Objective To elucidate the expression of Toll-like receptor 3 (TLR3) on dendritic cells(DCs) in patients with chronic hepatitis B (CHB), and to explore the correlation between hepatitis B virus (HBV) persistent infection and TLR3 expression. Methods Sixty CHB patients (CHB group) and 20 healthy controls (control group) were enrolled. The peripheral blood mononuclear cells (PBMCs) were isolated and CD14~+ monocytes were sorted by immunomagnetic beads. Immature DCs (imDC) were induced and proliferated in vitro and mature DCs (mDC) were obtained after the poly I:C stimulation. The expression of intracellular TLR3 mRNA was detected by real-time polymerase chain reaction (PCR), and surface markers [CD80 and human leucocyte antigen (HLA)-DR] were determined by flow cytometry after 48 h of stimulation. The comparison of quantitative data was done using t test. The qualitative data were compared using chi-square test.Results The mean fluorescence intensities (MFI) of intracellular TLR3 of imDC before poly I:C stimulation in CHB group and control group were 1212.05 ± 250.80 and 1192.95 ± 301.40,respectively, which were not significantly different (t = 0. 280, P>0. 05). While after stimulation,those were 1352.98± 313.67 and 1593. 00± 349. 65, respectively, the latter was significantly higher than the former (t = 2. 880, P<0. 05). The levels of TLR3 mRNA inside mDCs in both groups were increased after poly I:C stimulation, which were 0. 1204 ±0.0267 and 0. 1780 ± 0.0664, respectively in CHB group and control group, and that in control group was significantly higher (t = 3. 909, P<0.05). Furtherly, patients in CHB group were divided into HBeAg(+ ) and HBeAg( -) subgroups.After stimulation, the MFI and mRNA of TLR3 inside mDC were greatly elevated in both subgroups,but there were no difference between these two subgroups (t = 0. 366, P>0. 05). Conclusions The intracellular expressions of TLR3 in mDC in CHB group and control group are obviously increased after the poly I:C stimulation, but the increased level in CHB group is lower than that in control group. The results suggest that the insufficiency of TLR3 synthesis may be related to the HBVpersistent infection.

Objective To detect the expression of type Ⅰ interferon in monocyte-derived dendritic cells(MoDCs)after Toll like receptor(TLR)3 triggered in patients with chronic hepatitis B(CHB),and to evaluate immune responses of CHB patients and its roles in the mechanisms of persistent infection of hepatitis B virus(HBV)and chronicity of hepatitis.Methods Peripheral blood mononuclear cells(PBMCs)were isolated and purified using magnetic beads(plasma was saved simultaneously)from 26 CHB patients and 18 healthy volunteers(HV).Dendritic cells(DCs)were induced and proliferated in a culture medium with recombinant human granulocyte macrophage colony stimulating factor(rhGM-CSF)and recombinant human interleukin(rhIL-4).EX3s were stimulated with Poly Ⅰ:C and the supernatants were collected at 0 h and 24 h after stimulation.Type Ⅰ interferon(IFN-α and IFN-β)in plasma and supernatants were examined by enzyme linked immunosorbent assay (ELISA).Results The levels of type Ⅰ interferon in plasma were not significantly different in groups of HV and CH B.IFN-α and IFN-β expressions in supernatants before Poly Ⅰ:C stimulation were(80.00±16.15)ng/L,(36.39±13.90)ng/L in CHB group and(76.76±15.90)ng/L,(37.14±13.68)ng/L in HV group,respectively.And there were no statistical differences between two groups(t=1.651,t=0.178;both P＞0.05).IFN-α expressions in supernatants at 24 h after stimulation in two groups were both higher than those before stimulation(at 0 h),but there were no statistical differences(t=1.534,t=1.243;both P＞0.05).IFN-β expressions in supernatants at 24 h after stimulation in HV group was(54.57±16.80)ng/L,which was significantly higher than that at 0 h(37.14±13.68)ng/L(t=4.061,P＜0.05).However,there was no significant difference at 24 h than tht at 0 h in CHB group(t=1.796,P＞0.05).At 24 h after stimulation.IFN-β level was(54.57±16.80)ng/L in HV group,which was significantly higher than that[(41.64±12.57)ng/L]in CHB group(t=2.921,P＜0.05).Conclusions Functions of MoDCs from CHB patients are impaired and MoDCs could not express type Ⅰ interferon normally.Expression of type Ⅰ interferon after TLR3 triggered in CHB patients is mainly IFN-β.

Objective To investigate the biological properties of human amniotic mesenchymal stem cells (hAMSCs) which were preconditioned with phosphodiesterase-5 inhibitor (Vardenfil). Methods hAMSCs were in vitro isolated and cultured, hAMSCs were pre-treated with vardenfil in final concentration of 10 μmol/L. The morphology of Vard-hAMSCs was observed, and the immunological characteristics, proliferative capacity, and ability of anti-oxidative damage of hAMSCs and Vard-hAMSCs were analyzed by flow cytometry. Double labeling immunofluorescent staining was used to count the differences of differential potential between neural cells of hAMSCs and Vard-hAMSCs. Results (1)Flow cytometry revealed that both hAMSCs and Vard-hAMSCs positively expressed CD90、CD105 and CD73, and negatively expressed CD34、CD45、CD11b and HLA-DR. The SPF and PI in Vard-hAMSCs group were (0.57 ± 0.40)% and (2.20 ± 1.60)% respectively, there was no statistical significance compared with hAMSCs group; (2)After 4 hours treated by H2O2, the apoptosis rate in Vard-hAMSCs group were (7.67 ± 0.82)%,which were markedly lower than that in the hAMSCs group and specific blocker group; (3)Under the same induction condition, positive rates of MAP-2 and GFAP in Vard-hAMSCs group were (49.8 ± 6.42)%and (55.2 ± 6.10)% respectively detected by double labeling immunofluorescent staining, which were significantly higher than the control group. Conclusion The strategy that hAMSCs are treated with vandenfil can enrich the ability of anti-oxidative damage and the differential potential for neural cells in a certain time, and the morphology, immunological characteristics, proliferative capacity of Vard-hAMSCs have no significant change. It suggests that pre-treatment with vandenfil may provide a optimized experimental strategey for hAMSCs which were used to treat nervous system disease.

Academic Inspection is not only an important part of clinical teaching,but also a vital tache of improvement for medical treatment quality.Unlike internal medicine and surgery,acdemic inspection of ICU has its own characteristics.PBL teaching method on acdemic inspection of attending doctor in ICU is explored in this article.

ObjectiveTo investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.MethodsThe hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10％ fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results( 1 ) The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.( 2 ) At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67％ ± 1.53％,75.00％:1.00％,and 74.33％ ±1.53％,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were ( 331.62 ± 1.76 ),( 330.50 ± 1.22 ),and ( 331.65 ± 0.48 ) μIU/ml,respectively,but non-induced groups were negative.(3) PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.( 4 ) Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin ( all P ＞ 0.05 ).ConclusionThe hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes.

Objective To evaluate left ventricular myocardial function of rabbit after ischemiareperfusion using speckle tracking imaging (STI),and to explore the myocardial protect effects of ischemia postconditioning (I-PostC) on reperfusion injury.Methods 24 Japanese rabbits were divided into ischemiareperfusion group (group Ⅰ) and I-PostC group (group Ⅱ) randomly.The characteristic changes of left ventricular global and regional myocardial strain and twist function of two groups were evaluated quantitatively by STI and compared with pathological results.Results ① Global longitudinal systolic strain rate(GLSrsys),global longitudinal systolic strain (GLSsys) and global longitudinal peak strain(GLSp) decreased in both groups,longitudinal systolic strain rate(SrLsys),longitudinal systolic strain(SLsys) and longitudinal peak strain(SLp) in the lateral wall of left ventricle decreased significantly and negative peak of SrLivr and PSI increased after the left ventricular branch of coronary artery was occluded;The values of Ptw and untwR of left ventricle were smaller.② After the artery was released,GLSp recovered in group Ⅱ,which was not seen in group Ⅰ.The values of SrLsys,SLsys and SLp increased significantly and negative peak of SrLivr and PSI decreased in group Ⅱ.However,only Slsys and SLp rebounded in group Ⅰ.Between the 2 groups,SrLsys andSLp in group Ⅱ were higher than that of group Ⅰ (P＜0.05 or 0.01),and SrLivr was lower compared with group Ⅰ (P ＜0.05).In group Ⅱ,Ptw and untwR changed back to a normal range (P ＜0.05 or 0.01),and no index has changed in group Ⅰ ;Between the 2 groups,Ptw and untwR in group Ⅱ were higher than that of group Ⅰ (P ＜0.05).③ The sensitivity and specificity of GLSrsys,GLSsys,SrLsys,SLsys and Ptw to detect rabbit myocardial infarction were 81.3 ％ and 75.0 ％,62.5 ％ and 81.2％,87.5％ and 87.5％,93.8％ and 75.0％,81.3％ and 68.7％ respectively.Conclusions STI may provide a promising approach to evaluate the global and regional myocardial function.It also can observe the protect effects of I-PostC on myocardial reperfusion injury accurately.

Objective To explore the expressions and clinical significances of deleted in liver cancer-1 (DLC-1 ) and Rho associated coiledcoil forming protein kinase (ROCK )Ⅰ in non-small lung cancer (NSCLC).Methods The expressions of DLC-1 and ROCKⅠ in NSCLC and adjacent tissue of 48 patients with pathologically confirmed as NSCLC and undergone surgical resection were detected by immunohistochemis-try EnVision method.The correlations among DLC-1 protein,ROCKⅠ protein and the clinical pathological characteristics were analyzed.The prognostic value of DLC-1 in patients with NSCLC was studied.Results The expression of DLC-1 protein in NSCLC tissue was low or missing,and the positive rate was 33.3%(16/48),significantly lower than that in the tissue adjacent to carcinoma 70.8% (34/48),with statistical significance (χ2 =13.523,P<0.01).The positive expression rate of ROCKⅠprotein in NSCLC was 58.3%(28/48),higher than that of tissue adjacent to carcinoma 0(0/48),with statistical significance (χ2 =39.529, P<0.01).The expression of DLC-1 protein was correlated with tumor differentiation,lymph node metastasis and clinical stage,rather than with sex,smoking history and organization type.Through the correlation analy-sis,the expression of ROCKⅠin DLC-1 positive group was 37.5%(6/16),and the expression rate of ROCKⅠ in DLC-1 negative group was 68.8%(22/32).There was negative correlation between DLC-1 and ROCKⅠin NSCLC tissues (r=-2.214,P=0.039).The 3 year survival rate in DLC-1 protein high expression group was obviously higher than that in low expression group,with statistical significance (P=0.043).Conclusion Low or missing expression of DLC-1 and high expression of ROCKⅠ protein may play an important role in the occurrence and development of NSCLC.Detecting the expression of DLC-1 and ROCKⅠprotein may be useful for evaluating the biological behavior and prognosis of NSCLC.

Objective To analyse the relationship between cytochrome C release and the opening degree of the permeability transition pore (PTP) during in post cardiopulmonary resuscitation(CPR) rats. Method Adult male Sprague Dawley (SD)rats were randomly (random number ) divided into a surgical sham group (no CA/CPR) ( n = 8) and CA/CPR group ( n = 48). Animals in CA/CPR group was induced by asphyxiation and icecold 0.5 mmol/L KCI and killed by decapitation and processed for isolation of brain cortex mitochondria at 3 h,6 h,12 h,24 h,48 h,72 h after restoration of spontaneous circulation (ROSC). MPTP opening degree was based on the absorbance changes of the mitochondrial suspension at 540 nm. Western blot analysed the release of CytC from mitochondrial to cytoplast. Results Neural cells MPTP always remained opening post ROSC. The opening degree of MPTP was not reaching the peak instantly. While its' change depended on time: remaining low level within 6 h post ROSC,then rapidly opening, till 12 h reaching the largest degree,and at 24 h post ROSC slightly shrunken. All these suggested that mitochondria started to shink. While at 48 h the opening degree largen again, shrunken once more at 72 h,but not reaching the normal level ( P ＜ 0.05 or P ＜ 0.01). Cytochrome C could not be detected in the cytosol in the earlier phases of the sham-operated brain samples but appeared in the mitochondria.The amount of cytochrome C released was proportional after restoration of spontaneous circulation 3 h and 24 h,failed to detect the enzyme in the mitochondria fraction. Conclusions We draw the conclusion MPTP plays a key role in neurocyte apoptosis after CA/CPR, which leads to mitochondria release CytC.

Objective To understand the immune regulatory function of monocyte-derived dendritic cells (MoDC) in patients with chronic severe hepatitis B (CSHB) and its roles in the severe illness progression of chronic hepatitis B (CHB) by detecting surface phenotype of MoDC and expression level of cytokines in MoDC after polyl : C treatment. Methods The peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient separation from 37 patients with CSHB, 20 patients with CHB, and 20 healthy controls (NC). Purified PBMC were acquired using immunomagnetic anti-CD14-beads. Then PBMC were induced to immature dendritic cell (iDC) in vitro. PolyI : C was added to induce DC maturation. The mean fluorescence intensity (MFI) of the phenotype marker molecules including HLA-DR, CD83, CD86 and CD80 on surface of iDC and mature DC (mDC) were detected by flow cytometry. The supernatants of MoDC culture were collected at 12,24 and 48 h after polyI : C treatment, respectively and the release levels of interleukin (IL)-12, IL-6and tumor necrosis factor (TNF)-α were determined by enzyme linked immunosorbent assay (ELISA). Comparisons among groups were done by single factor analysis of variance and homogeneity of variance was tested. Results There were no significant differences of phenotype marker molecules on cell surface of iDC, including HLA-DR, CD83, CD86 and CD80 in CSHB, CHB and NC groups.However, the expressions of HLA-DR, CD83, CD86 and CD80 on cell surface of mDC in CSHB group were lower than those in CHB and NC groups (F=59.73, 13.95, 34.80 and 73.02, respectively; all P＜0. 05). The secretions of IL-12 at three time points of 12 h, 24 h and 48 h after polyI : C treatment in group NC were higher than those in CHB and CSHB groups (F= 151.34, 126.65 and 72.76, respectively; P＜0.05), and peaked at 24 h which were (48.2±7.6), (56.7±11.8) and (97.8±16.2) ng/L, respectively. The secretions of IL-6 at the above three time points were CSHB＞CHB＞NC (F=92.50, 86.89 and 64.57, respectively; all P＜0. 05) and peaked at 12 h which were (1698.3±340.4), (965.8±231.7), (697.8±213.6) ng/L, respectively. The secretions of TNF-αat the above three time points were CSHB＞CHB＞NC (F=58.66, 122.36 and 44.73, respectively;all P＜0. 05) and were (19 672. 7±4214. 7), (9946. 1 ± 2586 5), (6659. 2±955. 8) ng/L,respectively at 24 h after treatment. Conclusions MoDCs of CSHB patients show mature defection and abnormal cytokine secretion. The expression level of IL-12 which mediates cellular immune is low.Meanwhile, the productions of IL-6 and TNF-α which mediate inflammatory response are up-regulated. This may be one of the major factors which lead to exacerbation of liver inflammation and ultimately development of severe hepatitis.

Objective To explore the safety and clinical efficacy of central venous infusions of concentrated potassium chloride corrects hypokalemia by micro-pump in intensive care unit(ICU). Methods The data was analyzed retrospectively on 78 patients with hypokahmia in ICU. They were randomly divided into 2 groups: general group (39 cases) with potassium concentration 40 mmol/L and the rate 10-20 mmol/h,concentrated potassium group(39 cases) with potassium concentration 100-300 mmol/L and the rate 40-200 retool/h, calculating the whole potassium dosage respectively, examining the initial potassium concentration, urine volume per hour, the mean time to aimed potassium concentration and side effect.Results The initial potassium concentration and the whole potassium dosage were no significant difference between the two groups [(2.9 ± 0.2), (3.0 ± 0.2) mmol/L and (85.2 ± 8.7), (92.3 ± 7.6) mmol, respectively](P >0.05). It took longer time reaching the aimed potassium concentration in general group than that in concentrated potassium group [(17.25 ± 4.49) hours and (5.67 ± 0.75) hours, respectively] (P < 0.01).There were no comphcations such as hyperkalemia, fatal arrhythmia and phlebitis. Five patients were bloating in general group. Condusions Under meticulous monitoring, it is effective and relative safely to correct hypokahmia by central venous infusions of concentrated potassium chloride using micro- pump in ICU. The therapy is of clinical value in treating hypokalemia patients.