Objective To construct the bicistronic retroviral vectors containing GFP gene and human survivin gene(wildtype and T34A mutant). Methods Survivin fragments were amplified from eukaryotic expression vectors by PCR, then cloned into pMIG retroviral vectors. The constructed recombinant was identified by double digestion with restriction enzymes. The constructed retroviral vectors was transfected into Phoenix E package cells under mediation of liposome. The expression of mRNA was examined by RT-PCR and the transcription of gene was determined by FACS analysis of GFP. Results The target fragments were successfully bound to pMIG vector. After the vectors were transfected into NIH3T3 cells, the expression of mRNA was confirmed by RT-PCR and the expression of GFP was confirmed by FACS analysis. Conclusion Human survivin gene wildtype and T34A mutant retroviral vectors have been constructed successfully, which can provide strong molecular tools for further studies and a novel approach for selective cancer gene therapy.

Objective To study a-synuclein(SNCA)rs356200 and rs356165 loci polymorphism in the patients with Parkin son's disease(PD)and to investigate the correlation between SNCA gene polymorphism and PD cognitive impairment.Methods One hundred and twenty cases of PD in our hospital from June 2014 to December 2015 and 120 heaalthy people were selected as the research subjects.The PD cases served as the PD group and 120 healthy subjects as control group.The PD cases were also divided into the cognitive impairment group and non-cognitive impairment group according to the cognitive impairment.The rs356200 and rs356165 allele frequencies and genotype in each group were studied.Results The rs356200 allele frequency had statistical difference between the PD group and control group(P＜0.05),the rs356200 G/G,A/A and A/G genotype had statistical difference between the PD group and control group(P＜0.05).The rs356165 allele frequency had no statistical difference between the PD group and the control group(P＞0.05),the rs356165 G/G,A/A and A/G genotype had statistical difference between the PD group and control group(P＜0.05).The rs356200 and rs356165 allele frequencies and genotype had no statistical difference between the cognitive impairmentgroup and non-cognitive impairment group(P＞0.05).Conclusion The morbidity of PD is related to SNCA gene rs356200 and rs356165 sites polymorphism.PD cognitive dysfunction has no significant correlation with SNCA gene rs356200 and rs356165 loci polymorphism.

OBJECTIVE To evaluate distribution and resistance of pathogens in patients with pulmonary infection after abdominal operation by stages,and provide reference to select antibiotics in clinics.METHODS Forty five patients with pulmonary infection after abdominal operation were included from Jan 2004 to Sep 2007.Their course was divided into 3 stages: onset stage,middle stage and last stage,at every stage to identify pathogens and to test drug sensitivity and the sputa and broncho-alveolar lavage fluid(BALF) were sampled.RESULTS A total of 189 strains of pathogens were isolated,from them 110(58.2%)were Gram-negative bacilli including Pseudomonas aeruginosa,Escherichia coli and Klebsiella pneumoniae;and 59(31.2%)were Gram-positive cocci including Staphylococcus aureus and Enterococcus;and 20(10.6%)were fungi,Candida albicans was the main fungus.At onset stage the most pathogens were Gram-negative bacilli;at middle stage the Gram-positive cocci increased distinctly;and the fungi were detected at middle and last stages.The mixed infection rate was high.The result of drug sensitive test showed that there were high rates of multidrug resistance in P.aeruginosa.The resistance rates of all isolates of Enterobacteriaceae to imipenem or meropenem were 0.The resistance rates of all Gram-positive cocci to linezolid or vancomycin were 0.CONCLUSIONS Distribution of pathogens in patients with pulmonary infection after abdominal operation is different at each stage.The most are multidrug resistant,and rationl use of antibacterial drug in clinics must be based on the distribution and drug-resistance of pathogens.

Objective To prepare a redox-responsive doxorubicin-loaded nanoparticle,and to study its in vitro realease behavior and targeting effect on heptoma cells.Methods Cystamine was grafted on the side chains of hyaluronic acid with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide catalyst,and then β-cyclodextrin (β-CD) was conjugated on the amine groups of the cystamine by Schiff's base reaction to prepare β-CD modified hyaluronic acid (HACD).The HACD/DOX nanoparticles were prepared by encapsulating DOX into HACD using dialysis method.The drug loading,encapsulation efficiency,particle size and distribution,zeta potential and other physical and chemical properties,as well as in vitro drug release behavior of the HACD/DOX nanoparticles were characterized.The cytotoxicity of HACD/DOX nanoparticles to HepG2 cells was studied by cell counting kit-8 (CCK-8) method.The targeting effect of HACD/DOX nanoparticles on HepG2 cells was studied using flow cytometry and confocal laser scanning microscopy (CLSM).Results HACD were successfully synthesized,which could carry DOX to form uniform homogeneous nanoparticles.The drug loading of DOX in the nanoparticles was (16.1±0.2)％ and the encapsulation efficiency was (64.2±0.9)％.The transmission electron microscope images indicated that the shape of the HACD/DOX nanoparticles was homogeneous sphere.The results of granularity analysis showed that the average size of the HACD/DOX nanoparticles was (203.1 ±2.5) nm with a narrow size distribution (PDI =0.202).The zeta potential of the HACD/DOX nanoparticles was (-29.1±0.8) mV.The in vitro release behavior of the nanoparticles exhibited obvious redox-sensitivity.The results of in vitro cytotoxicity showed that the blank carrier material HACD had no obvious toxicity to hepatoma cells,and the HACD/DOX nanoparticles could effectively kill hepatoma cells with the 0.38 μg/ml half maximal inhibitory concentration (IC50) value at 48 h.Flow cytometry and CLSM results demonstrated that the HACD/DOX nanoparticles could target hepatoma cells through the mediating effect of hyaluronic acid.Conclusions The prepared HACD/DOX nanoparticles have suitable particle size,high drug loading and encapsulation efficiency,and can release DOX under the stimulation of reducing agent.These nanoparticles have obvious targeting effect on hepatoma cells,which is expected to be applied as the drug delivery system of hepatocellular carcinoma (HCC) therapy.

·AIM: To evaluate the function of primary human corneal endothelial cells(HCEC) serving as immunological cells. ·METHODS: Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells(PBMC) were cocultured with primary HCEC which were pre-treated with and without γ-IFN respectively; activation of lymphocytes was determined by FACS analysis.·RESULTS: In coculture system, T lymphocyte was activated by primary HCEC, HLA-DP, -DQ, -DR and CD40 expression were increased by γ-IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. ·CONCLUSION: Primary HCEC were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells(APC).

Objective To investigate the myocardial protective effects of trimetazidine by observing the changes of peripheral B-type natriuretic peptide (BNP),cardiac troponin I (cTnI) level in patients undergoing off-pump coronary artery bypass graft (OPCAB), and the clinical significance of peripheral cTnI and BNP in cardiac surgery. Methods One hundred and three OPCAB patients were divided into trimetazidine group (52 cases) and control group (51 cases) by random digits table. The serum levels of BNP and cTnI preoperatively,postoperatively of 24 h, 72 h and 7 d were detected. Results The serum levels of BNP [(224.5 ± 12.0), (331.2 ±22.6), (82.4 ±3.3) ng/L] and cTnI [(0.21 ±0.04), (1.32 ±0.49), (0.26 ±0.04) μ g/L] in trimetazidine group were lower than those in control group [(294.7 ± 11.8 ), ( 383.9 ± 28.3 ),( 112.4 ± 12.5 ) ng/L and ( 1.20 ± 0. 13 ), (2.35 ± 0.54), (0.75 :± 0.21 ) μ g/L] postoperatively of 24 h, 72 h and 7 d (P＜ 0.05 ). The serum levels of BNP and cTnI increasedd at 24 h after operation. The peak level was found at 72 h and remained higher level until 7 d after operation. The baseline levels of BNP were positively correlated with cTnI (r = 0.635,P ＜ 0.05), but negatively correlated with left ventricular ejection fraction (LVEF) (r =-0.674,P ＜ 0.01 ). Conclusion Trimetazidine can obviously reduce serum levels of BNP and cTnI in patients undergoing OPCAB. So the united application of serum BNP and cTnI may be as a monitor marker to reflect the cardiac function in patients after OPCAB.

Objective To find a proper number of implanted fiducials in order to reconcile both more accurate image tacking and less pain for the patients.Methods The phantom was made of two parts of different materials.The inner part was a ball-cube phantom developed by Accuray Corporation, which could be regarded as a rigid body, with 5 fiducials inside using as the reference to align the whole phantom in the experiment.The outer part was made of additional silicone rubber with two components to simulate soft tissue as a non-rigid body, which was implanted with 8 fiducials inside.All combinations of different number of fiducials were tracked to aquire the target location information, which were then compared with the reference position that was set by the inner 5 fiducials to obtain the deviations of the translation and the rotation parameters.Thus the impact of the number of fiducials on image target localization accuracy could be analyzed easily.Results When 4 fiducials are used for tracking, the decline of translation and rotation errors for every fiducial is largest (0.086 mm and 0.033°).The translation error was decreased by 0.343 mm from 1 to 4 fiducials, while by only 0.077 mm from 4 to 8 fiducials;the same decline was observed for rotation errors (0.131° for 3 to 4 fiducials , and 0.09° for 4 to 8 fiducials , respectively) .Conclusions When 4 fiducials are used for tracking, the decline of errors for every fiducial is maximum.When one more fiducial is adding, the image target localization accuracy can be enhanced obviously from 1 to 4 fiducials, but not as obvious for more than 4 fiducials.

Objective To observe the curative effect, safety and patient's satisfaction degree of combining compound betamethasone with anisodamine injection in the blocking treatment of eyebrow alopecia areata.Methods A total of 66 cases were randomly divined into two groups: patients were treated by compound betamethasone ( group A) and combining compound betamethasone with anisodamine ( group B). The drugs were injected from the middle of eyebrow alopecia areata to two sides by the standard of 0. 2 ml/cm2 in both groups one time a month. The curative effect of the all cases were observed at 1, 2 and 3 months after treatment. The safety of them were evaluated based on incidence rate of the side effect and their satisfactory degree were evaluated based on subjective reception 3 months after treatment. Results The effective rates in the group B were 58.82 %, 73.53 % and 88.23 % by turns at 1,2 and 3 months after treatment, and those in the group A were 31.25 %, 53.13 % and 71.88 %, respectively. The curative effect in the group B was better than that in the group A (P ＜ 0. 05), and the incidence rate of side effect in the group B was similar to that in the group A (P＞0.05) and the patient's satisfaction degree in group B was better than that in group A (P ＜0. 05) 3months after treatment. Conclusion The compound betamethasone combined with anisodamine injection in the treatment of eyebrow alopecia areata has superior curative efficacy, more safety and higher satisfactory degree in the patients.

Objective To explore the feasibility of culturing dermal papillae cells (DPC) of hu- man hair in a serum-flee medium,and to observe the growth characteristics of these cells.Methods Cell culture flasks (plates) were pretreated with fibronectin,and DPC (2nd passage) were incubated with Williams E serum-flee medium supplemented with insulin-transferrin-selenite (ITS).Cells were observed by an inverted phase-contrast microscope.Proliferation of DPC was evaluated with 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and by their growth curve.Results In a serum-free medium,2nd passage DPC adhered to the flask surface within two to four hours of incubation; two to three days later,confluence,of the cells was observed,without noticeable proliferation.Four days later,cell connection was interrupted,isolated cells or cell clusters were seen,and detachment of some cells from the flask surface was observed.One to two weeks later,most cells had died.After incubation with 4% bovine serum for ten hours,cell proliferation was observed surrounding the remaining viable cell colonies. DPC growth curve showed stagnant phase and slow growth phase;however,log growth phase was not ob- served.Conclusion DPC could be successfully cultured in serum-free medium.However,the culture con- dition needs to be further optimized.

The contents of ir-oxytocin (it-OT), ir-Arg-yasopressin (ir-AVP) and ir-?-endorphin (ir-?-EP) in hypothalamus, pituitary and plasma were measured by. radioimmunoassay (RIA) when the rats were swimming. It was showed that the ir-OT contents in hypothalamus, pituitary and plasma were significantly increased in the animals having swum 1, 5, 15 and 20min (P