated antigens with 100%positive rate. MTT assay showed that cell growth curves of 2, 4, and 5 generations of dermal microvascular endothelial presented the invertedSshape. These results suggest that abundant highly purified human dermal microvascular endothelial cells can be obtained through the adherent method and a small amount of short-term trypsin method.

Objective To investigate the clinical value of bile reinfusion combined with enteral nutrition before R0 resection of hilar cholangiocarcinoma.Methods The clinical data of 46 patients with hilar cholangiocarcinoma who underwent R0 resection at the Affiliated Drum Tower Hospital of Medical School of Nanjing University from January 2010 to January 2014 were analyzed retrospectively.The clinical data of 21 patients (the experimental group) who received bile reinfusion by preoperative percutaneous transhepatic cholangiography and drainage (PTCD) combined with enteral nutrition with nasojejunal tube were compared with 25 patients (the control group) who received neither PTCD nor enteral nutrition.The differences in the clinical indicators between the 2 groups were analyzed.Results In the experimental group,the levels of ALT,AKP,TBil and DBil decreased significantly after PTCD as compared with the original levels (t =4.433,4.547,5.648,4.681,P ＜ 0.05).The operation time and blood loss in the experimental group was reduced significantly when compared with the control group (t =-2.810,-2.047,P ＜ 0.05).The volume of postoperative albumin transfusion and the postoperative hospitalization in the experimental group was significantly reduced when compared with the control group (t =-3.083,t =-3.083,P ＜ 0.05).Conclusion Bile reinfusion combined with enteral nutrition facilitated the recovery of preoperative hepatic function in patients who underwent R0 resection of hilar cholangiocarcinoma,thus improving the safety of surgery and facilitated postoperative rehabilitation.

Objective To investigate the clinical value of enhanced recovery after surgery (ERAS) in patients undergoing postoperative early enteral nutrition (EEN) with radical resection for hilar cholangiocarcinoma.Methods The clinical data of 48 patients with hilar cholangiocarcinoma who were admitted to the Drum Tower Clinical Medical College of Nanjing Medical University from July 2006 to September 2014 were retrospectively analyzed.All the 48 patients underwent radical resection for hilar cholangiocarcinoma,including 24 patients receiving postoperative EEN (EEN group) and 24 patients receiving total parenteral nutrition (TPN group).The serologic indices and liver function were detected regularly after operation.Ten percent of albumin (Alb) 10 g was administered by intravenous infusion when Alb ＜ 30 g/L.The indexes of all the 48 patients were compared in the 2 groups at postoperative day 3 and 7,including the serologic indices and liver function,the exhaust time,the volume of Alb infusion,the complications (incisional infection,abdominal infection,pleural effusion,peritoneal effusion and bile leakage) and the duration of hospital stay.The patients were followed up by outpatient examination and telephone interview till September 2014.The measurement data with normal distribution were presented as x ± s,comparison between groups and count data were analyzed using the t test and chi-squared test,respectively.Results Patients in the 2 groups were cured successfully and discharged,and no patient died perioperatively.Patients in the EEN group had a good tolerance for EEN and no occurrence of EEN-related complications was detected.The level of the GGT was (108 ± 73) U/L in the EEN group,which was significantly lower than (225 ± 121) U/L in the TPN group at postoperative day 3 (t =4.041,P ＜ 0.05).The level of the GGT was (142 ± 86) U/L in the EEN group,which was no significantly different from (183 ± 107)U/L in the TPN group at postoperative day 7 (t =1.477,P ＞ 0.05).The postoperative time to anal exsufflation and the duration of hospital stay were (73 ± 18) hours and (15 ± 4) days in the EEN group,which were significantly different from (97 ± 21) hours and (18 ± 4) days in the TPN group,and the volume of Alb infusion was (44 ± 29)g in the EEN group,which was significantly lower than (101 ± 92) g in the TPN group (t =4.295,2.615,2.916,P ＜ 0.05).All the 48 patients were followed up for 1 to 71 months (mediantime,10 months),no patients received reoperation or re-admitted to the hospital due to complications.Conclusion The application of postoperative EEN in enhanced recovery of patients undergoing radical resection for hilar cholangiocarcinoma is safe and effective,it could accelerate the recovery of enteral function,shorten the postoperative duration of hospital stay and reduce the supplement of extrinsic Alb,which is helpful for the fast recovery of patients.

Objective To investigate the clinical efficacy of laparoscopic anatomical hepatectomy via Glissonean pedicle transection approach.Methods The clinical data of 68 patients who underwent laparoscopic anatomical hepatectomy by Glissonean pedicle transection approach at the Southwest Hospital of Third Military Medical University between March 2009 and December 2013 were retrospectively analyzed.All the patients received intravenous-inhalation general anesthesia.The splitting of liver was carried out after Glissonean pedicles were completely dissected and occluded under the laparoscope according to anatomical structure.The patients with hepatolithiasis underwent intraoperative bile duct exploration and stone removal with T-tube placement based on the conditions of extrahepatic biliary lesions.The fragmented specimens from benign lesions of liver were taken out through a Trocar hole with the diameter of 12mm.The complete specimens from malignancy tumors of liver were taken out through an enlarged Trocar hole or transverse incision at the symphysis pubis.According to the recheck results of benign and malignancy lesions,the patients were followed up by outpatient examination and telephone interview till September 2014.The measurement data with normal distribution were presented as (x) ± s.The survival curve was drawn by Kaplan-Meier method.Results Among the 68 patients,laparoscopic anatomical hepatectomy via Glissonean pedicle transection approach was performed on 64 patients and 4 patients were coverted to open surgery.Laparoscopic anatomical left hemihepatectomy was performed on 30 patients,anatomical right hemihepatectomy on 19 patients,anatomical right posterior lobectomy on 10 patients and anatomical right anterior lobectomy on 9 patients.The mean operation time,volume of intraoperative blood loss,rate of perioperative blood transfusion,time of postoperative gastrointestinal function recovery and duration of hospital stay were (224 ± 117)minutes,(413 ± 349) mL,5.9％ (4/68),(3.0 ± 0.5) days and (8.0 ± 3.0) days,respectively.There was no perioperative death,and 6 patients with postoperative complication were cured by symptomatic treatment.The tumor diameter and distance to resection margin in 29 patients with malignancy tumors of liver were (4.4 ± 1.6) cm and (2.0 ± 0.9)cm,respectively.The results of pathological examination showed that hepatolithiasis was detected in 22 patients,cavernous hemangioma of liver in 12 patients,hepatic focal nodular hyperplasia in 1 patient,hepatic adenoma in 1 patient,hepatic angiomyolipoma in 1 patient,hepatic multiple cysts in 1 patient,hepatic tuberculosis in 1 patient,hepatocellular carcinoma in 27 patients and cholangiocarcinoma in 2 patients.All the 68 patients were followed up for 6-60 months with a mean time of 24 months.No recurrence and residual stones or lesions in 39 patients with benign lesions of liver were detected during the follow-up.The 1-,3-,5-year overall survival rates and tumor-free survival rates in 29 patients with malignant tumors of liver were 92％,84％,60％ and 83％,59％,42％,respectively.Conclnsion Laparoscopic anatomical hepatectomy via Glissonean pedicle transection approach is safe and feasible,and is suitable not only for left hemihepatectomy,but also for right hemihepatectomy of high technical specification.

Objective To approach the proliferative activities of Hodgkin's disease cells were transfected with mtrⅡ gene.Methods The ki67 and PCNA expression of Hodgkin's disease cells and those which were transfected with mtrⅡ gene by immunohistochemical technique respectively.Results The expression rate of ki67 and PCNA in Hodgkin's disease cells which were transfected with mtrⅡ gene was higher than that weren't transfected(P