In this study, two expression vectors for recombinant human interleukin-6(rhIL-6):CSV-IL-6(1) and (2) were successfully constructed by routine gene manipulation, PCR amplification, sitespecific mutagensis, and optimionzation of translation. The constructed expression vectors for rhIL-6 were introduced into a simian cell line COS7 using gene transfer by DEAE-Dextran procedure. The expression level of rhIL-6 was determined by EL-6 ELISA using ELISA KIT for quantification of Human Interleukin-6, and the biological activity of rhIL-6 was assayed by 50% maximum incorporation of ~3H-TdR into 7TD1 cells, an IL-6-dependent hybridoma cell line. At 48h after transfection, the supernatant collected from the culture of COS7 cells transfected with CSV-IL-6 (1) contained rhIL-6 2573pg/ml, and at 72h after transfection it containde 1467pg/ ml; that for CSV-IL-6 (2)were 8261pg/ml and 7101pg/ml, respectively. The biological activity (hybridoma growth factor, HGF) at 48h after transfection from COS-CSV-TL-6 (1) was 10~4U/ ml corresponding to 3.9?10~9U/mg; and that from COS-CSV-IL-6 (2) was 6.3?10~4U/ml mcorresponeding to 7.6?10~9U/mg.

Dyslipidemia is one of the most important pathogenic factors of atherosclerotic cardiovascular disease.Detection of lipid and lipid metabolism related indicators is important in diagnosis and treatment for dyslipidemia,as well as risk assessment and prevention of cardiovascular disease.In recent years,with advances in technology and research,focuses on lipoproteins gradually extended from quantity changes toqualitative changes.The emphasis also turns to some lipid metabolism related proteins,susceptibility genes,DNA methylation,miRNA and lipidomics.Relevant researches have become a hot topic in this field.

Central nervous system ( CNS) injuries, such as cerebral ischemia, traumatic brain injury (TBI), and spinal cord injury (SCI), are often accompanied by complex pathological changes, and could lead to a variety of other neurological diseases.Neurons and glial cells are precisely regulated by many genes.MicroRNA ( miRNA ) are endogenous molecules discovered in recent years that regulate post transcriptional gene expression.They are highly expressed in the central nervous system and abnormal expressed under pathological conditions.They are involved in regulating variety of pathological processes after CNS injuries, and are CNS disease potential biomarkers.

OBJECTIVE:To study the spectrum-effect relationship of the antipyretic effect of Radix Bupleuri injection. METH-ODS:HPLC method was used to establish the fingerprint of Radix Bupleuri injection. The antipyretic effect of 10 batches of Radix Bupleuri injection on fever rats induced by dried yeast were determined respectively. The fingerprint peaks were screened,along with the temperature value of different time points(0,10,15,30,40,55,70 min). The principal components were extracted by principal component method and the indirect relationship between the fingerprint and antipyretic effect was analyzed,depending on the principal component correlation coefficient matrix. RESULTS:There were 39 common peaks in fingerprint(similarity>0.85), No. 8,12,14,19,26,31,34,35 and 39 common peaks with large peak area were included in the study. Four principal compo-nents were extracted by principal component analysis(87% of total variant). First principal component showed that the active com-ponents of the 12th peak may be related to the antipyretic effect of 6 to 13 hours. The second principal component showed that the active components of the 26th peak may be related to the antipyretic effect of 0.5 to 5 hours. The third principal component showed that the similar effect of the active components could be caused by 34th,35th and 39th peaks. The fourth principal component sug-gested that there were some similarities between the 14th and the 31st peaks. CONCLUSIONS:Radix Bupleuri injection have obvi-ous improvement for fever rats. There is certain corresponding relation between HPLC fingerprint and antipyretic effect of Radix Bu-pleuri injection .

Purpose To assess the feasibility of measuring maximal density of a region of interest (ROI) with contrast-enhanced CT in quantifying degrees of intestinal ischemia in patients with intestinal obstruction.Materials and Methods Abdominal CT images and reports of 160 patients with intestinal obstruction were retrospectively studied.All the data were reviewed by CT visual evaluation method and measuring maximal density of ROI respectively.The CT visual evaluation took the way of accumulated points,and divided the degrees of intestinal ischemia into five categories.The measuring maximal density of ROI quantified the degrees of bowel enhancement with a bar histogram on CT workstation.The results were compared with the pathological examination.The sensitivity,specificity,positive and negative predictive values and accuracy of the two methods were calculated respectively,and compared.Results The sensitivity,specificity,positive and negative predictive values and accuracy of CT visual evaluation method were 96.7％,72.9％,82.1％,94.4％ and 86.2％,respectively.The sensitivity,specificity,positive and negative predictive values and accuracy of the measuring maximal density of ROI were 68.8％,100.0％,100.0％,71.4％,82.5％,respectively.By measuring the area under the ROC curve,the ROI method (0.995) was more accurate than CT visual evaluation method (0.908) in the diagnosis of bowel ischemia.Conclusion Measuring the maximal density of ROI can quantize bowel wall enhancement.It is a reliable and useful method in the diagnosis of intestinal ischemia,and in accordance with pathological results.

Objective To investigate altered levels and clinical significance of serum miR-133a in patients with acute coronary syndrome ( ACS ) and stable coronary artery disease ( SCAD ) .Methods Retrospective study.Serum miR-133a levels were determined by TaqMan quantitative reverse-transcription PCR assay in 64 ACS, 62 SCAD patients who were admitted to Jinling Hospital from October 2011 to October 2012 and 70 normal controls who had contemporaneously visited Jinling Hospital for routine examination .The ACS and SCAD patients were diagnosed according to the European Society of Cardiology guidelines .Serum lipid/lipoprotein profiles , myonecrosis biomarkers and Gensini scores were also analyzed .The area under curve ( AUC) and 95%confidence interval ( CI) were calculated using ROC analyses .The odds ratio ( OR) and 95%CI were calculated using the multivariate logistic regression analyses .Results Compared with the controls [ΔCt:1.00 ±0.05], serum miR-133a levels were significantly increased in both ACS [ΔCt:2.34 ±0.24] (t=6.059, P<0.001) and SCAD [ΔCt:1.45 ±0.13] (t=3.265, P=0.001) patients.The miR-133a levels in ACS patients were significantly higher than in SCAD patients (t=3.133, P=0.002). Serum miR-133a were positively correlated with levels of creatine kinase MB ( CK-MB) ( r=0.402, P<0.001), cardiac troponin I (cTNI) (r=0.410, P=0.001) and Gensini scores (r=0.438, P<0.001). ROC curve analyses showed that the AUC of miR-133a for differentiating coronary artery disease (CAD) and controls was 0.717 (95%CI:0.645-0.788, P<0.001) and the AUC for differentiating ACS and SCAD was 0.667 (95% CI:0.573-0.761, P=0.001).Logistic regression analyses revealed that high miR-133a levels were closely associated with the presence of ACS ( OR=6.00, 95% CI:1.93 -18.67, P=0.002) and SCAD (OR=2.81, 95%CI:1.03-7.68, P=0.044), and also had statistical significance for differentiating ACS and SCAD (OR=2.13, 95% CI:1.20-3.78, P=0.010), after adjustment for the age, gender and serum lipid/lipoprotein levels.Conclusions Serum miR-133a levels were significantly elevated in CAD patients, and ACS patients exhibited the more significant increase .Serum miR-133a may be function as the potential biomarker for the disease assessment and judgement .

Objective:To map dominant antigenic determinants on SAK by gene-targeted fragmemt library . Methods:①The PcAbs specificly against SAK were produced by immunning BALB/C mice. Pu rified the antisera through a SAK-Sepharose 4B affinity chromatography column. The purified PcAbs were biotinylated for next step. ② After constructing SAK r andom epitope library we sequenced 12 isolated clones randomly to ensure its int egrity ,capability and randomness.③The library was screening by situ-clone hy bridization.④Constructed mSAK ,the A1 region deleted mutant of SAK ,and used Western-blot assay to identified its immunoreactivity. Results:①Got a dominant epitope at amino acid 71-89,called A1 region; ②Western-bl ot assay suggested that mSAK, a mutant SAK without A1 region., didn't combine to the anti-SAK PcAbs.Conclusion:A dominant epitope of SAK was mapped successfully with a simple,effective method . [

Objective To detect altered levels and clinical significance of serum miR-216a and prognosis monitoring in acute pancreatitis (AP) patients.Methods Serum miR-216a levels were determined by quantitative reverse-transcription PCR (qRT-PCR) assay among 80 mild acute pancreatitis (MAP) patients,80 severe acute pancreatitis (SAP) patients and 74 healthy controls.And amylase (AMY),lipase (LPS),Ca2+,glucose (Glu),hematocrit (HCT),triglyceride (TG),total cholesterol (TC) and C reactive protein (CRP) were measured by biochemical analyzer.The clinical usefulness of miR-216a for AP patients was assessed by ROC curve analysis and correlation analysis.Results Compared with healthy controls (11.12 × 10-5 [5.83 × 10-5,19.12 × 10-5],the serum miR216a levels were significantly increased in AP patients(38.49 × 10-5 [24.05 × 10-5,62.02 × 10-5],(U =-9.10,P ＜ 0.01) . The serum miR-216a levels in MAP and SAP patients were (36.46 × 10-5 [22.29 × 10-5,55.80 × 10-5] vs 40.44 × 10-5 [25.84 × 10-5,65.48 × 10-5]),there was no significant difference between MAP and SAP patients (U =-0.96,P ＞ 0.05).The areas under ROC curve (AUCROC) of miR-216a for differential healthy controls and AP patients was 0.870 (95％ CI:0.825-0.915),cut-off value is 0.61.AUCROC of miR-216a for differential healthy controls and MAP patients was 0.865 (95％ CI:0.808-0.921),cut-off value is 0.59.And AUCROC of miR-216a for differential healthy controls and SAP patients was 0.876 (95％ CI:0.822-0.930),cut-off value is 0.66.Moreover,after the clinical improvement of the patients,the levels of serum miR-216a were significantly lowered from (41.88 × 10-5 [24.24 × 10-s,64.44 × 10-5]) to (20.58 × 10-5 [11.01 × 10-5,41.91 × 10-5]),the differences was significant(U =5.24,P ＜ 0.0l).Correlation analysis showed that miR-216a was positively correlated with CRP (r =0.215,P =0.006) in AP patients.Conclusion The levels of miR-216a in serum of AP patients were increased,which is a potential biomarker for the diagnosis and prognosis monitoring of AP.

Objective To investigate the relationship between serum levels of homocysteine (HCy) and adiponectin (APN) in patients with type 2 diabetes mellitus(T2DM).Methods 65 patients with T2DM (diabetes mellitus group) and 25 healthy controls (control group) matched in the age and sex were recruited in the study.Serum HCy,APN,fasting plasma glucose (FPG),fasting insulin (FINS),total cholesterol (TC) and triglyceride (TG) were simultaneously measured.The homeostatic model assessment for insulin resistance(HOMA-IR) was calculated according to FPG and FINS.All the serum indicators were compared between the two groups.Results Serum level of HCy in T2DM group was (15.74 ± 2.76) μmol/L,which was significantly higher than (6.98 ± 1.94) μmol/L in the healthy control group (t =16.88,P ＜ 0.01).The serum level of APN in T2DM group was (8.14 ± 2.70) mg/L,which was significantly lower than (16.10 ± 1.93)mg/L in the healthy control group (t =13.44,P ＜ 0.01).Serum levels of FPG,HOMA-IR,TC,TG in T2DM group were significantly higher than those in the healthy control group (t =10.62,17.49,6.30,7.52,P ＜ 0.05).Serum level of APN in HCy ≥ 15μmol/L group was significantly decreased compared with HCy ＜ 15μmol/L group.Serum level of HCy was negatively correlated with APN in T2DM group after the influence of FPG,HOMA-IR,TG,TC were corrected in the partial correlation analysis.Conclusion In T2DM group,serum level of HCy was increased,but serum level of APN was decreased,serum HCy was negatively correlated with APN,higher serum level of HCy and lower serum level of APN are related with the process of insulin resistance and T2DM.

Objective To investigate the effect of human epidermal growth factor receptor-2 (HER2) silenced by lentivirus-mediated RNA interference (RNAi) on the growth of SKOV-3 ovarian cancer,and to explore the value of radioimmunoimaging in monitoring the biotherapy of RNAi.Methods The ovarian cancer cell line SKOV-3 was infected with lentivirus-mediated HER2-short hairpin (sh) RNA expression vector and scrambled control lentivirus vector,respectively.Both infected cells were inoculated into nude mice to establish two ovarian cancer xenograft models:knock down 1 (KD1) group and normal control (NC) group.The uninfected SKOV-3 xenograft model served as blank control (CON) group.The tumor formation rate,tumor generation time and tumor size at different time points were measured.The expression of HER2 protein was measured by immunohistochemistry.1n Ⅰ-Herceptin was injected before radioimmunoimaging,and the T/B ratios were acquired.One-way analysis of variance and the least significant difference (LSD)-t test were performed with SPSS 17.0.Results All mice models were constructed successfully (100％,15/15).The average time of tumor generation was (4.583±0.520) d,(4.567±0.284) d and (6.023±0.316) d in CON,NC and KD1 groups,respectively(F=13.946,P＜0.01).The tumor formation time of KD1 group was significantly longer than the other two groups (t=4.557,4.608,both P＜0.01),respectively.On the 28th day after the tumor cell implantation,the tumor size was significantly different among the three groups (F=26.343,P＜0.01).The tumor mass was (0.614±0.135) g,(0.558±0.190) g and (0.120±0.489) g in CON,NC and KD1 groups,respectively (F=225.026,P＜0.01).Both the tumor size (t=7.125,4.759) and tumor mass (t=19.158,16.977) of KD1 group were significantly less than those of CON and NC groups (all P＜0.01),respectively.Immunohistochemical results showed that the HER2 protein expression was inhibited in the KD1 group.The tumor could be visualized clearly on radioimmunoimaging at different time points.The T/B ratio of the KD1 group (0.208-4.427) was significantly lower than those of the other two groups at any intervals (0.576-5.508,0.640-5.695; F=9.197-39.375,all P＜0.05).Conclusions The growth of SKOV-3 could be inhibited remarkably by lentivirus-mediated HER2-siRNA.Radioimmunoimaging with 1nI-Herceptin might positively correlate with the expression of HER2 protein,which might have potential for monitoring the biotherapy of RNAi targeting HER2.