AIM: To construct the shuttle plasmid vector for thymidine kinase (tk) and EGFP fusion protein gene driven by IGF - Ⅱ P3 promoter, and investigate the specific killing effect of the HSV - tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro. METHODS: Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening, and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT -PCR. Cell killing after ganciclovir(GCV) application was determined by MTT. RESULTS: Identification of pDC316 -tkEGFP- P3 by enzyme digestion and sequencing analysis showed that the length, inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells, but not in HeLa cells. The results of RT -PCR showed that only two bands could be seen in the samples of pDC316 -tkEGFP- P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells. CONCLUSION: The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF - Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.

Objective To isolate and purify melanin from yeast cells of Penicillium mameffei (PM) and to analyze its physicochemical features.Methods PM yeast cells were cultured in minimal medium containing levodopa (L-dopa) with continuous shaking at 37 ℃.Melanin was isolated from the yeast cells of PM by cell walllysing enzyme,denaturant,concentrated hot acid,and purified by sodium hydroxide and concentrated hydrochloric acid.The physicochemical properties of isolated melanin were assessed on the basis of combined chemical analysis and spectroscopic methods including ultraviolet-visible (UV-vis),Fourier transform infrared (FT-IR) and electronparamagnetic resonance (EPR).Results Similar to synthetic dopa-melanin,the isolated melanin from PM was alkali soluble,acid-resistant,insoluble in water and most organic solvents,precipitated at or below PH 3,and could be bleached by hydrogen peroxide.UV-vis spectrophotometric analysis showed that the PM-derived melanin had a typical absorption peak at 205 nm and the absorbance decreased with the increase of wavelength.FT-IR spectroscopy displayed two absorption peaks at about 3 μm and 6 μm,which was characteristic of classic melanin.EPR spectroscopy revealed that the isolated melanin contained stable free radicals.Conclusion Yeast cells of PM can use exogenous L-dopa to synthesize dopa-melanin.

Objective To compare the successful ratio, efficacy and complications between ultrasound-guided and X-ray-guided endoscopic biliary drainage (EBD). Methods EBD was performed in 62 patients under ultrasound guidance and 54 patients under X-ray guidance. Serum bilirubin, the bile duct diameter and the changes of clinical symptoms were compared before and after the procedure. Results Tube placement was successfully achieved in 54 of 62 patients under ultrasound guidance and 51 of 54 patients under X-ray guidance. The serum direct bilirubin and the common bile duct diameter in patients with ultrasound guidance before and one week after procedure were (205.41±115.27) μmol/L vs. (106.47±82.16) μmol/L and (12.6±7.1) mm vs. (8.5±3.1) mm, respectively, with significant difference (all P values<0.05). Whereas they were (211.14±106.25) μmol/L vs. (110.89±59.47) μmol/L and (13.1±7.0) mm vs. (8.8± 3.2) mm, respectively, in patients with X-ray guidance (P<0.05). No complications such as abdominal pain, fever and elevated amylase were found in patients with ultrasound guidance, while 3 patients (5.9%) with X-ray guidence had above complications. Conclusions X-ray is a most effective method in guidance of EBD. However, ultrasound guidence, which may avoid unfavorable factors such as X-ray radiation and allergic contrast agent, has some advantages including real-time display, mobile convenience and emergency bedside application. It can instead of X-ray in performance of endoscopic nasobiliary drainage and endoscopic retrograde biliary drainage in patients with bile duct stone and mild narrow ducl caused by tumors.

AIM: To investigate the genetic and epigenetic alterations of p14~ ARF gene and mutation status of p53 gene in human primary colorectal carcinomas and to analyze the relationship between the two gene changes and the role of abrogation of the p14~ ARF -p53 pathway in colorectal carcinogenesis. METHODS: The homozygous deletions, mutations, methylation of 5′ CpG islands, mRNA expression of p14~ ARF gene and mutations of p53 gene were assessed by PCR, direct sequencing, methylation-specific PCR, and RT-PCR in the tumorous and matched adjacent normal colorectal tissues from 56 patients with colorectal carcinoma. RESULTS: ① p14~ ARF alterations were detected in 27% (15/56) of colorectal carcinoma tissues studied, of which 1 case showed homozygous deletion, 14 cases showed 5′ CpG island methylation, and no mutation was found in any tumor. ②15 colorectal carcinomas with p14~ ARF alterations indicated lack of (13 cases) or at low level of expression (2 cases) of p14~ ARF mRNA, while expression of the p14~ ARF transcript was detected in the remaining 41 colorectal carcinomas and any matched adjacent normal colorectal tissues. ③ The mutations of p53 gene were detected in 48% (27/56) of colorectal carcinomas investigated. ④ Of these 56 cases, 12 had p14~ ARF alterations alone, 24 had p53 mutations alone, 3 had both p53 mutations and p14~ ARF methylation, and 17 had neither. 70% (39/56) of the samples had either or both abnormalities of the two genes, and p14~ ARF hypermethylation was related to wildtype p53 (P

AIM: To clone P1 and P3 promoters of the human insulin-like growth factor Ⅱ(IGF-Ⅱ) gene. METHODS: According to the complete DNA sequence of IGF-Ⅱ gene, the nested primer PCR was performed for amplifying P1 and P3 promoter fragments of the gene from human L-02 cell line.These PCR products were analyzed by agarose gel electrophoresis, and cloned by using TOPO TA Cloning kit. The positive clones containing P1 and P3 fragments were selected and confirmed by sequencing.RESULTS: The DNA sequences of P1 and P3 promoters cloned were accordant with GenBank data. CONCLUSION: In this study P1 and P3 promoters of the IGF-II gene were cloned successfully.

Obesity is an established risk factor for endometrial cancer. Leptin, a secreted protein of the ob gene by white adipose tissue, plays an important role in the regulation of food intake and energy consumption in the brain and acts as a potential growth stimulator in normal and neoplastic cancer cells. However, a direct role for leptin in endometrial cancer has not been demonstrated. In the present study, the effect of leptin on the proliferation of Ishikawa endometrial cancer cells was investigated as well as the possible mechanism(s) underlying this action in endometrial cancers which express both short and long isoforms of leptin receptors. The expression of leptin receptor (ObRb) in Ishikawa cells was detected by RT-PCR and Western blotting. The cells after serum starvation, were treated by leptin with various concentrations (0, 10, 50, 100, 150 ng/mL) for different durations (6, 12, 24 h). The effect of leptin treatment on cell proliferation was examined by MTT assay. Meanwhile, inhibitory effect of Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) inhibitor AG490 or extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 on the proliferation of Ishikawa cells induced by leptin was also studied. Ishikawa cells were treated with 100 ng/mL leptin for various periods (0, 20, 40, 60 min), and the levels of STAT3 phosphorylation and ERK1/2 phosphorylation were examined by Western blotting. The results showed that leptin induced the phosphorylation of STAT3 and the activation of ERK1/2 in a time- and dose-dependent manner in the Ishikawa endometrial cancer cells. Blocking STAT3 phosphorylation with the inhibitor AG490, or blocking ERK1/2 activation by the specific ERK1/2 kinase inhibitor, PD98059, abolished leptin-induced proliferation of Ishikawa cells. In addition, leptin was found to potently induce the invasion of endometrial cancer cells in a Matrigel invasion assay. Leptin-stimulated invasion was effectively blocked by pharmacological inhibitors of STAT3 (AG490) and ERK1/2 kinase (PD98059). These results suggested that leptin promotes endometrial cancer growth and invasiveness by activating STAT3 and ERK1/2 signaling pathways and therefore blocking its action at the receptor level can be a rational therapeutic strategy.

Objective To determine the optimal pressure for facemask ventilation during induction of general anesthesia by real-time ultrasonographic measurement of antral cross-sectional area (CSA) in adult patients.Methods Sixty American Society of Anesthesiologists physical status Ⅰ or Ⅱ patients,aged 18-60 yr,with body mass index of 20-25 kg/m2,scheduled for elective operation under general anesthesia,were divided into 5 groups (n=12 each) using a random number table:P10 group,P13 group,P16 group,P19 group and P22 group.After induction of anesthesia,an oropharyngeal airway was inserted,and the patients were ventilated for a 2-min period in a pressure-controlled mode using the two-handed mask ventilation technique.The pressure for facemask ventilation was 10,13,16,19 and 22 cmH2O in P10,P13,P16,P19 and P22 groups,respectively.The antral CSA was measured using real-time ultrasonography before and after facemask ventilation.Respiratory parameters were recorded.Results Compared with group P1O,the number of patients in whom CSA＜340 mm2 after facemask ventilation was significantly decreased in P16,P19 and P22 groups,and the number of patients in whom the tidal volume ≥ 6 ml/kg was increased in P13,P16,P19 and P22 groups (P＜ 0.01).The number of patients in whom optimnal pressure for facemask ventilation was achieved was 2,10,6,4 and 1 in P10,P13,P16,P19 and P22 groups,respectively,with the most cases in group P13 (P ＜ 0.01).Conclusion The optimal pressure is 13 emH2O for facemask ventilation during induction of general anesthesia when determined by realtime ultrasonographic measurement of antral CSA,and it can ensure adequate oxygen supply and reduce gastric insufflation in adult patients.

Objective To explore influences of pre-tension and testing site on the radial compliance of vascular prostheses. Methods Three different textile vascular prosthesis samples (PET woven, PET/PPT woven and PET corrugate warp knitted) with the diameter of 8 mm were selected. Under various pre-tensions and testing sites, the pulsatile pressure and its corresponding radial diameter of these samples were measured by dynamic compliance testing instrument. The radial compliance was calculated according to the compliance formula in ISO 7198-1998. Results The radial compliance of all the three vascular prostheses increased along with the pre-tension increasing. When the pre-tension was 500~550 mN, all the radial compliance reached the maximum (1.03~1.72)%/100 mmHg (1 mmHg=0.133 kPa), and then began to decrease with pre-tension increasing. Furthermore, the radial compliance of corrugate vascular prostheses was slightly bigger at valleys than that at peaks, while the radial compliance at peaks was more stable than that at valleys. Conclusions The pre-tension and test site had vital effects on testing the radial compliance of vascular prostheses. Pre-tension of 500~550 mN should be right to apply to all the three kinds of textile vascular prosthesis at test. As for corrugate vascular prostheses, the radial compliance at peaks should be chosen as the test site to get more stable result and reliable data.

Objective To investigate the relationship of the expression of vascular endothelial growth factor (VEGF)and the mi-crovessel density (MVD)with multi-slice spiral CT perfusion imaging.Methods 80 patients with solitary pulmonary nodules under-went perfusion scan by 16-slice spiral CT.Among them,45 diagnosed as lung cancer by pathology were enrolled in the study.After surgery,the slice of the pecimen was selected similar to the corresponding slice of CT images,and the immunohistochemical staining was performed to determine the expression of VEGF and the MVD.Spearman correlation analysis was used to determine the rela-tionship of expression of VEGF and the MVD with CT perfusion parameters.Results There was more expression of VEGF and the MVD in NSCLC.There were positive correlations between VEGF,MVD and BF (both P<0.05).The peak enhancement image (PEI)and TTP have significant correlations with MVD (P<0.05).Conclusion Some parameters of lung CT perfusion imaging are correlated with MVD and VEGF.

Objective To compare the effect of treating cricopharyngeal achalasia in stroke survivors using transnasal or transoral balloon dilatation.Methods Thirty stroke survivors with cricopharyngeal achalasia were randomly divided into a transnasal and a transoral balloon dilatation group (group N and group O),each of 15.Both groups were given routine swallowing rehabilitation training as well as the transnasal or transoral balloon dilatation.Their heart rate was monitored during the dilatation.Nasal bleeding,mucous membrane swelling and pain were also observed.Their swallowing function was evaluated using the Fujishima Ichiro swallowing efficacy score (FISE) and videofluoroscopy (VFSS) before and after the intervention.Results After the treatment,the average FISE and VFSS scores of both groups had improved significantly comnpared to before the treatment but there were no significant differences between the groups.During the treatment,the average heart rate of group O increased significantly less than that of group N.The treatment acceptance of group O was 98.2％,significantly higher than that of group N (80.1％).One case of mucosal bleeding was observed in group O,and laryngeal edema occurred significantly less often than in group N (9 cases vs.7).The average pain score was also significantly lower in group O.Conclusions Balloon dilatation facilitates swallowing among stroke survivors with cricopharyngeal achalasia.The transoral approach can help to reduce the occurrence of complications such as mucosal bleeding,laryngeal edema and pain,and has better patient acceptance.