Objective: To construct a recombinant lentivirus harboring RNAi sequence targeting rat nogo receptor gene and to observe its infection efficiency of 293T cells. Methods: Lentivirus shuttle plasmid containing siNgR199 cDNA was constructed by gene engineering and was used to transfect 293T cells in the presence of packaging plasmids. Forty-eight hours later the supernatant was collected and the titer of virus was determined. The recombinant lentivirus and the standard lentivirus were used to transfect 293T cells at 1 MOI,3 MOI,5 MOI,10 MOI and 20 MOI. Polymerase chain reaction (PCR) was used to detect the recombinant vector; enhanced green fluorescent protein (EGFP) expression was used to determine the titer and the infection rate of the recombinant lentivirus under fluorescent microscope. Results: Restriction endonuclease and PCR analysis confirmed that the siNgR199 cDNA was successfully inserted into the lentivirus vector. The titer of the recombinant lentivirus harboring siNgR199 was 1×108 ifu/ ml and the best MOI was 3. Conclusion: The recombinant lentivirus containing siNgR199 gene has been successfully constructed, which lays a foundation for future axon regeneration in treatment of spinal cord injury.

OBJECTIVETo evaluate the effects of lentiviral vector-mediated RNA interfere gene Nogo receptor (NgR) of rat cortical neurons in repairing spinal cord injury.

METHODSThe recombinant-lentiviral vector with small inferring RNA siNgR199 which had been constructed was transfected into rat cortical neuron cells in vitro in 3 multiplicity of infection (MOI). The infection rate was determined with fluorescent microscope, and the target gene was detected by PCR analysis. Then, the recombinant was injected into the cortical motor area of the rats with severe spinal cord injury, and the saline was also injected into other rats with severe spinal cord injury as a match control. The functional recovery of the rats' hindlimb was assessed using BBB score and the nerve fiber of the injured region was observed by nerve tracing.

RESULTSThe rate of recombinant infecting rat cortical neuron in vitro exceeded 99%. PCR analysis confirmed that the effect of lentiviral vector-mediated RNA interfering gene NgR of rat cortical neurons in vitro was 61%. Although all rats with spinal cord injury were observed to have the hindlimb functional recovery, these rats injected with recombinant had better hindlimb functional recovery than others showing by more BBB score (P < 0.01). Moreover, it was found that some nerve fiber passed the injured spinal cord region of the rats which were injected with recombinant.

CONCLUSIONThe recombinant lentiviral vector with siNgR199 which had been constructed is able to promote the growth of nerve fiber and the functional recovery of the rats' hindlimb.

Animals ; Cells, Cultured ; Disease Models, Animal ; GPI-Linked Proteins ; Genetic Vectors ; Hindlimb ; physiopathology ; Lentivirus ; genetics ; Myelin Proteins ; Nerve Regeneration ; Neurons ; Nogo Receptor 1 ; RNA Interference ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; Receptors, Peptide ; genetics ; Spinal Cord Injuries ; genetics ; physiopathology ; therapy ; Spinal Cord Regeneration ; Transfection

Objective To investigate the accuracy of low-dose dual-source computed tomography (DSCT) coronary angiography in the step-and-shoot (SAS) mode for the diagnosis of coronary artery stenosis in comparison with conventional coronary angingraphy (CCA).Methods Prospective multiple-center study, 46 patients[mean age(58±9) years;bedy mass index(BMI) (25±3) kg/m2]underwent both DSCT in the SAS mode and CCA within 14 days.The inclusion criteria for contrast-enhanced CT: (1) heart rate less than 65 times/rain (bpm).(2) regular sinus rhythm, heart rate fluctuations within the range of 6 bpm. (3) holding breath well, breath-hold time is about 12-15 s.The exclusion criteria:(1) allergy to iodinecontaining contrast medium, nephropathy (serum creatinine level 120 μmol/L), heart failure and serious arrhythmias.(2) patients with coronary stents or bypass grafts.(3) heart rate can not be controlled very well (4)the patient could not take nitroglycerin.(5)BMI 30 kg/m2.(6) other heart disease: carcliomyopathy, valvular disease etc.Sensitivity, specificity, negative (NPV) and positive predictive value (PPV) were determined with CCA as standard of reference.The Kappa value between the two modalities and the two observers was calculated.Radiation dose values were measured.Results Mean heart rate during scanning was (61±6)bpm.99.19% (614/619) coronary segments were depicted with a diagnostic image quality. The vessel-based sensitivity, specificity, PPV, and NPV for the diagnosis of coronary artery stenosis were 96.2% (75/78), 88.2% (60/68), 90.4% (75/83), and 95.2% (60/63), respectively.The Kappa value between the two modalities was 0.848 (P=0.000).The mean effective dose of the SAS-CTCA was (2.95± 0.96) rosy(1.26-4.32 mSy).Conclusion In selected patients, DSCT coronary angiography in the SAS mode have good image quality, which allows for the accurate diagnosis of coronary stenosis at a low radiation dose.

This article presents the design of a bioreactor using hollow fiber membrane and, isolated hepatocytes of suckling pigs, and the experimental study of its efficacy in vitro. Liver cells were harvested from suckling pigs with collagenase perfusion in situ, and parenchymal and non-parenchymal hepatocytes were cocultured in a hollow fiber module which was rotated sporadically. Bioartificial liver(BAL) was developed using this bioreactor,and the BAL was perfused with ascites of patients suffering from liver cirrhosis. The yield of viable hepatocytes was (6.29 +/- 0.37) x 10(8) cells, and cell viability was greater than 84%. Hepatocytes aggregated to multi-cells spheroids after being rotated every thirty minutes for three hours. The hepatocytes in the bioreactor could synthesize urea. Total billirubin was decreased, and AST was significantly increased in the group of bioreactor, as compared with that in the control group. Glucose decreased in the group of bioreactor,whereas there was no significant descent in the control group; and the difference between the two groups was significant. The above results demonstrate that this bioreactor is effective for decreasing total bilirubin and glucose.
Animals ; Animals, Newborn ; Bioreactors ; Cell Separation ; Cells, Cultured ; Hepatocytes ; cytology ; Liver, Artificial ; Swine

OBJECTIVETo investigate the role of postoperative chemoradiotherapy (CRT) as a multimodality treatment option for locally advanced thoracic esophageal squamous cell carcinoma (ESCC) by a prospective comparison between surgery alone and postoperative CRT.

METHODSUsing preoperative computed tomography (CT)-based staging criteria, 158 patients with ESCC (stage II-III) were enrolled in this prospective study. With informed consent, the patients were randomized into two groups: postoperative CRT (78 cases) and surgery alone (S, 80 cases). After a few minor adjustments to the enrolled patients, the actual patients of postoperative CRT group and S group were 74 cases and 77 cases, respectively. Comparison of the complications, local recurrence rate, distant metastasis rate, survival rate and progression-free survival in the two groups was carried out.

RESULTSWith a median follow-up of 37.5 months, the 1-, 3-, 5-, 10-year overall survival (OS) rates were 91.0%, 62.8%, 42.3%, 24.4% and 87.5%, 51.3%, 33.8%, 12.5% for the postoperative CRT and S arm, respectively. A significant difference in OS was detected between the two arms (P = 0.0276). There was a significant difference of progression-free survival (PFS) between the two arms (P = 0.0136). The local recurrence rates in the postoperative CRT group and S group were 14.9% and 36.4%, respectively (P < 0.05). No significant difference was detected between the complications of the two groups (P > 0.05). Toxicities of chemoradiotherapy in the postoperative CRT arm were moderate, which can be relieved rapidly by adequate therapy.

CONCLUSIONRational application of postoperative chemoradiotherapy can provide a benefit in progression-free survival and overall survival in patients with locally advanced esophageal squamous cell carcinoma.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Squamous Cell ; pathology ; therapy ; Chemotherapy, Adjuvant ; Cisplatin ; administration & dosage ; Disease-Free Survival ; Esophageal Neoplasms ; pathology ; therapy ; Esophagectomy ; methods ; Female ; Follow-Up Studies ; Humans ; Lymph Node Excision ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Paclitaxel ; administration & dosage ; Prospective Studies ; Radiotherapy, Adjuvant ; Radiotherapy, High-Energy ; Survival Rate