Objective To compare the accuracy of stroke volume variation (SVV),central venous pressure (CVP) and puhnonary arterial wedge pressure (PAWP) in monitoring the changes in blood volume in the patients undergoing renal transplantation.Methods Sixteen patients with chronic renal failure,of American Society of Anesthesiologists physical status Ⅱ or Ⅲ,aged 18-55 yr,scheduled for elective allograft renal transplantation under general anesthesia,were enrolled in the study.SVV was continuously monitored with the FloTrac/Vigileo monitor,and CVP,PAWP and stroke volume index (SVI) were continuously monitored with the volumetric pulmonary artery catheter during surgery.The parameters of hemodynamics were recorded at 30 min after induction of anesthesia,5 min before renal artery opening,5 and 30 min after renal artery opening,and at the end of surgery.Hydroxyethyl starch 130/0.4 electrolyte solution 6 ml/kg was infused over 15 min via the central venous catheter to perform fluid responsiveness starting from 30 min after induction of anesthesia.Positive fluid responsiveness was defined as the change in SVI ≥ 15％.The relationship between SVV and CVP and between SVV and PAWP was analyzed using the Pearson correlation analysis.The receiver operating characteristic curve for CVP,SVV and PAWP in monitoring the changes in blood volume was drawn,and the area under the curve was calculated.Results Compared with the value at 5 min before renal artery opening,SVV was significantly increased after renal artery opening (P＜0.05),and no significant change was found in CVP and PAWP after renal artery opening (P＞0.05).SVV was negatively correlated with CVP,and r=-0.82 (P＜0.01);SVV was negatively correlated with PAWP,and r=-0.77 (P＜0.01).The area under the curve of SVV in monitoring the changes in blood volume was 0.87,and of CVP and PAWP was 0.69 and 0.66,respectively.Conclusion SVV provides better accuracy than CVP and PAWP in monitoring the changes in blood volume in the patients undergoing renal transplantation.

Objective To investigate the pathologic mechanism of radiofrequency ablation ( RFA ) combined with intravenous infusion of thermosensitive liposome encapsulated vinorelbine (TL-Vin) in treating liver tumors, and to analyze the effect of combination therapy on the long-term survival rate. Methods H22 liver adenocarcinoma tissue was subcutaneously implanted into ICR mice to establish the animal models. At the first experimental period, 40 mice were randomly and equally divided into 5 groups to receive different therapeutic scheme (using different TL-Vin concentrations). Twenty-four hours after the treatment the tumor specimens were collected, the necrotic areas were measured separately, and the optimal TL-Vin concentration was determined. At the second experimental period, 13 mice were randomly selected to receive treatment. Half an hour after the treatment the tumor tissues were collected and the TL-Vin concentration within the tumor was determined. At the third experimental period, 32 mice were randomly and equally divided into 4 groups, and 90 days after treatment the tumor growth curve was drawn. The survival rate was compared between each other of the groups. Results Compared with pure RFA group, TL-Vin + RFA significantly increased tumor coagulation extent (P<0.01). But free-VIN+RFA had similar tumor necrotic extent as that produced by RFA alone (P>0.05). Tumor coagulation area in TL-Vin + RFA group was bigger than that in free-VIN + RFA group at the concentration of 10 mg/kg [(341.8 ± 65.4)mm2 vs (225.3 ± 25.4)mm2, P < 0.01]. In TL-Vin group the coagulation margin was clear. The mean intratumoral Vinorelbine accumulation in TL-Vin + RFA group was 10 folds of that in free-Vin group [(1 156.5 ± 158.3)ng/ml vs (194.5 ± 52.3)ng/ml, P = 0.005]. TL-Vin +RFA had better survival result than that of RFA alone (37.6 ± 20.1 days vs. 23.4 ± 5.0 days, P=0.015), as well as than that of free-Vin + RFA [(37.6 ± 20.1)days vs (23.3 ± 1.2)days, P = 0.016]. Conclusion Thermosensitive liposomal chemotherapies (Vinorelbine) can be selectively delivered at the edge of RFA coagulation area and thus effectively increase RFA-induced tumor coagulation and prolong the end-point survival in experimental mice.

Objective To study the quantity change and significance of myeloid‐derived suppressor cells(MDSCs) in patients withpost‐traumaticmultipleorgandysfunctionsyndrome(MODS).Methods 66patientswithMODS,34patientswithnon‐system‐ic inflammatory response syndrome(SIRS)and 37 healthy volunteers were enrolled in this study .Peripheral blood was collected and CD14-CD11b+ CD33+ were used as markers of MDSCs .The percentage of MDSCs was determined by flow cytometry and serum interleukin‐10(IL‐10) and tumor necrosis factor‐α(TNF‐α) levels were determined by ELISA .The MODS scoring system was used to assess patients′disease severity .The relationship was analyzed between MDSCs and TNF‐αand MODS score .Results The per‐centage of MDSCs in peripheral blood of healthy volunteers was(1 .18 ± 0 .22)% .after MODS ,the percentage of MDSCs in periph‐eral blood was(11 .84 ± 2 .18)% and(6 .52 ± 0 .37)% in patients with non‐MODS ,the percentages of MDSCs in three groups showed significant differences(P<0 .05) .Serum IL‐10 and TNF‐αin patients with MODS group and non‐MODS group were signif‐icant differences(P<0 .05) .The correlation was found between MDSCs percentage in peripheral blood and MODS score and TNF‐α(r=0 .342 6 ,0 .387 9 respectively ,P<0 .05) .Conclusion The increase proportion of MDSCs in peripheral blood correlates with the onset of infection in patients with MODS ,indicating that the expansion of MDSCs in peripheral blood may play important roles in immune dysfunction after MODS .

Objective To study the activation of alveolar macrophage β (AM) and the expression of co-stimulatory molecule CD40 in transfusion-related acute lung injury (TRALI) model in order to illustrate the pathogenesis of TRALI.Methods Sixty SD rats were randomly (random number) divided into normal control group (n =15) with sham operation using normal saline instead of LPS and plasma,positive control group (n =15) with ALI induced by intravenous infusion of 5 mg/kg lipopolysaccharide (LPS) in equivalent volume of whole blood drawn out),and TRALI group (n =15) treated by intra-peritoneal injection of 2 mg/kg LPS 2 h before the transfusion of human plasma (1 mL whole blood about 10％ of total blood volume drawn out and replaced with 1 mL plasma),LPS control group (n =15) treated by intra-peritoneal injection of 2 mg/kg LPS 2 h before saline infusion in equivalent volume of blood drawn out.The pathologic changes of rat lung tissue were observed by HE staining.The expression of TLR4 was examined by RT-PCR.The activation of NF-κB in AM was measured by electrophoresis mobility shift assay (EMSA).The expression of CD40 mRNA and CD40 molecule were analyzed by Northern blot and flow cytometry respectively.ELISA was performed to detect the concentration of TNF-α,MIP-2 and IL-1 β in broncho-alveolar lavage fluid (BALF).Results Broken alveolar septa,hyperemia,and massive infiltration of inflammatory cells including the neutrophils were observed in lung tissues of TRALI group.The expression of TLR4 gene was detected in activated macrophage phi (AMφ) of TRALI group rats.The activation of NF-κB was increased in TRALI group rats.The expression of CD40 in AMφ was higher in rats of TRALI group than that in rats of control group and LPS control group.The concentration of TNF-αt,MIP-2 and IL-1β were enhanced significantly in BALF of TRALI group rats.Conclusion The activation of AM and up-regulation of costimulatory molecule CD40 induced release of some inflammatory cytokines.It suggested that AM activation may play an important role in the pathogenesis of TRALI.

Objective To estimate the activity of the phosphatidylinositol3-kinase (PI3K) signaling pathway in peripheral blood T cells from patients with atopic dermatitis (AD),and to investigate its clinical significance.Methods T cells were isolated by using the Rosettsep T cell purification kit from the peripheral blood of 38 patients with AD and 38 healthy human controls,and classified into several groups to be treated with anti-CD3 monoclonal antibody,anti-CD28 monoclonal antibody,and LY294002 (an inhibitor of PI3K) respectively.The activity of PI3K signaling pathway in T cells was estimated by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA).Western blot was performed to measure the expressions of total Akt and phosphorylated Akt in T cells,methyl thiazolyl tetrazolium (MTT) assay to examine the proliferation of T cells,and ELISA to determine the levels of interleukin 6 (IL-6) and IL-10.Results The activity of PI3K and Akt was significantly higher in freshly isolated patient-derived T cells than in control-derived T cells (both P ＜ 0.05).However,the difference in the activity of PI3K and Akt between patient-derived and control-derived T cells disappeared (both P ＞ 0.05) after 24-hour in vitro culture.The activity of PI3K and Akt in control-derived T cells was significantly increased after 24-hour incubation with sera from the patients with AD (both P ＜ 0.05).In addition,compared with patient-derived T cells treated with patients' sera or anti-CD3/CD28 monoclonal antibody alone,those treated with the combination of LY294002 and patients' sera or anti-CD3/CD28 monoclonal antibody showed a significant decrease in the proliferative activity (63％ ± 11％ vs.123％ ± 25％,125％ ± 22％ vs.195％ ± 28％,both P＜ 0.05),supematant levels of IL-6 ((168 ± 33) vs.(265 ± 46) ng/L,(431 ± 64) vs.(823 ± 128) ng/L,both P＜ 0.05) and IL-10 ((56 ± 14) vs.(98 ± 25) ng/L,(120 ± 21) vs.(213 ± 35) ng/L,both P＜ 0.05).Eczema area and severity index (EASI) was unassociated with the activity of PI3K or Akt in fresh T cells from patients with AD (both P ＞ 0.05).Conclusions The PI3K signaling pathway is abnormally activated in peripheral blood T cells from patients with AD,which is associated with the proliferation of,as well as secretion of cytokines by,T cells,suggesting that there exist serum factors activating this pathway in peripheral blood of patients with AD.

Objective To investigate the effects of miRNA-146a on the differentiations and functions of CD4+ T lymphocytes in patients with psoriasis vulgaris.Metbods Thirty patients with psoriasis vulgaris and twenty heathy subjects were enrolled in this study.Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of miRNA-146a in CD4+ T lymphocytes isolated from the peripheral blood samples.The levels of IFN-γ and IL-4 in serum samples were determined by using enzyme-linked immunosorbent assay (ELISA).Mononuclear cells were isolated from human peripheral blood samples by using Ficoll-Hypaque density-gradients centrifugation,from which the CD4+ T lymphocytes were separated by magnetic-activated cell sorting.The CD4+ T cells (2× 106/ml) were seeded in culture plates with 6 wells.The CD4+ T lymphocytes were divided into 3 groups including the control group,miRNA-146a group and miRNA-146a inhibitor group.The numbers of Th1 and Th2 cells were measured by flow cytometry analysis (FACS).The expression of IFN-γRα,T-bet and GATA-3 at mRNA and protein levels were measured by using RT-PCR and Western blot assay,respectively.The levels of IFN-γ and IL-4 in culture supernatants of CD4+ T lymphocytes were detected by using ELISA.Results In comparison with the normal control group,there were significant increases in the expression of miRNA-146a in CD4+ T lymphocytes and the level of IFN-γin serum samples from patients with psoriasis vulgaris [(2.43±0.94) vs (1.05±0.23),(27.69±7.64) ng/L vs (9.75±2.81) ng/L,all P＜0.01].A positive correlation between the expression of miRNA-146a and the level of IFN-γ in serum was observed (r=0.837,P＜0.01).Results of the in vitro culture of CD4+ T lymphocytes showed that the number of Th1 cells,the expression of T-bet at mRNA and protein levels and the level of IFN-γin culture supernatant were significantly increased,while the expression of IFN-γRα protein was decreased in the miRNA-146a group in comparison with those of the control group (all P＜0.01).No significant differences in the number of Th2 cells,the expression of GATA-3 protein,the expression of GATA-3 and IFN-γRα at mRNA level and the level of IL-4 in culture supernatants were found between the control and miRNA-146a groups (all P＞0.05).The miRNA-146a inhibitor could effectively attenuate the effects of miRNA-146a on Th1 cells.Conclusion The miRNA-146a could promote the differentiation and enhance the function of Th1 cells.It might play an important role in the pathogenesis of psoriasis vulgaris.

Objective To evaluate changes of Rho kinase (ROK)activity in peripheral blood T lymphocytes from patients with atopic dermatitis (AD), and to analyze their clinical significance. Methods Eight milliliters of heparin-anticoagulated blood samples were collected from 60 patients with AD and 60 healthy human controls followed by separation of T lymphocytes and sera from these blood samples as well as culture of isolated T lymphocytes with 10% fetal bovine serum for 24 hours. Both patient- and control-derived T lymphocytes were classified into two groups to be cultured with patient- or control-derived sera. In addition, some patient-derived T lymphocytes were classified into 4 groups:Y27632 group treated with the Rho kinase-specific inhibitor Y2763, CD3/CD28 group treated with anti-CD3/anti-CD28 monoclonal antibodies, Y27632 + CD3/CD28 group treated with Y27632 and anti-CD3/anti-CD28 monoclonal antibodies, and control group treated with patient-derived sera. Subsequently, Western-blot analysis was performed to evaluate ROK activity in cells, methyl thiazolyl tetrazolium(MTT)assay to evaluate proliferative activity of T lymphocytes, and ELISA to measure interleukin 6 (IL-6)and IL-10 levels in supernatants of T lymphocytes. Results ROK activity was significantly lower in fresh T lymphocytes from patients than in those from healthy controls (2.47% ± 0.89% vs. 0.65% ± 0.35%, t =2.729, P < 0.05). After 24-hour culture with 10% fetal bovine serum in vitro, ROK activity was significantly decreased in patient-derived T lymphocytes compared with those before culture (0.70% ± 0.38% vs. 2.47% ± 0.89%, t = 2.658, P <0.05), but no significant difference was observed between patient- and control-derived T lymphocytes(0.70% ± 0.38% vs. 0.63% ± 0.32%, t = 1.010, P > 0.05). Compared with T lymphocytes cultured with control-derived sera, those cultured with patient-derived sera showed significantly increased ROK activity (F = 8.22, P < 0.001). Concretely speaking, ROK activity was significantly higher in patient-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera (2.41% ± 0.87% vs. 0.76% ± 0.41%, P < 0.05), and higher in control-derived T lymphocytes cultured with patient-derived sera than in those cultured with control-derived sera(2.17% ± 0.85% vs. 0.64% ± 0.33%, P< 0.05)at 24 hours. Y27632 could significantly inhibit the proliferation of as well as secretion of IL-6 (F = 18.68, 22.95, respectively, both P < 0.001)by patient-derived T lymphocytes, but had insignificant effects on secretion of IL-10. The cellular proliferative activity and IL-6 supernatant level were significantly lower in the Y27632 group than in the control group, and lower in the Y27632 + CD3/CD28 group than in the CD3/CD28 group (all P < 0.05). Conclusion Aberrant activation of ROK exists in T lymphocytes from patients with AD, which may play a certain role in the pathogenesis of AD.

Objective To evaluate regulatory effects of miRNA-146a on peripheral blood CD4 + T lymphocytes from patients with psoriasis vulgaris, and to investigate the role of miRNA-146a in the pathogenesis of psoriasis vulgaris. Methods Totally, 30 patients with psoriasis vulgaris and 30 healthy human controls were enrolled into this study. Venous blood samples were obtained from these subjects, and CD4 + T lymphocytes were isolated from these samples by using magnetic activated cell sorting (MACS). Real-time quantitative PCR (RT-PCR)was performed to measure the expression of miRNA-146a in peripheral blood CD4+ T lymphocytes, and enzyme-linked immunosorbent assay(ELISA)to determine plasma levels of interferon-γ(IFN-γ)and interleukin 4(IL-4). Some CD4+ T lymphocytes were divided into 3 groups to be transfected with 50 nmol/L negative control miRNA (control group), miRNA-146a mimics(miRNA-146a group)or miRNA-146a inhibitor (miRNA-146a inhibitor group). After 24-hour additional culture, flow cytometry was conducted to determine the number of Th1 and Th2 cells, Western-blot analysis and RT-PCR were performed to measure the protein and mRNA expressions of IFN-γ receptor α (IFN-γRα), T-box expressed in T cells (T-bet)and GATA-binding protein-3 (GATA-3)respectively, and ELISA was carried out to determine the levels of IFN-γ and IL-4 in supernatants of CD4 + T lymphocytes. Results Compared with the healthy control group, the patient group showed significantly increased miRNA-146a expression in peripheral blood CD4 + T lymphocytes (243.81% ± 94.32% vs. 105.74% ± 22.93%, t = 6.653, P < 0.01)and plasma IFN-γ level (27.69 ± 7.64 ng/L vs. 9.75 ± 2.81 ng/L, t = 4.237, P <0.01). Moreover, miRNA-146a expression was positively correlated with plasma IFN-γ level in the patients(r = 0.837, P <0.01). After 24-hour culture in vitro, there was a significant increase in the number of Th1 cells, protein and mRNA expressions of T-bet, and supernatant level of IFN-γ, but a significant decrease in the protein expression of IFN-γRα in the miRNA-146a group compared with the control group (all P < 0.01). However, no significant differences were observed in the number of Th2 cells, mRNA or protein expressions of GATA-3, or supernatant level of IL-4 among the control group,miRNA-146a group and miRNA-146a inhibitor group (all P > 0.05). Conclusion miRNA-146a may play an important role in the pathogenesis of psoriasis vulgaris by participating in the regulation of peripheral blood CD4+ T lymphocytes via affecting Th1 cell differentiation and function.

BACKGROUND:There is no clear understanding about the effect of let-7f and interleukin-6 (IL-6) on the proliferation of bone marrow mesenchymal stem cels and their relationship.
OBJECTIVE: To explore the effects of expression levels of let-7f and IL-6 on the proliferation of bone marrow mesenchymal stem cels and their relationship.
METHODS:(1) LV-rno-let-7f-up and LV-rno-let-7f-down were constructed and transfected into bone marrow mesenchymal stem cels of Sprague-Dawley rats, respectively. Then, there were four groups in the study: transfection upregulation group transfected with LV-rno-let-7f-up), transfection inhibition group (transfected with LV-rno-let-7f-down), negative control group (transfected with FU-RNAi-NC-LV), and untransfected group. The expression level of let-7f in each group was detected by qRT-PCR. The proliferation ability of cels and expression levels of IL-6 when let-7f expression was at different levels were detected by MTT, flow cytometry and ELISA. The expression of Cyclin D1 at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. (2) To predict the potential target gene of let-7f, the wild-type/mutant IL-6 3’UTR reporter gene vectors were constructed, and cotransfected with let-7f/let-7f inhibitor respectively into the 293T cels to measure the luciferase.
RESULTS AND CONCLUSION: Compared with the negative control group, the proliferative and cloning capacities of cels in the transfection upregulation group were higher; the number of cels was significantly decreased at G1 stage and increased at S stage, and the apoptotic cels were reduced in number (P < 0.05). However, the transfection inhibition group had opposite results. The expression level of IL-6 in the transfection upregulation group was lower than that in the untransfected group and negative control group (P < 0.05); while in the transfection inhibition group, the expression level of IL-6 was significantly increased (P < 0.05). The expression of Cyclin D1 at mRNA and protein levels was up-regulated in transfection upregulation group (P < 0.05) and down-regulated in the transfection inhibition group (P < 0.05), but there was no significant difference between the negative control group and untransfected group (P > 0.05). Luciferase activity of cels transfected with wide-type IL-6 3’UTR and let-7f was significantly reduced (P < 0.05). These findings indicate that up-regulation of let-7f can promote the proliferative and cloning capacities of bone marrow mesenchymal stem cels and reduce cel apoptosis, but downrelation of let-7f exhibits an inhibitory effect. Overexpression of IL-6 can suppress the proliferation of bone marrow mesenchymal stem cels, which is considered to be a target gene of let-7f, and let-7f may suppress the expression of IL-6 to promote the cel proliferation.

BACKGROUND:MicroRNA plays an important role in the process of growth and aging of living body. To know the role of let-7d in inducing bone marrow mesenchymal stem celldifferentiation into neurons can promote the stem celltransplantation. OBJECTIVE:To investigate the role of let-7d in inducing bone marrow mesenchymal stem celldifferentiation into neurons. METHODS:(1) The lentiviral vector of let-7d was constructed and transfected into rat bone marrow mesenchymal stem cells. The cells were divided into non-transfected group, negative control group (transfected with FU-RNAi-NC-LV), transfected enhancement group (transfected with let-7d-LV), transfected inhibition group ( transfected with let-7d-inhibition-LV). (2) Rat bone marrow mesenchymal stem cells were treated with fasudil as an inducer for triggering the cells to differentiate into neurons. The expression of neuron-specific markers, neuron-specific enolase and microtubule-associated protein 2, were measured by immunocytochemical method. The mRNA expression of microtubule-associated protein 2 was detected by RT-PCR. The viability of bone marrow mesenchymal stem cells was determined by MTT method. RESULTS AND CONCLUSION:Under inverted fluorescence microscope, the cells were successful y transfected with let-7d. Fasudil induced bone marrow mesenchymal stem cells to differentiate into neurons. The transfection efficiency and expression levels of neuron-specific enolase and microtubule-associated protein 2 in transfected enhancement group were higher than those in the negative control group (P<0.05);while in the inhibition group, they were lower than those in the negative control group (P<0.05). These findings indicate that let-7d can promote the differentiation of bone marrow mesenchymal stem cells into neurons induced by fasudil, and by control ing the expression of let-7d we can influence the differentiation efficiency from bone marrow mesenchymal stem cells to neurons.