Autophagy is a metabolic pathway that widely presents in eukaryotic cells through the lysosomal mechanism to degrade its components. Autophagy regulates cell death not only by activating classic autophagosomal-lysosomal pathway, but also by influencing the occurrence and development of apoptosis and necrosis. Currently, the effect of autophagy in neuronal injury after acute cerebral ischemia/hypoxia and its specific mechanisms remain unclear. Studies have demonstrated that the autophagy after ischemia/hypoxia has a neuroprotective effect, such as maintaining neuronal homeostasis and reducing neuronal death; but other studies have also suggested that autophagy may aggravate neuronal injury after ischemia/hypoxia by activating multiple pathways, and even induce neuronal death.

Objective To study the transformation mechanism of triterpenes in processing of Alisma orientalis. Methods The triterpene transformations of A. orientalis pre and post-processing were comparatively analyzed by techniques of HPLC and Packed Column Supercritical Fluid Chromatography (SFC). Results In baked processing (70 ℃) of A. orientalis, little alisol B 23-acetate was transformed into alisol A 24-acetate and alisol B.However, more alisol B 23-acetate was transformed into alisol A 24-acetate and alisol B, then both of them were further transformed into alisol A in processing under high temperature (160-200 ℃). Conclusion Transformation of alisol B 23-acetate has two routes when A. orientalis is processed under high temperature: For one, alisol B 23-acetate is rearranged into alisol A 24-acetate which could be deacetylated into alisol A; for the other; it is deacetylated into alisol B first, then transformed into alisol A.

Objective To observe the aging,apoptosis,cell cycle arrest and oxidative stress in human skin fibroblast(HSF)induced by UVB,and to detect the expression profiles of p66Shc,a determinant of oxidative stress response and life span,in this process.Methods HSF cells were exposed to UVB at a subcytotoxic dosage twice a day for three days.The cells without exposure served as control.After another 24-hour culture,SA-β-Gal staining was performed to evaluate the senescence state of the cells,flow cytometry to observe cell apoptosis;cell cycle arrest was detected by serum starvation and flow cytometry:ELISA was applied to detect intracellular levels of superoxide dismutase(SOD)and malondialdehvde(MDA),and Western blotting to analyze the expression of p66Shc protein.Results The percentage of cells positive for SA-β-Gal staining increased from 0 to 98.3% after UVB radiation,which strongly suggested an aging state of HSF cells.The percentage of apoptotic cells increased from 0.96% to 37%.and 80.07% of the HSF cells were arrested in G0/G1 phase following the irradiation.Intracellular SOD activity decreased from(52.35±4.97)ng/g to(7.81±0.68)ng/g(P＜0.01).while intracellular MDA was found to increase from(3.52±0.34)ng/g to(33.91±3.20)ng/g(P＜0.05).The p66Shc protein was found to be weakly expressed in HSF in 24 hours following the exposure to UVB,and a stronger expression was noted 48 hours later.Conclusions HSF cells are induced into a state of senescence associated with oxidative stress after UVB irradiation,which may be applied as an in vitro model in aging research.The expression of p66Shc is increased in HSF during this process,and further studies are needed to explore the relation between p66Shc and oxidative stress as well as cellular aging.

Objective To study the process of removing bacterial endotoxins by ultrafiltration technology in dextran 40 injection. Methods Dextran 40 solution was ultrafiltrated by 100,200,and 300 kDa aperture ultrafiltration membranes with composite, PES and PVDF materials. In order to optimize ultrafiltration process,the content of effective component and endotoxins were detected by HPLC and kinetic-turbidimetry,respectively,and the change of particle size distribution in dextran 40 solution was analyzed before and after ultrafiltration. Results The transmittance of dextran 40 was close to the same MWCO and different membrane material. When MWCO reached 300 kDa,the transmittance was above 91%,which met the requirement of filtration. The endotoxin removal rates by 100-300 kDa composite ultrafiltration membranes were more than 99%. But the endotoxin removal rates of both of PES and PVDF membranes were less than 40%,which were unable to guarantee the removal efficiency of the endotoxin in dextran 40 solution. The particle size declined after ultrafiltration by 300 kDa composite membrane, and level of the insoluble particles decreased. Conclusion The 300 kDa composite ultrafiltration membrane can effectively remove endotoxin in dextran 40 solution with less main components loss. The material can meet requirements for producing dextran 40 injection.

Objective To investigate the presence of type 2 diabetes dyslipidemia,and the association with HDL levels and vascular complications in diabetes.Methods Of 524 cases of type 2 diabetes,fasting blood lipids,blood glucose,glycated hemoglobin results,blood basic data,the level of lipid controlling and based data of the patients were observed.The relevance between abnormal blood lipids and vascular atherosclerosis was analyzed.Results Type 2 diabetic patients have lipid metabolism,in 524 type 2 diabetes patients,58.4％ had lipid disorder;The serum level of HDL in atherosclerosis group[(0.8 ±0.26)mmol/L]was significantly lower than that in non-atherosclerotic group[(0.95 ± 0.43)mmol/L](t =1.648,P ＜ 0.05).Conclusion The decreasing of HDL had relationship to vascular atherosclerosis in patients with type 2 diabetes.

Objective To construct the eukaryotic vector that expressing hepatitis B virus (HBV) S and the single fragment of variety chain (ScFv) of monoclonal antiboy against cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4) and to analyze the immunological activity of recombinant S-ScFv protein.Methods The oringially constructed pSect2/ScFv4F10 and pSect2/S were double enzyme digested by Sfi I and Hind Ⅲ,respectively.Then the HBV S gene was cloned into the pSect2/ScFv4F10 vector.The pSect2/ScFv4F10 and pSect2/S-ScFv4F10 were expressed in Chinese hamster ovary (CHO) cells,and the expressed proteins were verified through sodium dodecyl sulfatepolyacrylamide gelelectrophoresis(SDS-PAGE)andWesternblotting.Afterultrafiltration concentration and affinity chromatography,the biological affinity of the expressed ScFv4F10 and S-ScFv4F10 proteins were examined by competitive enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology.The comparison between groups was done by One-way ANOVA.ResultsThe eukaryotic expression vector of pSect2/S-ScFv4F10was successfully constructed,and relative molecular mass of the expressed protein of S-ScFv4 F10 was about 52 000 that analyzed by SDS-PAGE and Western blotting.With the fixed concentration of 4F10-mAb against CTLA-4,the A570 value of the mixed reaction with purified CTLA-4 antigen gradually increased with the decrease of ScFc fusion protein proportion; when the molar ratio of ScFv,S-ScFv4F10∶4F10=2∶1,the competitiveinhibitionratesagainst 4F10conjugatedantigenwere72.6％and64.5％,respectively.The affinity constants of association kinetics for CTLA-4 mAb,ScFv4F10 and S-ScFv4F10 with CTLA-4 antigen were 7.29 × 108 mol/L,9.52 × 106 mol/L and 2.04 × 106 mol/L,respectively,and the dissociation constants of KD were 1.40 × 10-9 mol/L,1.05 × 10-7 mol/L and 4.91 × 10-7 mol/L,respectively.ConclusionsThe eukaryotic expression vector of pSect2/S-ScFv4F10is successfully constructed,and the recombinant protein of S-ScFv4 F10 has a fairly high affinity with CTLA-4 antigen.

Objective To analyze the association between mtDNA mutations and photodamagc after ultraviolet B (UVB) irradiation. Methods Primary human skin fibroblasts (HSF) and primary human epi- dermal keratinocytes of adult (HEKa) were irradiated by sub-lethal doses of UVB thrice a day for 4-5 days. Thereafter, genomic DNA was extracted from irradiated cells and conventional PCR was applied to detect the frequency rates of 4977 bp and 3895 bp mtDNA deletion. To quantitatively analyze the mutation levels, SYBR Green real-time PCR method was performed. Results In both cell lines, the frequency rates and relative copy number of deletions increased with the cumulative doses of UVB exposure (P<0.05). The prevalence rate of 3895 bp deletion peaked 53.3% and and relative copy number reached (49.63±4.38)×10-5, showing a more intense response to the accumulation of UVB radiation than 4977 bp deletion. In HSF, the minimum cumu- lative dose of UVB radiation was 150 mJ/cm2 for the induction of 3895 bp deletion, and 200 mJ/cm2 for the induction of 4977 bp deletion. It seemed that mtDNA deletion was more readily to be induced by UVB radia- tion in HSF than in HEKa. Conclusions The development and accumulation of mtDNA mutation are intimately related with cumulated UVB dose received by skin cells, and the 3895 bp deletion is more reliable in moni- toring the photodamage caused by UV than 4977 bp deletion. Therefore, the 3895 bp deletion may serve as a biomarker for the detection of photodamagc in skin cells. HSF appear to have an increased susceptibility to UVB radiation, which results in a higher frequency and level of mtDNA mutations compared with HEKa.

Objective To study the pharmacokinetic features of silibinin from Jiqi Injection(JI) in Beagle dogs,and to observe the effect of Panax notoginseng saponins,another active component in JI,on pharmacokinetics of silibinin. Methods The Beagle dogs received intravenous injection of JI and silibinin,and then its plasma sample was collected in different time. The plasma samples of Beagle were prepared by hydrolysis with sulfatase-? glucuronidase complex enzyme and liquid-liquid extraction with aether. A high-performance liquid chromatographic method was developed to determine the plasma concentration of silibinin,and the pharmacokinetic parameters were performed by BAPP2.3 program. Results The pharmacokinetics of two tested preparations met with two-compartment model. There were not significant differences between pharmacokinetic parameters of JI and that of Silibinin Injection. Conclusion The silibinin in Jiqi Injection has a fast in-vivo clearance rate after intravenous injection,and Panax notoginseng saponins have no effect on its pharmacokinetic parameters.

Objective To explore the value of echocardiography in the diagnosis of anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) in children.Methods The echocardiographic images of 14 patients with ALCAPA confirmed by operation and 18 patients with endocardial fibroelastosis (EFE) diagnosed by clinical were compared and analyzed.Results Both ALCAPA and EFE exhibited obvious dilated left ventricle,decreased left ventricular systolic function,thick endocardium and mitral regurgitation of different degree.The former additionally showed dilated right coronary artery(RCA) with normal origin,left coronary artery(LCA) emerging from the root or wall of the pulmonary artery(PA),the retrograde flow into PA in LCA and abundant collateral vessels in myocardium.However the later exhibited normal diameter of LCA and RCA and no collateral vessels.Conclusions Color Doppler echocardiography not only demonstrates left ventricular systolic function,endocardium,mitrial regurgitation and collateral vessels in myocardium,but also shows the origination and courses of LCA clearly,which provide exact informations to diagnose ALCAPA.The echocardiography can be used as a powerful tool of ALCAPA diagnosis and preoperative assessment.