Objective:To investigate the role of mTOR in regulation of ICOS expression in human blood regulatory T cells. Methods:Isolation of Treg cells from human PBMC using MACS beads. We detected the ICOS expression on purified Treg cells and Treg cells viability using flow cytometry in anti-CD3 plus anti-CD28 ( antibody or beads) or anti-CD3 plus ICOSL-Fc for 3 days and 7 days. CFSE labeling human PBMC cells and in vitro cultured Treg mixed, Treg contact inhibition activity was detected by flow analysis. Results:After in vitro stimulation of Treg cells in the presence of anti-CD3+anti-CD28 for 3 days, there was no significant statistic difference in viability between ICOS+(92. 00±2. 69)% and ICOS-(90. 30±3. 53)% Treg-cells. After cultured for 7 days,the decreased ICOS+ Treg cells percentage within total Treg cells from ( 40. 20 ± 1. 83 )% to ( 11. 60 ± 1. 10 )% compared with that of 3 days. Further more,the ICOS expression level between stimulated with anti-CD28 or ICOSL-Fc condition group,compared with the ICOS MFI in the condition of anti-CD3 plus anti-CD28 treatment for 3 days was (2410. 0±746. 4) obviously higher than (403. 30±74. 42), that of the group treated with anti-CD3 plus ICOSL-FC. Rapamycin could partially suppress Treg cells ICOS expression,but unaffected the Treg suppression ability. Conclusion:ICOS expression level may not important for in vitro cultured human PBMC Treg cells survival although mTOR signling is important for regulation ICOS expression on in-vitro cultured Treg cells,but the ICOS expression on Treg regulated by multiply signaling pathways. CD28 signaling is the key stimulation factor for ICOS upregulation on in-vitro cultured Treg cells compared to ICOSL signaling.

Objective To investigate the relationship between values of blood calcium, serum urea nitrogen (BUN), D-dimer, C-reactive protein (CRP), fibrinogen and amylase with the severity of the disease in patients with acute pancreatitis (AP). Methods There were 70 patients with mild AP (MAP group), 18 patients with moderate AP (MSAP group), 26 pa?tients with severe acute pancreatitis (SAP group) in 114 AP patients. The laboratory indexes were compared between these groups. The correlation between indexes and the acute physiology and chronic health evaluation systemⅡ (APACHE Ⅱ) score was analysed. The diagnostic sensitivity of SAP using CRP, D-dimer and fibrinogen was analysed by ROC curves. Re?sults Compared with MAP group, values of BUN, CRP, D-dimer,fibrinogen and APACHEⅡscore were significantly increased in SAP group (P<0.05), but serum calcium level was significantly decreased (P<0.05). The APACHEⅡscore were significantly higher in SAP group than that of MSAP group (P<0.05). There were no significant differences in level of amylases between three groups. There was a positive correlation between APACHEⅡscore, CRP, D-dimer and fibrinogen (r=0.407, 0.404 and 0.245, P<0.05). There was a negative correlation between APACHEⅡscore and serum calcium level (r=-0.333, P<0.05). The area under the ROC curve showed a maximum CRP curve for diagnosing SAP 0.752 (95%CI=0.644-0.860). The cut-off value was 74.45 mg/L. The sensitivity was 86.4%. And the specificity was 68.2%. Conclusion Combining with monitoring BUN, blood coagulation index, CRP, serum calcium level and other laboratory parameters was useful to overall evaluate AP patients and improve the prognosis.

Purpose To prepare streptavidin-tagged mouse interleukin-4(mIL-4-SA)bifunctional fusion protein and to study on its bioactivity.Methods The mIL-4 gene was cloned by RT-PCR and cloned into pET21 vector to get mIL-4-SA-pET21 expression plasmid.The mIL-4-SA fusion protein was expressed in BL21 (DE3)host bacteria and purified through the Ni-NTA affinity chromatography and refolded by dilution and dialysis.The effect of mIL-4-SA fusion protein on mouse thymocytes proliferation was evaluated by MTY.Flow cytometric analysis was performed to detect the mIL-4-SA fusion protein on the biotinylated B16F10 tumor cells.Results The mIL-4-SA-pET21 vector was successful by constructed and the mIL-4-SA fusion protein was expressed in BL21(DE3)at about 35%of total bacterial proteins.The purity of mIL-4-SA Was about 95% through Ni-NTA.The mIL-4-SA fusion protein exhibited bifunctional activities,i.e.,stimulative effect for mouse thymocyte proliferation and SA-mediated high-affinity binding to biotinylated cell surfaces(anchoring modified rate Was about 96.69%).Conclusion The mIL-4-SA fusion protein was expected to be developed for the treatment of tumors.

AIM： On the basis of in vitro study， a preliminary survey of the stability of diclofenac lysine sustained-release tables(DLST) was carried out to identify the period of validity.METHODS： To observe the changes of DLST under high temperature， blaze， high humidity and room temperature(for long period) and to perform accelerating test for 3 months.RESULTS： The drug release was accelerated under high temperature， and the tablet was apt to absorb moisture because of its hydrophilic matrix and the drug release became faster at day 10 under blaze， and therefore DLST should be stored away from high temperature， high humidity and blaze.CONCLUSION： DLST is stable and the period of validity is tentatively fixed at 2 years.

Objective To compare the clinic significance of four clinical scoring systems in evaluating prognosis of acute pancreatitis: bedside index for severity in acute pancreatitis(BISAP), acute physiology and chronic health evaluation (APACHEⅡ), Ranson’s scoring system, computed tomography severity index (CTSI) in AP. Methods Patients visited our clinic with AP (n=114) in recent 2 years were retrospectively analyzed. BISAP and APACHEⅡscores were obtained at 24 hours after admission; Ranson ’s score was obtained at 48 hours after admission and CTSI are obtained was obtained at 72 hours after admission. Results of four scoring system were compared under different causes and different severity of the dis?ease. Correlation between BISAP score and the other three scores were analyzed and the predicative value of all four scoring systems for severity of AP and death were also compared. Results The mean values of four scoring systems show no signifi?cant difference in AP patients with different etiology (P>0.05). The BISAP score is positively correlated with APACHE-Ⅱ, Ranson ’s score and CTSI score (P<0.01). The four scoring systems all present good predictive value on the severity of AP and death (P<0.01). Conclusion The four scoring systems can all be applied to grading and prognosis for AP of various causes. BISAP is a simple, prompt, economical scoring system in clinical practice.

To establish a method to detect cardiac troponin I by using time-resolved fluoroimmunoassay ( TRFIA) and apply to the clinic.Methods:The assay were measured by TRIFA and double antibody sandwich method .Standard protocols were evaluated with the standard curve , the limit of detection , stability, precision and cross reaction .Healthy reference populations and clinical serum specimens were measured to established the reference interval and evaluated the perspective of the clinical application . Results:The standard curve was Y=7485 .878+1400.924 X with a correlation coefficient of 0.999.The limit of detection was 0.052 ng/ml.The intra-and inter-assay coefficients of variation ( CV) were all <10%.Reference values was <0.14 μg/L.The AUC of ROC curve was 0.971 while the sensitivity was 96.45%,the specificity was 91.43% and the accuracy was 95.69%, with 98.45% of positive predictive value and 82.05%of negative predictive value.The correlation coefficient was 0.993 between our proposed method and the commercially available CLIA kits.There was no significant difference in statistics compared with ECG , CK-MB and cTnT ( P>0.05 ).There was significant difference in statistics compared before and after treatment with AMI ( P<0.001 ) .Conclusion: The TRFIA method for detecting cTnI achieves clinical application standards and may be used for the diagnosis and serosurveillance of acute myocardial infarction patients.

AIM:To investigate the correlation between matrix metalloproteinase-9(MMP-9),tissue metalloproteinase inhibitor-1(TIMP-1),MMP-9/TIMP-1 and carotid atheromatous plaque stability in cerebral infarction patients.METHODS:80 patients with cerebral infarction were categorized as microemboli-negative group(n=70)and microemboli-positive group(n=10),20 normal human were served as control group.The MMP-9 and TIMP-1 levels in plasma were determined by mean of ELISA in 3 groups.RESULTS:The levels of MMP-9 and TIMP-1 in plasma were significantly higher in cerebral infarction patients than those in control group(P

We discussed the function of four pairs of genes in the toxin-antitoxin system of Mycobacterium tuberculosis,providing theoretical foundation and scientific basis for studying the transmission mechanism of Mycobacterium tuberculosis.Four pairs of genes which belong to VapBC family,including four VapC genes (Rv1720c,Rv2103c,Rv2494,Rv3408) and four VapB genes (Rv1721c,Rv2104c,Rv2493,Rv3407) were chosen.We constructed a serial of arabinose-induced hybrid plasmid system in Escherichia coli and a serial of acetamide-induced hybrid plasmid system in Mycobacterium smegmatis respectively,in order to observe the potential inhibition effect of VapC and the release inhibition of homologous VapB.Results showed that only one toxin gene(Rv2103c) showed the function of bacteriostasis in both E.coli and M.smegmatis and the homologous antitoxin gene(Rv2104c) could release the inhibition of growth.We built the inducible systems of VapBC family in both E.coli and M.smegmatis respectively and found only a pair of toxin and antitoxin genes(Rv2103c,Rv2104c) had the function of inhibition and release for the growth of bacteria.And two pairs of toxin genes(Rv1720c,Rv2494) did not have the function of inhibition for the growth of both E.coli and M.smegmatis.Whereas,another toxin gene VapC47(Rv3408) also did not have the bacteriostastic activity,only this result was not consistent with the existing literature.We speculated that the reason for this kind of difference may be the different inducible systems we used.Cause the other three results were consistent with all existing literature and the doubtful result also appeared in other reports,so our protocol could be confirmed as reliable,and we would use it to build inducible systems and make further functional identification of certain toxin and antitoxin genes that we are interested in.

Objective To investigate the alteration and significance of inflammatory factors in hypertension patients complicated with acute myocardial infarction(AMI).Methods Eighty seven AMI patients were grouped as normotensive group(n=43) or hypertensive group(n=44),25 healthy subjects served as control.Plasma level of IL-1?,IL-6,IL-8,TNF-? and IFN-? were determined at admission of hospitalization,24 h,48 h,5 d,7 d and 14 d after myocardial infarction using ELISA method.Results 1)Compared with the control group,the level of all inflammatory markers were elevated significantly in 2 weeks after myocardial infarction and showed a pattern of dynamic changes.2)Compared with normotensive group,IL-6 in hypertensive group was increased more significantly on 5th day(P=0.019) to 7th day(P=0.005)after AMI.Conclusion Inflammation was involved in the course of AMI since the early stage.Hypertension seems to exaggerated the inflammatory reaction after myocardial infarction.

Objective To establish a novel method by integrating immunomagnetic bead enrich-ment with immunochromatography for the detection of influenza A virus. Methods The immunomagnetic beads were prepared by using EDC/NHS method and then coupled with monoclonal antibodies against influ-enza A virus. A direct immunomagnetic beads-based immunochromatography for the detection of influenza A virus was developed by using double-antibody sandwich method and immunochromatography, which was fur-ther combined with immunomagnetic separation to establish the novel integrated method of immunomagnectic bead enrichment and immunochromatography. Clinical throat swab samples collected from patients with influ-enza A virus infection and healthy subjects were analyzed by the novel method and the results were compared with those by using the conventional colloidal gold immunochromatography to evaluate the specificity, sensi-tivity and positive coincidence rate of this established method. Results The direct immunomagnetic beads-based immunochromatography and the colloidal gold immunochromatography showed no significant differences in specificity and sensitivity and could be used to identify influenza A virus-positive samples with cycle threshold ( Ct) values less than or equal to 22 obtained by real-time PCR assay. The integrated method could identify positive samples with Ct values less than or equal to 28, indicating that the novel method was more sensitive. Conclusion The novel method by integrating immunomagnetic bead enrichment with immunochroma-tography was successfully established and suitable for the rapid and on-site detection of influenza A virus.