AIM: To purify murine yolk sac endothelial cells (mYS-EC) and investigate the cytokines mRNA expression in mYS-EC. METHODS: The murine yolk sacs were digested with 0.1% collagenase, resuspended in DMEM and counted after digestion and centrifugation. The yolk sac adherent cells were cultured in DMEM containing 15% FBS with 10% mBMEC-CM or 5?g/L VEGF, ECGF and bFGF. The phagocytose function and expression of vWF were evaluated via particle phagocytosis and immunohistochemistry method. Atlas cDNA expression array was used for analysis of cytokine expression in mYS-EC. RESULTS: Colonies consisting of pure yolk sac endothelial cells were obtained in liquid culture system containing 15% FBS and 10% mBMEC-CM or 5?g/L VEGF, ECGF and bFGF. For complete purification of the endothelial cells, subsequent passage was also necessary. Cellular cord formed during passage culture. The endothelial cells were round or oval sharp in morphology, positive in phagocytosis and factor VIII related antigen (von Willebrand's Factor,vWF). The mRNA expressions of cytokines, such as TGF-?2, TNF-?, IFN-?, FL, BMP-4, MIP-1?, BMP-2A, FLT2, endothelin 2, thymosin ?10, IL-6, IL-13, IL-9, SCYA5 and ACBP were detected in mYS-ECs. CONCLUSION: mYS-EC was purified and expanded in vitro . The mRNA expression of 15 kinds of cytokines was detected in mYS-ECs by Atlas arrays.

Ubiquitination on target proteins is the signal of cellular protein degradation.Ubiquitin ligase E3 is one of the key enzymes in ubiquitination,it recognizes a specific substrate protein and recruits an ubiquitin conjugating enzyme E2,mediating the ubquitin transfer from the E2 to the substrate protein.Ubiquitin ligase E3 can be divided into two subfamilies according to their different structure characters and function mechanisms,the HECT(homologous to E6AP C terminus) family and the RING-finger family.Members of the HECT E3 share the common HECT catalytic domain,which can bind to an E2 and load the ubiquitin on themselves before catalyzing the transfer of ubiquitin to the target proteins.While the RING-finger E3 all contain an similar E2 binding domain and a unique substrate binding part,mediating direct ubiquitin transfer from the E2 to the substrate.The most recent progresses in the stuctural and functional studies of these two E3 famlies were summarized.

Objective To investigate antifungal activities of AMB, ICZ, VRC, CBF against 72 strains of filamentous fungi in vitro. Methods Based on CLSI M38-P and M38-A scheme, MIC of antifungal drugs were determined. The growing inhibitory concentration of 100%, 100%,≥80%, for AMB, VRC ,ICZ act as respective MIC. For caspofungin, the minimal effective concentration (MEC) was determined as the lowest drug concentration showing morphology change of filaments. The fractional inhibitory concentration (FIC) was used to evaluate the effect of combination therapy. FIC was calculated by the following equation: FIC = MICcombination/MICA drug alone+ MICcombination/MICB drug alone. Results MIC90 of AMB, ICZ, CBF, VRC against 72 isolates of filamentous fungi were 8 μg/ml, 4 μg/ml, 2 μg/ml, 8 μg/ml, respectively. MICs range of combined AMB + ICZ, AMB + VRC, ICZ + VRC were 0. 125-16. 97, 0. 2452-1.25, and 0.0625-8. 25 μg/ml respectively. The percent of synergistic interaction of AMB + VRC against filamentous fungi (20.0%-88.9% ) was higher than those of AMB + ICZ ( 10.0% -62.5% ) and ICZ + VRC ( 20.0% - 44.4% ) ( P=0.007 <0.05 ). Conclusions The antifungal activities of four kinds antifungal drugs against 72 strains of filamentous fungi vary in vitro. The therapy of AMB combined with VRC is maybe better than AMB + ICZ and ICZ + VRC for severe fungi infection.

AIM: To find out the gene structure and diversity of protate specific membrane antigen(PSMA) alternative spliced variants, and probe into the pathogenesis of prostate cancer.METHODS: 5'-RACE and 3'-RACE methods were used to amplify the 5' and 3'end of alternative spliced variant and then those viariants were sequenced for analyzing the gene stucture and diversity of PSMA alternative spliced variants of prostate cancer tissues.RESULTS: Four new alternative spliced variants of PSMA were discovered from prostate cancer tissues.Compared with reported PSMA alternative spliced variants,different insertions and deletions existed in different sites of those new variants.CONCLUSION: The discovery of the new variants confirms the diversity of PSMA spliced variants and provides the clues for seeking the target of diagnosis and therapy of prostate cancer.

Objective:To study the correlation of β-fibrinogen-854G/A,-455G/A,-249C/T,-148C/T,448G/A and BcI-1G/A polymorphisms, functional expression of plasma fibrinogen concentration, molecular reactivity, and the type of cerebral infarction.Methods: A casecontrol study was used to analyze 54 patients with main-think cerebral infarction(MCI), 106 patients with penetrating-arterial cerebral infarction (PCI)and 160 heathy cases as control group in Kailuan Hospital between July 2002 and June 2003.Gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Fg concentration, fibrin monomer polymerized velocity(FMPV), absorbance maximum(Amax), FMPV/Amax and biochemistry factors including TG were measured, Results: Fg concentration, FMPV, FMPV/Amax in the MCI group and TG, VLDL and FMPV in the PCI group were higher than in the control group(P＜0.05).The frequencies of854A and Bcl-1A alleles had significant difference among three groups,and the frequencies of GA and AA genotypes in the MCI and PCI groups were higher than in the control group(P＜0.05), however, no different genotypes and allele frequencies of the remaining sites were found in the three groups(P＞0.05).Fg concentration and FMPV of allele T carriers in the MCI group were less than that of-249C/C homozygous ones(P ＜0.05); FMPV/Amax of allele T carriers in the PCI group was higher than that of-148C/C homozygous ones(P＜0.05);with allele A carriers, Fg concentration of control group and FMPV of PCI group were higher than that of Bcl-1 wild homozygote(P＜0.05).Conclusion: Bβ-249 C/T polymorphism in the 5-flanking promoter region can influence the expression of plasma FMPV, Bβ-148 locus is the main regulation location of Fg molecular conglomerate function.Bcl-1 locus in the 3-flanking region is an important gene regulator of plasma Fg concentration, moreover,people with its mutated genotypes are susceptible to MCI.The abnormal plasma Fg concentration, FMPV/Amax and FMPV simultaneously are important risk factors for MCI, and only abnormal FMPV and TG are prone to PCI.

Objective:To study the influence of thrombus aspiration combined tirofiban on patients with acute ST seg-ment elevation myocardial infarction (STEMI)after primary percutaneous coronary intervention (PCI).Methods:A total of 98 patients,who received primary PCI because of STEMI in our hospital from Jan 2012 to Mar 2013,were selected.They were divided into thrombus aspiration group (n=48,received pure thrombus aspiration)and com-bined treatment group (n = 50,received thrombus aspiration combined intracoronary tirofiban injection during PCI).Coronary angiography (CAG)instantly after PCI and follow-up condition during hospitalization and six months after discharge were compared between two groups.Results:(1)Compared with thrombus aspiration group after PCI,there were significant rise in TIMI blood flow grade [(2.3±0.6)grades vs.(2.7±0.3)grades],per-centage of TIMI flow grade 3 (72.9% vs.90.0%)and ST segment regression >50% rate within 90min after PCI (52.1% vs.74.0%),P < 0.05 or < 0.01,and significant reduction in percentage of postoperative no-reflow (18.8% vs.4.0%,P =0.038)in combined treatment group in hospital;(2)After six-month follow-up,left ven-tricular ejection fraction (LVEF)of combined treatment group was significantly higher than that of thrombus aspi-ration group [(58±6.3)% vs.(51±5.6)%,P <0.05].Conclusion:Thrombus aspiration combined tirofiban can effectively reduce coronary thrombus burden and improve cardiac function in STEMI patients during primary PCI.

Objective: To improve the economic and technical indicator’s evaluation system and the essential medicines’ centralized bidding procurement practice in China. Methods:By using the literature analysis, comparative analysis and field survey, we collected and analyzed the implementation plans and regulations for the essential medicines’ centralized bidding procurement in 30 provinces. Results: The quality level classification lacks in preci-sion. The economic and technical indicator’s concentration grade is low, the score and content in each indicator un-reasonably fluctuates in different provinces and these indicators are of low efficiency in bond with their structures for the drug quality evaluation. The quality level indicator lacks in the distinction degree and the government’s unreason-able interference exists in competition. Conclusions and suggestions: The quality levels’ indicator type and number should be simplified. The economic and technical indicators’ function, content, score, weight value and the structure should be normatively and scientifically set to improve the efficiency during the drug quality evaluation and the gov-ernment should strive to play their role in the market.

Objective:The paper aims to provide recommendations for improving the essential medicines’ cen-tralized bidding procurement and quantity-based pricing policy. Methods: Based on the documents and literature on essential medicines’ centralized bidding procurement, we analyzed the factors which have a great impact on implemen-tation of the quantity-based pricing in essential medicines’ centralized procurement using the text research, semi-structured interview questionnaire and on-phone interviews. Results:The quantity-based pricing needs to define a ge-neric name and specific dosage form of drugs in the essential medicines’ centralized procurement. Its implementation was mainly influenced by the following factors:the procurement area accessibility, the pharmaceuticals category, dis-ease and drug alternative procurement methods and cycle, the payment and settlement time, and irregularities in the procurement process. Suggestions:During this implementation, we also need to clearly predict the quantity and pro-curement method, set up a proper policy environment for a quantity-based pricing, cancel the price linkage mecha-nism, strictly put into practice the payment deadline, employ a unique billing method and strengthen the information construction for the provincial centralized procurement platform. Some medicines’ quantity-based pricing should be carried out in the chosen pilots for laying a good foundation for its promotion.

Objective]To discuss the effect of sciatic nerve injury on the expressions of tumor necrosis factor-alpha(TNF-α), interleukin-1β(IL-1β)and interleukin-10(IL-10)in anterior cingulate cortex(ACC),and further to explain their roles resided in the development of neuropathic pain.[Method]With use of the methods of behavioral test,western blot and immunohistochemistry, we examine the effects of spared sciatic nerve injury(SNI)on the expressions of TNF-α,IL-1β,and IL-10 in ACC,and observe the effect of the neutralizing antibody of TNF-α,IL-1β on the rat mechanical allodynia.[Result]In present experiment ,SNI increased the protein levels of TNF-α,IL-10,but not IL-1β in ACC. Increased TNF-α-IR and IL-10-IR in ACC is located in neurons ,but not astrocytes and microglia at 7 d following L5-VRT. Pre-treatment with anti-TNFα antibody but not anti-IL-1βantibody into ACC significantly increased the rat paw withdrawal threshold to von Frey hairs.[Conclusion]These data suggested that the increased TNF-αin ACC neurons might be responsible for the development of neuropathic pain.

Objective To study the inhibitory effect of fluorouracil combined with DDP on the growth of human osteosarcoma cell line MG-63,and to explore its influence on the expressions of transient receptor potential vanilloid 5 (TRPV5 )and transient receptor potential vanilloid 6 (TRPV6 )proteins.Methods The MG-63 cells were cultured by the density of 5 × 104 mL-1 .Fluorouracil group,DDP group,fluorouracil+ DDP group and control group containing 10% FBS were set up.The inhibitory rates of growth of MG-63 cells at different time were detected by CCK-8 assay.The apoptosis of MG-63 cells after treated with different drugs was determined by Hoechst staining Kit.The immunocytochemical staining was used to treatent to detect the expressions of TRPV5 and TRPV6 before and after treatment.Results Fluorouracil and DDP both inhibited the growth of MG-63 cells in a time-and dose- dependent manner.There were a lot of black particles in the MG-63 cells and the cells were smaller,aging or death when they were exposed to fluorouracil or DDP.Compared with 24 h group,the inhibitory rates of proliferation of MG-63 cells after treated with the sigle drug of fluorouracil or DDP for 48 and 72 h were increased significantly (P <0.05).Compared with control group,the apoptotic rates of MG-63 cells in fluorouracil group and DDP group 24,48,and 72 h after treatment were increased (P < 0.01)in a time-dependent manner. The expression levels of TRPV5 and TRPV6 in MG-63 cells 72 h after treatment of fluorouracil and DDP were decreased significantly compared with before treatment (P < 0.05 ). Conclusion Fluorouracil, DDP and fluorouracil combined with DDP could significantly inhibit the proliferation of MG-63 cells,induce the apoptosis, and decrease the expression levels of TRPV5 and TRPV6.