Objective To construct eukaryotic expression vector of hepatitis C virus (HCV) cytotoxic T-lymphocyte (CTL) epitopes, and to establish stable transfected CHO cell-lines.Methods The HCV CTL epitopes of different genotypes and the mouse H2 complex were predicted by bioinformatics, then synthesized and inserted into eukaryotic expression vector pEGFP-N3.The recombinant vector was transfected into CHO cells by lipofectamine 2000.After screening with G418, stably transfected CHO cell line was established.The expression of HCV multi-epitopes was identified by RT-PCR and western-blot and immunofluorescence.Results The eukaryotic expression vector was constructed successfully.The stable transfected CHO cell line was established.Conclusion The establishment of stable transfected CHO cell line and the expression of the target gene provide solid foundation for further experimental studies.

Intrinsic or acquired chemo-resistance is the main reason for chemotherapy failure, and thus finding ways to reverse chemo-resistance has become an interesting topic for research. Studies have revealed that immunomodulatory molecules are involved in cancer chemo-resistance. Hence, interventions that target immunomodulatory molecules to reverse chemo-resistance have attracted a great deal of concern from domestic and foreign scholars. Immunomodulatory molecules, such as PD-L1, B7-H3, HMGB1, TRAIL, MyD88, and Cytokines (TNF-α, IFN-α, IL-6), have been proven to take part in regulating immune function and tumor drug-resistance characteristics, thereby providing new ideas to the reversal of tumor chemo-resistance. This artide reviews the progression of immuno-modulatory molecules with the change in cancer chemotherapy sensitivity to provide a theoretical basis for the application of new thera-peutic regimen of bio-chemotherapy.

Objective To construct and evaluate the real-time fluorescence quantitative polymerase chain reaction for detecting IL-2R? mRNA in ankylosing spondylitis(AS) based on TaqMan technique.Methods A pair of primers and a TaqMan probe were designed by sequence in GenBank.Total RNA isolated from the fresh peripheral blood monocytes (PBMC) of homo sapiens was amplified by the real-time FQ-RT-PCR.The product was collected by agarose gel electrophoresis and sequencing analysis then ligated with pUCm-T.The recombined plasmid was transcribed to cRNA by T7 RNA polymerase in order to prepare serial standard materials.A new method was created to quantify IL-2R? mRNA in ideal condition.Sensitivity, reproducibility and efficiency were evaluated and used, combined with sIL-2R,for clinical application of AS.Results The linear range was (7~107) cells/ml.The intra-and-inter-assay coefficient variation was 8.4% and 9.6% respectively. Recombined plasmid contained the target fragment was specific and accurate by BLAST.Standard reference was constructed successfully.The RT-PCR product in AS with HLA-B27 positive groups was higher than that with HLA-B27 negative groups and health controls (P0.05).The sensitivity of IL-2R? mRNA was 96.7%.Conclusions Real Time FQ-RT-PCR of IL-2R? mRNA is constructed successfully.This is an easy,rapid,sensitive, accurate and reliable method for quantifing IL-2R? mRNA. There is highly statistical significance, compared with sIL-2R, on the expression of IL-2R? mRNA and inflammatory states between AS and control group and effective information for administration of patients.

Objective To observe inhibitory effect of recombinant human endostatin on the proliferation of vascular tumor endothelial cells and to investigate its possible mechanism.Methods Hemangioma endothelial cells were cultured in vitro and different concentrations of endostatin on hemangioma endothelial cell proliferation inhibition were detected by MTT method.Effect of recombinant human endostatin on endothelial cell cycle was detected by flow cytometry.The expression of VEGF, KDR mRNA in hemangioma endothelial cell were detected by Real-time RT PCR. Results Recombinant human endostatin concentrations in 24 h, 48 h and 72 h after three period during which the hemangioma endothelial cells inhibited significantly( P <0.01 ) , and there was a clear dose dependence, IC50 was 355 μg/mL.Recombinant human vascular endostatin ( 250 μg/mL ) intervented for 24 hours, the proportion of cells in G0/G1 phase(94.23 ±1.66)%, compared with control group (90.63 ±1.14)%, had significantly difference (P<0.05).Compared with the control group, the difference of the expression of vascular endothelial growth factor (VEGF) was statistically significant (P<0.05) as well as Flk-1 (P <0.05).Conclusion Recombinant human endostatin does not only have inhibitory effect of hemangioma endothelial cell cycle, but also can inhibit the expression of VEGF and FLK-1.

Objective To analyze the risk factors of Gram‐negative bacterial blood‐stream infection in ICU for conducting the risk evaluation and guiding medication .Methods The inpatients were diagnosed with Gram‐negative bacterial blood‐stream infec‐tion in ICU of the Renmin Hospital of Wuhan University from January 2013 to December 2014 were retrospectively surveyed and analyzed .The risk factors of blood‐stream infection caused by Gram‐negative bacteria were analyzed and screened by the Logistic re‐gression analysis method .Results A total of 172 case‐times of blood‐stream infection occurred in ICU during this period ,including 93 case‐times of Gram‐negative bacterial infection .The Gram‐negative pathogenic bacteria were Acinetobacter baumanii ,Klebsiella pneumoniae ,Acinetobacter calcoaceticus baumanii ,E .coli ,pseudomonas aeruginosa ,etc .Except E .coli was mainly originated from community acquired infection ,other bacteria were mainly originated from nosocomial infection .In order to differing from other pathogenic bacterial blood stream infection ,the Logistic regression analysis results showed that the independent risk factors of G‐negative bacterial blood‐stream infection in ICU had serum PCT levels over 10 .0 ng/mL (OR= 60 .52 ,P= 0 .001) ,receiving the therapy of carbapenem and third generation cephalosporins (OR=16 .09 ,P=0 .03) ,hospitalization duration less than 2 weeks be‐fore suffering from disease (OR=13 .79 ,P=0 .03) and digestive system basic disease(OR=12 .94 ,P=0 .01) .Conclusion Gram‐negative bacterial blood‐stream infection in ICU of the Renmin Hospital of Wuhan University is mainly caused by multi‐drug resist‐ant bacteria .Serum PCT level over 10 .0 ng/mL ,hospitalization duration less than 2 weeks before infection ,receiving the therapy of carbapenem and third generation cephalosporin and basic diseases of digestive system are the independent risk factors influencing the occurrence and diagnosis of blood‐stream infection .

Objective To study the mechanism of spontaneous rupture of hepatocellular carcinoma(HCC).Methods Articles have been reviewed to find out the theory of spontaneous rupture of HCC.Results Researchful results suggested that the injury of small arteries was usually followed in patients of spontaneous rupture of HCC.In this review,the immune complex,which composed of hepatitis B virus e antigen,complement C1q and immunoglobulins,was found deposited in the elastic membrane of arteries.Likely as a result of immune complex deposition,vascular injury occurs mainly in the small arteries where the deposition of immune complex was present.The small arteries in which immune complex deposited are readily injuried and cause hemorrhage and rupture of HCC during vascular load increase. Conclusion We would conclude that immune complex deposition in vessel wall led to the small arteries injury may be the factor involved in the pathogenesis of spontaneous ruptured HCC.

Objective:To develop and apply a method for real-time quantifying IL-6 mRNA.Methods:By Ficoll-Hypaque density gradient centrifugation, human peripheral blood mononuclear cells(PBMC) were isolated from whole blood treated with ethylenediaminetetraacetic acid(EDTA). Total RNA extracted from the PBMC in trizol solution was reverse transcribed to cDNA, which used as templates for fluorecent real-time quantitative PCR amplification of IL-6. PCR system consists of IL-6 primer, SBY Green Ⅰ and so on.Results:The method is wide linear range, sensitive, specific, reproducible and simple.Conclusion:The method can accurately quantify IL-6 mRNA.

Objective To sbserve the effect of sirolimus-eluting stent in actual clinical practice. Methods The study included 263 patients who had implanted the cypher stents from December 2002 to May 2004. The incidence of MACE was followed up in all patients 8?2 months after stent implantation by means of telephone or out-patient department visit. Evaluation of the therapeutic effect was also made in the diabetes subgroup and different types of lesions, including left main, bifurcation, chronic total occlusion, diffuse disease, in-stent restenosis, littlie vessel, acute occlusion, ostial lesions, and just A-type lesions. The rate of restenosis was observed in all of the lesion. Results Among 246 patients, all patients were successfully implanted with the cypher stents, 93.5% patients had been followed up, 10 (3.8%) patients had MACE. 139 (52.6%) patient were revised by angiography. The total rate of restenosis was 10.1%, and among it 12.9% was diabetes mellitus patients, 11.1% with chronic total occlusion, 11.3% with little vessel lesion, 10.0% with diffuse lesion, 22.2% with left main lesion, 18.1% with bifurcation, 11.1 with CTO 9.1% with ostial lesions, and 8.7% with A-type lesion lesion. Conclusion Cypher stent implantion was safe and effective in actual clinical proctice. The incidence rate of MACE was low in lesions such as chronic total occlusion, diffuse disease, in-stent restenosis, littlie vessel, acute occlusion, ostial lesions, and A-type lesion. It may be beneficial for patients who had left main, bifurcation, in-stent restenosis.

Objective To study the effects of hypoxia on adhesion and migration of ovarian carcinoma cell line Caov-3 in vitro. Methods The ovarian carcinoma cell Caov-3 was cultured under normoxic condition or in hypoxic condition. In vitro adhesion of Caov-3 on extracellular matrix fibronectin and matrigel was detected using MTT colorimetric assay. In vitro migration of Caov-3 on extracellular matrix matrigel was assayed with cellular migration test. Results In vitro cell adhesion experiment showed that, compared with the control cells, hypoxia increased cell adhesion to extracellular matrix matrigel and fibronectin by 17.1% and 15.2%, respectively. There were significant differences between the normal conditions and hypoxia (P

Objective:To investigate and assess the systemic functional training in the prevention of lower limb trauma fracture surgery knee stiffness effect. Methods: Select General Hospital of PLA (301 Hospital) traumatic lower extremity Department of orthopedics from 2009 since April admissions of fractures in patients with 126 cases, according to the nursing methods of postoperative patients were divided into experimental group 63 cases and control group 63 cases, control group was given routine nursing after traumatic fracture of lower limb, the experimental group received training rehabilitation care plan and patient in the control group. Postoperative follow-up, the time of 5 months-2 years, two groups of patients were muscle strength, extension and flexion degree and the patients with complications were analyzed statistically, and the comprehensive evaluation of knee joint function recovery of patients. Results:The postoperative knee flexion strength and degree than the control group;experimental group, the complication rate was 15.9%in the control group of patients swelling after seven cases, the complication rate was 28.6%; comprehensive evaluation knee function two groups of patients in the experimental group was 82.5% excellent (52/63), good rate of the control group was 65.1% (41/63);said comparison items were statistically significant differences (x2=12.6, P<0.05). Conclusion: Lower limb trauma fracture surgery rehabilitation for patients with systemic functional training rehabilitation care, joint function recovery is remarkable, scientifically valid methods, should be further promoted.