To construct, express and purify fusion protein containing HBcAg and HBsAg preS1 epitope peptide for the purpose of investigating a novel HBV vaccine with both prophylactic and therapeutic functions. Using DNA recombinant technology, prokaryotic expression plasmid pBTcs1 expressing HBcAg and HBsAg pre-S1 epitope peptide fusion protein was constructed. After expressed in E.coli. HB101, the production BTcs1 was purified by sucrose density gradient ultracentrifugation and identified by SDS-PAGE, SEC, Western-blot and electron microscope. The results indicated that expression plasmid pBTcs1 was constructed successfully, and 20~25 mg purified BTcs1 fusion protein was obtained from 1L LB culture. Result of DOT-BLOT indicated that the distribution of BTcs1 was mainly in 30~50% sucrose, the purity of BTcs1 was greater than 95% by SDS-PAGE and SEC analysis. BTcs1 could probe with specific antibodies at 28 kDa by Western-blot, BTcs1 could also self assemble VLP by electron microscope analysis, its diameter was 30~34 nm approximately. The present study lay a foundation for further research functions and applications of BTcs1.

AIMTo develop a novel non-viral gene delivery systems lipid-polycation-DNA complexes (LPD) and investigate their transfection efficiencies in vitro.

METHODSLPD were prepared as follows by first mixing the plasmid DNA and protamine together, then the resulted polyplexes were incubated for 10 min at room temperature, followed by addition of preformed cationic liposomes. The morphology of LPD was observed by transmission electron microscopy. The diameter and surface charge of LPD were measured by photon correlation spectroscopy (PCS). The nuclease protection ability of LPD was evaluated by agarose gel electrophoresis. Estimation of transfection efficiency was performed by galactosidase assay in Chang, HepG2 and SMMC-7721 cells.

RESULTSThe average diameter and the zeta potential of LPD were 143.5 nm and 32.6 mV, respectively. LPD could protect the plasmid DNA from nuclease degradation after 2 hours incubation at 37 degrees C while the naked DNA degraded rapidly. The average transfection efficiencies were (69 +/- 6)%, (43 +/- 7)% and (96.2 +/- 1.8)% in Chang cells, HepG2 cells and SMMC-7721 cells respectively.

CONCLUSIONLPD could be prepared easily with small particle sizes and high transfection activities. LPD may be good non-viral vectors for applications in gene delivery.

Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cells, Cultured ; DNA ; Genetic Vectors ; Hepatocytes ; cytology ; Humans ; Liposomes ; Liver Neoplasms ; pathology ; Particle Size ; Protamines ; Transfection

Animals ; Bacterial Toxins ; administration & dosage ; Biological Transport ; Cell Membrane Permeability ; Drug Delivery Systems ; Endocytosis ; Liposomes ; chemistry ; Peptides ; administration & dosage ; Viral Envelope Proteins ; administration & dosage

Objective To report the outcome of emergency repair of severe complex defect in forearm by transplantation of free flap and simultaneous functional reconstruction.Methods From Mar.1994 to Aug.2003,4 cases with severe complex defect in forearm was repaired by transplantation of free skin flap, free skin flap combined with fibula flap,or fibula osteocutaneous flap in emergency.Simultaneously the flexion and extension function were repaired by muscle transfer and/or tendon grafting,tenonectomy.Results All the cases were successful.Follow-up period ranged from 1 to 3 years postoperatively.The blood-supply,tex- ture and elasticity of transferred flaps were excellent with good bone healing.Opposition of thumb with four fin- gers was good.Sensory recovery of the hand was satisfactory.Conclusion Transplantation of free flap com- bined with simultaneous functional reconstruction is an ideal method in emergency repair of severe complex de- fect in forearm.

This study is to report the preparation of complexes of Ad5 and anionic liposomes (AL-Ad5), the amplification of adenoviruses with enhanced green fluorescent protein (eGFP) reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect (CPE) method, hexon capsid immunoassay and quantitative-PCR (Q-PCR), separately. The prescription and experiment conditions were optimized by central composite design (CCD). The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Additionally, the bicolourable fluoresce-labeled complexes (F(labeled)-AL-Ad5) were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy (CLSM). The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 +/- 10 nm and a zeta potential of -41.2 +/- 2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5.
Adenoviridae ; genetics ; ultrastructure ; Animals ; Anions ; Cytopathogenic Effect, Viral ; Dogs ; Drug Compounding ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; chemistry ; HEK293 Cells ; Humans ; Liposomes ; chemistry ; pharmacokinetics ; ultrastructure ; Madin Darby Canine Kidney Cells ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Particle Size ; Polymerase Chain Reaction ; methods ; Recombinant Fusion Proteins ; ultrastructure

OBJECTIVETo evaluate if 99mTc-HYNIC-annexin V may be used to detect the early chemotherapeutic effect and to determine the best timing for detecting apoptosis in vivo.

METHODSAnnexin V was labeled with 99mTc using HYNIC as a bifunctional agent. Normal Kunming mice received inoculation of Ehrlich ascites cells into the right upper limb. After the tumor reached 1 cm in diameter, the mice were randomly divided into saline treatment group as control and cyclophosphamide (150 mg/kg injected intraperitoneally) treatment group. 99mTc-HYNIC-annexin V was injected intravenously at 1 h and 24 h after treatment. Region of interest technique (ROI) from the SPECT images taken at different time was used to get the ratio of tumor/limb in each group. TUNEL staining was used to detect apoptotic cells and the rates of positive stained cells were calculated.

RESULTSAfter treatment with saline, only little amount of the radiolabeled tracer could be seen in the tumor and showed weak image of the tumor. But after 24 h of treatment with cyclophosphamide, clear image on the tumor could be seen. 24 h after the treatment of cyclophosphamide, the ratio of tumor/limb was (6.27 +/- 0.24) which was much higher than that at 24 h after treatment with saline (2.36 +/- 0.18) and that at 1 h after cyclophosphamide treatment (4.00 +/- 0.38). At 24 h after cyclophosphamide treatment, TUNEL staining showed a significantly higher rate of apoptotic cells in the mice.

CONCLUSION99mTc-HYNIC-annexin V can be used as an apoptosis-imaging agent to detect and evaluate the early curative effect after chemotherapy. The effective detection of apoptotic response in tumor with 99mTc-HYNIC-annexin V requires a 24 h interval after chemotherapy. SPECT images can be obtained at 60 min after injection of the imaging agent. It suggests that 99mTc-HYNIC-annexin V may become a promising agent for apoptosis-imaging in clinical application.

Animals ; Annexin A5 ; pharmacokinetics ; Antineoplastic Agents, Alkylating ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Ehrlich Tumor ; diagnostic imaging ; drug therapy ; pathology ; Cyclophosphamide ; therapeutic use ; Male ; Mice ; Neoplasm Transplantation ; Organotechnetium Compounds ; pharmacokinetics ; Radiopharmaceuticals ; pharmacokinetics ; Random Allocation ; Tissue Distribution ; Tomography, Emission-Computed, Single-Photon

Objective To compare the effect of storage autologous blood component transfusion versus storage autologous whole blood transfusion on the cellular immune function and hemorheology in the patients undergoing spinal surgery.Methods Forty patients of both sexes,aged 32-60 yr,of American Society of Anesthesiologists physical status Ⅰ or Ⅱ,undergoing elective multilevel spinal surgery,were divided into 2 groups (n =20 each) using a random number table:stored autologous whole blood transfusion group (group A) and stored autologous blood component transfusion group (group B).Before blood sampling (T0),immediately after blood sampling (T1) and at the end of surgery (T2),arterial blood samples were collected for determination of red blood cell count (RBC),hemoglobin (Hb),hematocrit (Hct),erythrocyte aggregation index (EAI) and erythrocyte rigidity index (ERI).Venous blood samples were collected at T0,T2 and on day 6 after surgery (T3),the distribution of T lymphocyte subsets (percentage of CD3+,CD4+ and NK cells) was measured,and CD4+/CD8+ ratio was calculated.Results Compared with the baseline at T0,the percentage of CD3+,CD4+ and NK cells and CD4+/CD8+ ratio were significantly decreased at T2,3 in group A and at T2 in group B,and RBC,Hb and Hct were significantly decreased at T1,and EAI and ERI were decreased at T1,2 in two groups (P＜0.05).Compared with group A,the percentage of CD3+,CD4+ and NK cells and CD4+/CD8+ ratio were significantly increased at T3 (P＜0.05),and no significant change was found in RBC,Hb,Hct,EAI or ERI at each time point in group B (P＞0.05).Conclusion The effect of storage autologous blood component transfusion on cellular immune function is mitigated than that of storage autologous blood transfusion and the effects on hemorheology are comparable in the patients undergoing spinal surgery.

Breast Neoplasms ; pathology ; Cell Culture Techniques ; methods ; Cells, Cultured ; Female ; Humans ; Immunohistochemistry ; methods ; Quality Control

Objective:To explore the efficacy of preperitioneal tension-free hernia repair by lower abdominal median incision in incarcerated inguinal hernia.Methods:The clinical data of 126 patients with incarcerated hernia who underwent emergency operation from January 2010 to August 2016 were retrospectively analyzed.According to the surgical incision,they were divided into two groups:the observation group (41 cases) had the lower abdominal midline incision,and the control group (85 cases) had the traditional inguinal incision.The time of operation,the postoperative hospital stay,perioperative complications and the main long-term complication were compared between the two groups.Results:In the observation group (41 cases),35 cases underwent preperitoneal tension-free repair (85.4％),1 case recurred and was repaired again,and 2 cases underwent intestinal resection and anastomosis on account of intestinal necrosis.Of the 85 cases in the control group,8 cases recurred and 7 cases underwent reoperation,33 cases underwent first-stage tension-free repair and 1 cases was repaired again,and 3 cases underwent intestinal resection and anastomosis on account of intestinal necrosis.The rate of herniorrhaphy was significantly higher in the observation group than that in the control group (85.4％ vs 38.8％,P＜0.001).The incidence of total surgical complications (including chronic pain,foreign body sensation,hernia recurrence,incision infection,and local serum swelling) in the observation group was lower than that in the control group (5.7％ vs 24.2％,P=0.031).There was no significant differences between the two groups in terms of operation time,hospital stay,perioperative acute pain,and urinary retention.Conclusions:The tension-free repair of preperitoneal hernia through lower abdominal midline incision is simple and can improve the rate of first-stage tension-free repair of incarcerated hernia,and the complication rate is low.It is suitable for clinical application.

OBJECTIVETo explore the potential risk factors of intrahepatic cholangiocarcinoma (ICC) in China.

METHODA case-control study including 317 patients with pathologically confirmed ICC and 634 healthy individuals was conducted. The cases and controls were matched in age, sex and inhabitancy. Data were statistically analyzed by Chi-square test and conditional logistic regression.

RESULTSUnivariate analysis showed significant difference in HBsAg seropositivity, liver cirrhosis, hepatolithiasis, choledocholithiasis and schistosomiasis between ICC patients and healthy controls (P < 0.05). Multivariate analysis confirmed that HBsAg seropositivity, liver cirrhosis, hepatolithiasis and hepatic schistosomiasis were associated with ICC, and their adjusted odds ratio (95% confidence interval) were 10.265 (6.676-15.783), 13.101 (5.265-32.604), 18.242 (3.580-92.958), 18.435 (1.930-176.082), 15.102 (4.607-49.499) and 11.820 (3.522-39.668), respectively. The incidence of hepatic cyst, cholecystolithiasis, hepatic hemangioma, fatty liver, diabetes mellitus, smoking and drinking were not significantly different between ICC patients and controls.

CONCLUSIONSThe HBV infection, liver cirrhosis, hepatolithiasis and hepatic schistosomiasis may be the risk factors for ICC in China.

Adult ; Aged ; Bile Duct Neoplasms ; epidemiology ; etiology ; Bile Ducts, Intrahepatic ; Case-Control Studies ; Cholangiocarcinoma ; epidemiology ; etiology ; Cholelithiasis ; complications ; epidemiology ; Female ; Hepatitis B ; complications ; epidemiology ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Humans ; Liver Cirrhosis ; complications ; epidemiology ; Liver Diseases ; complications ; epidemiology ; Logistic Models ; Male ; Middle Aged ; Odds Ratio ; Risk Factors