To investigate the optimum transfection condition in transfecting human keratinocytes with plasmid-liposome complexes in vitro,the cultured human keratinocytes at 60% ～ 100% confluences were exposed to the eukaryotic expression plasmid,pCMV*SPORT-β-gal,coated with LipofectAMINE in different DNA/liposome mixing concentration ratios.After cultured for another 48 hours following the ends of 6 ～ 24 hours exposures to the DNA-liposome complexes,the transfected human keratinocytes were visualized by β-galactosidase staining.Then,the transfection efficiency was determined by calculating the rate of β-galactosidase staining positive cells.β-galactosidase expression was showed clearly in human keratinocytes transfected with the DNA-liposome complexes.The highest efficiency was achieved with cultured cells at 80% and 90% confluences,demonstrating by the transfection rates of (31.35±1.35)% and (32.32±2.47)% respectively.Meanwhile,the essential transfection conditions for these efficiencies were in coating pCMV*SPORT-β-gal DNA of 1.5μg/100μL with LipofectAMINE of 12.5μL/100μL,and exposing the cells to the DNA-liposome complexes for 8 hours.These results indicate that LipofectAMINE could effectively transfer eukaryotic expression plasmid into human keratinocytes in vitro,for which the optimization of transfection conditions involve in cells growth state,DNA/liposome mixing concentration ratio,and exposure time of cells to DNA-liposome complexes.

Objective To construct an eukaryotic expression vector carrying human glutathione-S-transferase(GST)A1gene and to provide study materials for toxicology and pharmacogenetic.Methods The GST A1cDNA was amplified and separated from human liver total RNAs by RT-PCR approach and recombined with eukaryotic expression vector pcDNA3.The recombined plasmid pcDNA3-hGSTA1was verified using PCR,restriction analysis and sequencing determination.Results Human GST A1gene was recombined correctly with pcDNA3,compared with Genbank,in code152T→C,amino acid Met→Thr.Conclusion The eukaryotic expression vector pcDNA3-hGSTA1was constructed for research on toxicology and pharmacogenetics.

ObjectiveTo investigate the effects of bFGF on human hypertrophic scar in a nude mouse model. MethodsHuman hypertrophic scars excised in operation for burned patiens were grafted onto the back of nude mice subcutaneously and the mice were randomly divided into: control group, collagenase group, bFGF group, and bFGF plus collagenase group. Two weeks after grafting, preparations were injected into the scars for three times. The size, hardness, and morphological changes of the scars were examined. Biopsies were done 3 weeks after first injection and the histological changes were examined. ResultsIt was found that in bFGF group the size of the grafted scars reduced to 1/2～1/3 of their original size, the majority of the coarse and dense scar collagen bundles resolved and became soft loosed lump. In bFGF plus collagenase group, two thirds of the grafted scar disappeared. ConclusionTopical injection of bFGF into hypertrophic scar degrades scar collagen.

Objective To evaluate the safety and efficacy of pancreatic duct precutting by arciform knife combined with needle knife in difficult cannulation of ERCP.Methods The clinical data of 38 patients who were treated with pancreatic duct precutting by arciform knife combined with needle knife in our hospital from June 2005 to May 2012 were retrospectively reviewed.Results The success rate of ERCP was 86.8％ (33/38) without any complications of bleeding or perforation,while 4 cases were presented with hyperamylasemia after ERCP,4 with pancreatitis and 1 with cholangitis who all got recovered after 5 days with conservative treatment.Conclusion The application of pancreatic duct precutting by arciform knife combined with needle knife can improve the success rate of ERCP and is a kind of effective operation technique.

Objective To evaluate the safety and efficacy of endoscopic intraductal radiofrequency ablation combined with biliary metal stent and biliary metal stent only for the treatment of biliary malignant tumor.Methods Data of 48 patients with biliary malignant tumor were reviewed.All patients were examined by ERCP for the range of obstruction.Twenty-four patients received combined therapy (combination group),while 24 others received stent placement only (control group).In the combination group,stenotic segments were treated with intraductal radiofrequency ablation,then metal stents were placed into the biliary duct.In the control group,only metal stents were placed.Changes of serum total bilirubin (TB),surgical complications,days of stent patency and median survival time were studied.Results In the combination group,TB level [median (min-max)] was 238.15 μmol/L (22.10-410.70 μmol/L) before treatment and it decreased to 113.40 μmol/L (9.80-243.70 μmol/L) after,while in the control group it was 239.70 μmol/L(112.30-558.50 μmol/L) before and 121.10 μmol/L (52.10-329.30 μmol/L) after respectively,significantly different in both groups (P＜0.05).The time of stent patency was 156 days (44-530 days) in the combination group and 110 days (14-462 days) in the control group (P＜0.05).The median survival time was 173 days (44-550 days) and 133 days (14-490 days) respectively(P＜0.05).Conclusion The combination therapy of endoscopic intraductal radiofrequency ablation with biliary stent is safe and feasible for biliary malignant tumor.It can prolong the duration of stent patency and median survival time as well as improve postoperative survival quality.

The performance of CRA directly affects the quality of clinical trials. In the appraisal of the CRA performance, satisfaction of clinical institutions plays a vital role. This article presents the outcome of a satisfaction survey on 16 clinical research centers in Shandong province, and the analysis of 503 survey samples regarding their exploratory factor and confirmatory factor respectively. The purpose is to identify the main factors for the satisfaction and to build a model to evaluate the satisfaction of hospitals for CRA.

Objective:To compare the clinical effect of domestic and imported omeprazole injection in the treatment of peptic ulcer bleeding.Methods:According to the random sequence generated by the computer,118 patients who were treated in digestive department of our hospital from April 2013 to May 2015 were randomly divided into 2 groups,59 cases in each group.The control group was given treatment of imported drug losec,while the study group was given treatment of domestic drug aoxikang,and two groups were treated continuously for 7 days.The incidences of gastrointestinal reactions,dizziness and palpitations,the average treatment time,and clinical therapeutic effects were compared,in addition,the costs of treatment in the two groups were analyzed.Results:There were no significant differences in the incidences of gastrointestinal reactions,dizziness and palpitations and the average treatment time between the study group and the control group (P＞0.05).There was no significant difference between the two groups in the treatment effect after rank sum test (P＞0.05).The total effective rates (96.61％ vs 94.91％) were equivalent,the differences were not statistically significant (P＞0.05),but the costs of using domestic drugs were lower than that of imported drugs.Conclusion:The clinical curative effects of domestic aoxikang and imported losec in treatment of peptic ulcer bleeding have no significant difference,which can have a good therapeutic effect,but the economy of aoxikang is more better.

Objective To investigate the impact of pancreatic duct stenting as a preventive measure for post-ERCP pancreatitis in patients with repeatedly non-selective pancreatic duct cannulation.Methods Clinical data of 64 patients with biliary tract disease from January 2008 to December 2015 was prospective analyzed. All the patients were randomly divided into observation group and control group. Patients in observation group received pancreatic stent placement, while patients in control group was not received pancreatic stent placement and nasal duct. Postoperative monitoring items included abdominal pain, blood amylase. Then record and compare the incidence of acute pancreatitis, pancreatitis severity rating, abdominal pain score, Ranson score, amylase recovery time between the two groups.Results Observation group had ifve cases of PEP, the rate was 15.6 %, including mild four cases (12.5 %), medium one case (3.1 %); the control group had 13 cases of PEP, the rate was 40.6 %, including mild six cases (18.8 %), medium four cases (12.5 %), severe three cases (9.4 %). PEP observation group was signiifcantly lower than the control group (P < 0.05). Mild, medium and severe PEP were lower than the control group, in which the severe PEP was signiifcantly lower than the control group (P < 0.05). Ranson score of the observation group was (1.2 ± 0.4), significantly lower than the control group (2.5 ± 1.2) (P < 0.05); the observation group amylase average recovery time was (3.0 ± 0.6) d, it is also signiifcantly lower than the control group (5.8 ± 1.4) d (P < 0.01). No bleeding and perforation complications occurred.Conclusion Pancreatic stenting can effectively prevent the incidence of PEP, reduce postoperative pain, improve patient recovery.

OBJECTIVETo observe the effects of adipose-derived mesenchymal stem cells (ADSCs) with continous over-expression of glial cell line-derived neurotrophic factor (GDNF) on the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

METHODSFive SD rats were collected to prepare ADSCs with over-expression of GDNF. One hundred and fifty SD rats were divided into normal control group (N), GDNF-ADSCs group (GA), ADSCs group (A), GDNF group (G), and physiological saline group (P) according to the random number table, with 30 rats in each group. Rats in group N were routinely fed without treatment, and rats in the other 4 groups were inflicted with electrical injury on sciatic nerve of thigh of the right hind leg. Rats in groups GA, A, G, and P were respectively injected with 100 µL suspension of ADSCs with over-expression of GDNF (1 x 10(7) cells per mL), 100 [µL ADSCs suspension (1 x 10(7) cells per mL), 100 µL GDNF solution (100 mg/L) , and 100 µL physiological saline to the surface of the injured nerves immediately after injury. Six rats of each group were collected for measuring hind limb stride from post injury week (PIW) 1 to 8, and morphology of the sciatic nerves was observed in PIW 8. In PIW 4, the protein expression of GDNF of sciatic nerves of the rest rats in each group was determined with Western blotting. Data were processed with one-way analysis of variance, analysis of variance of repeated measurement, and SNK test.

RESULTSCompared with that of group N, the hind limb stride values in groups GA, A, G, and P were significantly lower at each time point (with P values below 0.05). Compared with those of group P, the hind limb stride values in group GA from PIW 3 to 8, in group A in PIW 3, 5, and 7, and in group G in PIW 3, 5, 7, and 8 were significantly longer (with P values below 0.05). The hind limb stride values in group GA from PIW 4 to 8 were respectively (10.83 ± 0.97), (13.25 ± 1.40), (12.86 ± 1.42), (14.06 ± 1.50), and (15.09 ± 1.17) cm, which were significantly longer than those in group A [(8.90 ± 0.82), (9.03 ± 0.57), (9.27 ± 0.36), (9.86 ± 0.36), and (9.52 ± 0.58) cm] and group G [(8.87 ± 0.69), (8.51 ± 1.18), (9.34 ± 0.87), (9.76 ± 0.67), and (9.50 ± 1.22) cm], with P values below 0.05. Compared with that of group N, the number of myelinated nerve fibers of sciatic nerves was obviously decreased in group P but obviously increased in groups GA, A, and G; the diameter of axons was obviously shorter, and the myelin thickness was obviously increased in groups GA, A, G, and P in PIW 8 (with P values below 0.05). The number of myelinated nerve fibers in group GA was 31.2 ± 0.8, which was significantly higher than that in group A (23.7 ± 2.7), group G (22.3 ± 2.7), or group P (9.3 ± 2.8), with P values below 0.05. The diameter values of axons among groups P, A, G, and GA were similar (with P values above 0.05). The myelin thickness of rats in group GA was (3.41 ± 0.34) µm, which was significantly thicker than that in group A [(2.64 ± 0.37) µm] or group G [(2.41 ± 0.34) µm], with P values below 0.05. In PIW 4, the protein expression of GDNF of sciatic nerves was significantly higher in groups P, A, G, and GA than in group N (with P values below 0.05), and the protein expression of GDNF in group GA was significantly higher than that in group P, A, or G (with P values below 0.05).

CONCLUSIONSADSCs over-expressing GDNF protein can obviously promote the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

Adipose Tissue ; Animals ; Electrophysiology ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; metabolism ; Nerve Crush ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; physiology

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