Objective To investigate the clinical application value of fluorescence PCR in vitro diagnosis(IVD)reagent kits in the HLA-B27 detection by comparing 2 kinds of HLA-B27 IVD reagent kit approved by CFDA.Methods A total of 573 clinical blood samples were collected and detected for HLA-B27 by the approved reagent kits based on the fluorescence PCR technique and the flow cytometry.The samples with inconsistent testing results by the two kits were further confirmed by the PCR sequencing.At the same time,about 5% samples of the positive results detected by the fluorescence PCR method were extracted for conducting the re-testing.Results Among 573 samples,191 samples were HLA-B27 positive and 382 cases were HLA-B27 negative by flow cytome-try;the same samples had 194 cases of HLA-B27 positive and 379 cases of HLA-B27 negative by real-time PCR.With flow cytome-try as reference of the final results,the positive coincidence rate of the two kinds of kit was 96.33%(184/191),the negative coinci-dence rate was 94.76%(362/382),27 samples had inconsistent results from the two kinds of assay(accounting for 4.71% of the to-tal number of samples),the total coincidence rate was 95.29% [(184+362)/573],the Kappa value was 0.896(P =0.02);the chi-square test P =0.021,the two kinds of testing method had the high consistency,but the differences existed in the testing results. The re-testing results by PCR sequencing(including 27 samples with inconsistent results by two kinds of kit)were entirely consist-ent with the fluorescence PCR testing results.Conclusion Compared with the authority method flow cytometry for HLA-B27 tes-ting in clinic,the fluorescence PCR kit may present more accurate judging ability for the HLA-B27 testing on the basis of ensuring the higher consistency of the testing results,is easier compared with the sample preparation and operating procedures,and has the stronger clinical application value and prospects s.

Objective To investigate the relationship of TCM constitution and syndrome elements based on patients with hyperhomocysteinemia. Methods A survey was conducted among 1316 patients with diabetes, coronary disease, hypertension and cerebral infarction. Totally 326 patients with hyperhomocysteinemia were incorporated in diagnostic data and their constitutions were determined. At the same time, syndrome elements identification method for syndrome elements was applied for judgment, and consistency of the constitution and syndrome elements was analyzed. Results The survey showed that prevalence rate of population with hyperhomocysteinemia was 24.77%;there were 6 cases with mild nature, and 320 cases with biased constitution;phlegm dampness, blood stasis, qi deficiency of constitutions 110 cases, 73 cases and 59 cases, respectively;blood stasis, phlegm, qi deficiency, and dampness were the main syndrome elements of hyperhomocysteinemia, of 100 cases, 98 cases, 73 cases and 59 cases, respectively;the consistency of phlegm dampness constitution and syndromes of phlegm was the most obvious (Kappa=0.89, P<0.05). Conclusion Phlegm dampness, blood stasis, and qi deficiency were the most common constitutions of hyperhomocysteinemia;blood stasis, phlegm, qi deficiency and dampness were the main syndrome elements of hyperhomocysteinemia patients. The two results were consistent, and the consistency of phlegm syndrome and phlegm dampness constitution was the most obvious.

Objective To investigate the changes in cathepsin B (CatB) expression in acutely photodamaged human dermal fibroblasts (HDFs) and their significance.Methods HDFs were isolated from the foreskin of children,and subjected to primary culture and subculture.The fourth-to eighth-passage HDFs were used in the following experiment.HDFs were divided into two groups to receive irradiation with different doses of ultraviolet A (UVA) for different durations (acutely photodamaged group) or remain unirradiated (control group).Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferative activity of HDFs after irradiation with UVA at 5,10,15,20 and 25 J/cm2 respectively.Western blot and quantitative real-time reverse transcription PCR were performed to measure the protein and mRNA expressions of CatB respectively in HDFs at 24,48 and 72 hours after exposure to UVA at 10 J/cm2,and at 48 hours after exposure to UVA at 10,15,20 and 25 J/cm2.Statistical analysis was carried out by analysis of variance and least significant difference (LSD) test using the SPSS 13.0 software.Results UVA radiation induced a decrease in the proliferative activity of HDFs.When the dose of UVA was ≤ 10 J/cm2,the survival rate of HDFs maintained higher than 85％,and significant differences were observed in cell survival rate between unirradiated and irradiated HDFs at 24,48 and 72 hours (all P ＜ 0.05).Western blot showed that the gray value of CatB protein in the acutely photodamaged group irradiated with 10 J/cm2 UVA was significantly higher than that in the control group at 24 hours (0.76 ± 0.14 vs.0.35 ± 0.01,P ＜ 0.05),48 hours (1.34 ± 0.38 vs.0.45 ± 0.12,P＜ 0.05) and 72 hours (0.82 ± 0.09 vs.0.61 ± 0.06,P＜ 0.05).Increased mRNA expressions of CatB were also observed in the acutely photodamaged group compared with the control group at 24 hours (0.149 ± 0.009 vs.0.089 ± 0.015,P ＜ 0.05),48 hous (0.173 ± 0.009 vs.0.091 ± 0.010,P ＜ 0.05) and 72 hours (0.185 ± 0.158 vs.0.111 ± 0.017,P ＜ 0.05) after UVA radiation at 10 J/cm2.The gray value of CatB protein was 0.99 ± 0.07,1.49 ± 0.14,1.89 ± 0.08,2.07 ± 0.06 in HDFs at 48 hours after exposure to UVA of 10,15,20 and 25 J/cm2,respectively,significantly higher than that in the control group (0.60 ± 0.05,all P ＜ 0.05).Similarly,the mRNA expression of CatB was up-regulated in HDFs at 48 hours after UVA radiation at 10,15,20 and 25 J/cm2 compared with the unirradiated HDFs.Conclusion The protein and mRNA expressions of CatB are up-regulated in acutely photodamaged HDFs induced by UVA radiation.

AIM: To evaluate effects of Pinaverium Bromide on different segments and layers of colonic smooth muscle in wrap restraint stress (WRS) rats and explore its possible therapeutic mechanism on different types of irritable bowel syndrome (IBS). METHODS: Adult SD rats were randomly divided into model group (wrap restraint stress group) and control group. Colonic smooth muscle strips were made from different segments and layers in two groups. The spontaneous contraction activities of colonic longitudinal/circular muscle (LM/CM) strips of rats were observed with organ bath system before and after addition of series concentrations of pinaverium. RESULTS: Pinaverium Bromide caused concentration-dependent inhibition of colonic smooth muscle, the inhibitory effect of pinaverium in model group was significantly stronger than that in control group(proximal colon: 28.54±4.82 vs 7.48±1.65,21.75±1.00 vs 12.56±3.15; distal colon: 15.71±5.27 vs 3.89±1.16, 20.16±3.16 vs 7.56±1.96 )(P<0.05). Compared with that of distal colon, inhibitory effect of pinaverium was significantly higher of proximal colon (P<0.05). For the inhibition of pinaverium, there was no significant difference between LM and CM strips in the same intestinal segments (P>0.05). CONCLUSION: Effects of Pinaverium Bromide on different colonic muscle layers and segments in WRS rats is probably related with its therapeutic mechanism on different types of IBS.

Objective To explore the abnormally functional brain region in resting state in first-episode major depression disorder patients with function magnetic resonance imaging.Methods 51 patients diagnosed with first-episode major depression disorder according to DSM-Ⅳ and 50 gender-,age-,and education-matched healthy controls completed resting state fMRI scan.The severity of depression,and unpredicted homodynamic responses across the whole brain were analyzed using Hamilton depression scale,regional homogeneity and amplitude of low-frequency fluctuation,respectively.Results Compared with control group,the right medial frontal gyms (BA6,MNI:3,-3,63,K=34) and left medial frontal gyrus(BA9,MNI:-9,36,30,K=10) (P＜0.001,uncorrected) in the case group showed higher regional homogeneity,with statistical significance.Compared with control group,right medial frontal gyrus (BA6,MNI:3,-3,63,K =35) and right posterior cingulated gyrus (BA31,MNI:3,-36,36,K =11) (P＜ 0.001,uncorrected)in the case group showed higher amplitude of low-frequency fluctuation,with statistical significance.Conclusion First-episode major depression disorder patients in resting state had several abnormally functional brain regions,which might be related to the pathological mechanism of depression disorder.

ObjectiveTo investigate the effects of chitosan/PVA nerve conduits which used for repairing sciatics nerve defect in rats.MethodsTwenty-seven rats were divided into 3 groups randomly,with 9 rats in each group. Firstly, the 15mm defects in the left sciatic nerves were made in the rats and were respectively repaired with chitosan/PVA conduits graft (group A), the silicon conduits graft (group B),and autografts (group C). At 12 weeks after the operations, the left sciatic nerves were taken out, and the comparative evaluation was made on the repairing effects by wet weight of gastrocnemius and soleus muscles, histological examination,computerized imaging analysis and True Blue retrograde tracing. ResultsThe wet weight of gastrocnemius and soleus muscles showed no significant difference between the chitosan/PVA graft and autograft groups (P ＞ 0.05). The wet weight of gastrocnemius and soleus muscles in significant difference between the chitosan/PVA graft and the silicon group at 12 weeks after the operation(P ＜ 0.05). The nerve fiber density showed no statistically significant differences between the chitosan/PVA and autograft groups(P＞ 0.05).The regenerative nerve fiber in group B had normal morphological and structural characters under transmission electron microscope.True Blue-labeled neuron cell bodies were found within both anterior horn of gray matter in the spinal cord and dorsal root ganglions (DRGs) ipsilateral to the operated side of the tested rats on illumination with ultra-violet light 1 week after the injection of True Blue.Conclusion Chitosan/PVA nerve conduit can effectively promote the nerve regeneration and myelinization of rat sciatic nerve, which is expected to substitute for autograft to repair nerve defects succesfully.

Objective To evaluate the effect of isoflurane on the expression of matrix metalloproteinase 2 (MMP-2) in human tongue cancer cell.Methods Tea8113 cells at the logarithmic growth phase were seeded into 96-well plates (200 μl/hole) with the density of 5 × 103/ml,6-well plates (5 ml/hole) with the density of 1 ×105/ml,culture dishes (5 ml/dish) with the density of 5 × 105/ml or Transwell chamber (200 μl cell suspension for upper chamber with a density of 5 × 105/ml,10 ％ blood serum medium 500 μl for lower chamber).The cells wererandomly divided into 3 group (n =30 each):control group (group C),2％ isoflurane 2 h group (group Ⅰ1) and 2％ isoflurane 4 h group (group Ⅰ2).2％ isoflurane was added to the culture medium and the cells were incubated for 2 and 4 h in groups Ⅰ1 and Ⅰ2,respectively.The cell viability was detected by MTT assay.The cell apoptosis was assessed by flow cytometry.The migration and invasion of the cells were measured by cell wound scratch assay and Transwell chamber assay,respectively.The expression of MMP-2 and MMP-2 mRNA was detected by immunocytochemistry and RT-PCR,respectively.Results The cell viability and migration of cells were significantly increased,the apoptotic rate was decreased,and the expression of MMP-2 and MMP-2 mRNA was up-regulated in groups Ⅰ1 and Ⅰ2,and the invasion of the cells were increased in group Ⅰ2 as compared with group C (P ＜ 0.05).Compared with group Ⅰ1,the cell viability and migration and invasion of the cells were significantly increased,the apoptotic rate was decreased,and the expression of MMP-2 and MMP-2 mRNA was up-regulated in group Ⅰ2 (P ＜0.05).Conclusion Isoflurane enhances the migration and invasion of the cancer cells by up-regulating the expression of MMP-2 in human tongue cancer Tea8113 cells.

ObjectiveTo explore the effects and possible mechanisms of VCR combined with low dose cyclophosphamide(CTX) intermittently to treat severe systemic lupus erythematosus(SLE).It is assumed that this might be a new combination therapy for SLE and expected to improve the overall prognosis and outcome of SLE.MethodsMurine chronic graft-versus-host disease(cGVHD) model were developed for study.They were randomly divided into the control group,vincristine (VCR) pulse therapy group,CTX pulse therapy group,CTX every other day(EOD) group,VCR+CTX combination group.One way ANOVA and repeated measure variance analysis were used for statistical analysis.Results① Six weeks after cGVHD models were set up,the average 24-hour urine protein quantification was(5.02±0.88) mg,anti-dsDNA antibody was positive,and Ⅳ LN pathology could be observed histologically in the model murine.So cGVHD models were successfully developed.② Significantly difference in decreasing of 24-hour urine protein quantification was found in the CTX EOD group,VCR+CTX combination group and other groups (P＜0.01).Significant decrease in Cr,ALT,anti-dsDNA,was found in the CTX EOD group,VCR+CTX combination group,CTX pulse therapy group and other groups(P＜0.05).Decrease in urine MCP-1 and TGF-β1 could be detected,and statistical significant difference in these parameters could be found in the CTX EOD group,CTX pulse therapy group,VCR+CTX combination group and other groups (P＜0.01).MCP-1 and TGF-β1'expression in model kidney were reduced in the CTX EOD group,VCR+CTX combination group and had statistical significant difference in the CTX EOD group,VCR+CTX combination group,VCR pulse therapy group,and CTX pulse therapy group.③ VCRand CTX combination treatment was effectivein 24-hour urine protein quantification,blood Cr,ALT,anti-dsDNA and urine MCP-1,as well as urine TGF-β1 (P＜0.05).Conclusion ① The combination of VCR and CTX is synergistic in decreasing 24-hour urine protein quantification,Cr,and the expression of MCP-1,TGF-β1.② The adverse effect of VCR+CTX combination group is similar to VCR pulse therapy group and CTX pulse therapy group.

Objective To examine whether IFN-λ has the ability to inhibit HIV-1 infection of blood monocyte-derived macrophages and its mechanism(s). Methods Macrophages were pretreated with IFN-λ/ IFN-λ2 for 24 h before infected by HIV-1 R5 strains (Bal, Jago, and JRFL). And then the culture supernatants were detected HIV-1 reverse transcription (RT) activity and p24 protein expression by HIV-1 BT assay and ELISA. The expressions of IFN-λ receptor, CD4, CCRS, CXCR4 were evaluated by real-time PCR. Results Both IFN-λ1 and IFN-λ2, when added to macrophage cultures, inhibited HIV-1 infection and replication. This IFN-λ-mediated anti-HIV-I activity is broad, as IFN-λ could inhibit infection by both laboratory-adapted and clinical strains of HIV-1. Investigations of mechanism(s) responsible for the IFN-λ action showed that although IFN-λ had little effect on HIV-1 entry receptor CD4 and co-receptor CCR5 and CXCR4 expression, IFN-λ inhibited HIV-I infection of macrophages through connecting with IFN-λ recep-tor. Conclusion IFN-λ could inhibit HIV-I replication in macrophages. These findings indicate that IFN-λ may have a therapeutic value in the treatment of HIV-1 infection.

Objective To study the effect of inosine monophosphate dehydrogenase inhibitor (IMPDHI) on chemotaxis, migration and endocytosis of human peripheral myeloid dendritic cells (MDCs). Methods Freshly isolated peripheral blood mononuclear cells(PBMC)collected from healthy volunteers (N=15) and the study group were treated with IMPDHI. CC chemokine receptors on MDCs were analyzed by flow cytometry. The study group, control group and different chemokines were added via trans-well approach for different chemokines, stained by Lin-1/CD11c/HLA-DR and counted by flow cytometry. The migration index was calculated as a percentage of MDC migrated in response to the tested chemokine. After isolation of blood dendritic cell antigen-1+ (BDCA-1+ ), mannose receptor-mediated endocytosis was measured as the cellular uptake of FITC-dextran by the flow cytometry. Results (1) Compared to the control group, the expression of CCR1 in the study group was up-regulated significantly(17.02±3.23～30.63±9.13, P＜0.05) and the expressions of CCR3(10.26±2.25～5.81±0.97 P＜0.05) and CCR7 (9.56± 1.84～5.18±0.60 P＜0.05)were downregulated significantly. MDCs in the study group showed enhanced migratory response to inflammatory chemokine CCL2, CCL3, CCL4, CCL7 and CXCL12 (P＜0.05). (2)The endocytosis capacity in the study group was significantly higher than that in control group (P ＜0.05). Conclusion IMPDHI enhances the endocytotic capacity of MDCs and impairs the migratory response of peripheral MDCs to lymphocytic tissue by up-regulating the expression of chemokine receptor in MDCs and enhancing migratory response to inflammatory chemokines.