Objective:Making single chain antibody(scFv)-alkaline phosphatase(Ap)fusion protein.Methods:An expression vector pSTE2-C66-Ap was constructed by sequentially inserting the Ap coding region into plasmid pSTE2-8E5 and replacing the VH-VL fragment of 8E5 by VH-VL fragment of C66.The fusion protein scFvC66-Ap was expressed in E.coli.and analysed by SDS-PAGE and immunoblotting.Results:Have obtained the scFvC66-Ap fusion protein with a molecular weight of 75 kD.It bound a 60 kD molecule from KG1a cell proteins on immunoblotting membrane detected directly by Ap enzymatic activity.Conclusion:A method permits the production of scFv-Ap conjugates in E.coli.which can replace conventionally prepared Ap-labeled antibodies in immunoassays.

Objective To investigate the relationship between insulin level and mild cognitive impairment(MCI) of type 2 diabetes and analyze the risk factors for MCI. Methods We collected 101 type 2 diabetes subjects who were divided into two groups:MCI and normal cognition(NC).All subjects were evaluated with cognition scores of MMSE,GDS,CMS,ADL.Insulin,glycosylated hemoglobin,blood routine test,blood pressure,ECG,brain CT were detected.Independent t-test,correlation and multiple regression analysis were performed. Results 1. The diabetic patients with MCI showed the higher levels of insulin at fasting, 2hr and 3hr after meal than did NC(P

Objective:To investigate whether the IgG from patients with antiphospholipid syndrome(APS) is capable of stimulating blood monocytes to express tissue factor(TF) activity and to explore the mechanisms possibly concerned.Methods:Freshly isolated peripheral blood monocytes were cultured in media containing purified APS IgG or normal IgG or RP-1 [a monoclonal anti-?_2 glucoprotein I(?-?_2GPI)antibody] and/or other agonists and the TF activity on monocytes was investigated by measuring factor Ⅶa-dependent generation of factor Xa.Results:APS IgG as well as RP-1 significantly increased monocytes TF activity,comparing to normal IgG;exogenous ?_2GPI enhanced the effects as the intact IgG molecule.Conclusion:That certain antiphospholipid antibodies,mainly anti-?_2GPI antibodies,induce monocytes TF activity in an antigen-specific manner,not via Fc receptor pathway,is contributed to the thrombotic diathesis in APS.

Objective:To establish bacterial endotoxin test for brozopine. Methods: Interference pre-test and interfering factors test were conduced on 3 batches of samples from 2 manufacturers to confirm the applicability of bacterial endotoxin test and the non-in-terfering concentration. The bacterial endotoxin test was carried out based on the method described in the second part of Chinese Phar-macopeia (2010 edition) and relevant standards and guidelines. Results: The three batches of brozopine showed no interference in bacterial endotoxin test at the concentration less than or equal to 2. 5 mg·ml-1 . The bacterial endotoxin test of the three bathes of samples all met the requirements. Conclusion:Bacterial endotoxin test can be used for the quality control of brozopine.

AIM:To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms .METHODS: CFs were cultured using myocardial tissue with dry method .Hypoxic environment was established for CFs by continuous nitrogen supplement .Type I and type III collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents .The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining .RESULTS:Un-der hypoxic condition , TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-de-pendent manner in the CFs .At the concentration of 5μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01).RXR agonist 9-cis-retinoic acid (9-cis-RA;10 -9 ~10 -6 mol/L) decreased TGF-β1 (5μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic con-dition.The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01).Smad2 inhibitor ( 20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF -β1 under hypoxic condition.Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly in-creased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05).CONCLU-SION:Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition .

Objective To investigate the impaired characteristics of working memory in Parkinson's disease patients with depression and the recovery status of working memory after antidepressant treatment.methods Totally 60 cases were enrolled in this study and divided into three groups:30 Parkinson's disease patients with depression (PD-D),15 Parkinson's disease patients without depression (PD) and 15 persons without Parkinson's disease as normal controls.All subjects were evaluated with Wechsler Memory Scale (WMS-R) audio-visual span,Tower of Hanoi (TOH) and Wisconsin Card Sorting Test (WCST).30 PD-D patients were randomly divided into two subgroups:routine treatment subgroup (8 weeks levodopa therapy) and antidepressant combined treatment subgroup (8 weeks levodopa + citalopram therapy).The evaluation of Hamilton Depression Scale (HAMD),WMS-R audio-visual span,TOH and WCST were performed on these two groups before and after treatment.Result s① There were significant differences comparing PD-D and PD groups with the normal control group in scores of WMS-R,TOH and WCST indicators (P<0.05).There were significant differences in WMS-R visual span,accuracy rate and speed of TOH,as well as the percent errors and percent perseverative responses of WCST comparing PD-D group(11.88±5.91,0.420±0.345,0.408±0.334,0.882±0.253,0.565±0.229) with PD group (15.87±5.21,0.768±0.167,0.634±0.232,0.493±0.161,0.327±0.122) (P<0.05).② Before and after treatment in PD-D routine treatment subgroup,there were significant differences in digital memory and audio-visual memory of WMS-R((6.73±3.72,5.95±3.13) vs (3.77±2.16,1.91±1.58)),accuracy rate of TOH(0.45±0.26 vs 0.23±0.13),as well as percent errors((-0.58±0.17) vs (-0.37±0.14)),percent perseverative responses((-0.32±0.15) vs (-0.14±0.09)),percent conceptual level responses(0.38±0.09 vs 0.13±0.07) and number of categories completed(3.27±1.56 vs 1.06±0.91) of WCST(P<0.05).③ After 4-week treatment and 8-week treatment,there were significant differences in HAMD score comparing PD-D antidepressant combined treatment group(16.33±2.72,10.27±2.66) with PD-D routine treatment group(21.73±2.28,18.4±2.47) (P<0.05).Conclusion Comparing Parkinson's disease patients without depression,the impaired working memory is more serious and extensive in Parkinson's disease patients with depression.Antidepressant treatment can improve the working memory of Parkinson's disease patients with depression.

Case teaching method was used to undergraduates’specialized courses of clinical laboratory medicine such as the clinical laboratory hemotology and basic clinical laboratory medicine. This teaching method achieved satisfactory effect in clinical ability culture and obtained the positive opinion of students.

Objective:To detect the effect of EphrinA1-Fc on the phosphorylation of EphA2 and extracellular signal-regulated ki-nase (ERK) in 786-O renal carcinoma cells (RCCs). Methods:The soluble ligand EphrinA1-Fc was used to inhibit the 786-O RCCs in vitro. Western blot analysis was used to examine the phosphorylation of EphA2 and ERK1/2 in the 786-O RCCs at different time points. Results:After the intervention with EphrinA1-Fc for 5, 10, 30, and 60 min, the expression of p-EphA2 increased (F=9.392, P=0.025) as well as that of p-ERK (F=4.428, P=0.041). No p-EphA2 and p-ERK expression was observed in the pre-intervention group. Conclusion:One of the possible mechanisms of the inhibitory effect of EphrinA1-Fc on tumor metastasis and recurrence involves the phosphorylation of EphA2 by EphrinA1-Fc, leading to the degradation of EphA2.

Objective To study the methylation status of secreted frizzled-related protein (SFRP) 1 and SFRP2 genes in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) and the relationship between the methylation status of the two genes and the development of HCC.Methods Using methylation-specific polymerase chain reaction (MSP) to detect methylation status of SFRP1 and SFRP2 genes of 45 specimens of HCC tissue and adjacent non-tumorous liver tissue from HCC patients during operations,and 6 normal liver tissues from patients with cholecystolithiasis or hepatic hemangiomas. The data were analyzed by chi-square test and Fisher exact test. Results SFRP1 gene methylation was detected in 28 HCC tissues and 16 adjacent non-tumorous liver tissues,accounted for 62.2% and 35.6%,respectively;and SFRP2 gene methylation was detected in 23 HCC tissues and 13 adjacent non-tumorous liver tissues,accounted for 51.1% and 28.9%,respectively;while no methylation was detected in 6 samples of normal liver tissues. There was no significant difference between the methylation of SFRP1 and SFRP2 genes in HCC tissues and gender,age,HBV serum markers,types of adjacent non-tumorous liver tissues,metastasis and pathological stage (P＞0.05).The abnormal methylation status between SFRP1 and SFRP2 genes was linear correlated in HCC tissues (r=0.381,P=0.01).Conclusion Hypermethylation of SFRP1 and SFRP2 genes frequently occurs in HBV-related HCC,which may be an important molecular biomarker for prediction of hepatocarcinogenesis in the future.

Objective To detect the integration of hepatitis B virus (HBV) DNA in HBVrelated human hepatocellular carcinomas (HCC). Methods Extracted DNA from the liver tissue samples and amplified by nested polymerase chain reaction (PCR) with specially designed U-base primers. According to the known genes and human Alu repeat sequences (Alu repeat) , primers were designed respectively. Integrated clones combined target HBV DNA and the adjacent cell gene sequences were established by PCR and products were sequenced by biotechnology companies.Accurate locations of HBV genes integrated in the human genomes were analyzed by national center for biotechnology information (NCBI) basic local alignment search tool (BLAST) and Map Viewer search. Results In 24 HBsAg positive HCC samples, 15 cases showed the integrations of HBV fragment. And the other 8 samples didn't show any evidence of integration. Among 14 samples with integration, forward insertions of HBV DNA into the host chromosomal DNA were found in 10 samples and reverse insertions were found in 8 samples while both forward and reverse insertions were found in 5 samples. Analysis from viral-cellular junctions suggested that the integrations were all happened with truncated viral DNA and could be in any locus of X gene. Conclusion HBV DNA integration is not distributed evenly throughout the host genome.