Objective To construct recombinant lentiviral vector pSin-EF2-Puro-DAB2IP transfect prostate carcinoma PC3 cells,and to observe its transfection rate and expression level in PC3 cells.Methods The cDNA sequences specifically targeting the DAB2IP gene were designed and cloned into lentiviral vector PC3-pSin using DNA recombinant technique.Using PC3-pSin-EF2 as control,the 293T cells were transfected by Lipofectamine reagent for lentiviral particles packaged, and viral titer was determined. The DAB2IP mRNA and protein expression levels were examined by RT-PCR and Western blotting methods. Results The PCR and sequencing analysis results confirmed that the DAB2IP gene sequence was consistent with the sequence in GeneBank. The number of DAB2IP gene copy in PC3-pSin-EF2-DAB2IP cells and its control PC3-pSin-EF2 cells were 0.001±0.000 and 0.158 ± 0.013,respectively.Compared with control cells,the overexpression of mRNA in PC3-pSin-EF2-DAB2IP cells upregulated (179.370 ± 15.891)times,the difference was statistically significant (P<0.001). Compared with internal reference and control cells, the expression levels of DAB2IP protein in PC3-pSin-EF2-DAB2IP cells upregulated (2.431 ± 0.892)times and (2.415 ± 0.961)times respectively,the differences were statistically significant (P<0.001).Conclusion The lentiviral vector of the DAB2IP gene pSin-EF2-Puro-DAB2IP is successfully constructed,and its targeted gene is stably expressed in the targeted cells,which provides a basis for the further functional study of DAB2IP.

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BACKGROUND: Under certain condition, circulating endothelial progenitor cells (EPCs) can differentiate into endothelial cells, and further participate in angiogenesis.OBJECTIVE: The goal of this study was to investigate the feasibility and efficacy of peripheral blood-derived EPCs in promoting angiogenesis in the ischemic myocardium, in order to provide a new cell implanting method for the treatment of coronary heart disease.DESIGN: A randomized controlled experiment.MATERIALS: Sixty male Sprague-Dawley (SD) rats, of clean grade, weighing (340±20) g, were provided by Qingdao Laboratory Animal Center. These animals were randomly divided into 2 groups with 30 rats in each: experimental group and control group. In each group, ten rats were separately observed 2, 4 and 8 weeks after EPCs being injected. The protocol was conducted in accordance with animal ethics guidelines for the use and care of animals.METHODS: This study was carried out in the Qingdao Key Laboratory of Medical Biological Technology between May 2003 and September 2004. After SD rats in the experimental group were anesthetized, peripheral blood was taken.Mononuclear cells were harvested by density gradient centrifugation. CD31, CD34, Flk-1 and von Willebrand disease factor immunofluorescence staining positive EPCs were harvested by adding the defined media of vascular endothelial growth factors and basic fibroblast growth factor. Myocardial ischemia was induced by ligation of murine left anterior descending coronary artery. Autologous EPCs isolated from the peripheral blood of each animal were infused to ischemic myocardium. In the control groups,cell culture media were infused, and the other procedures were the same as those in the experimental group. Two, four and eight weeks after ligation, all animals were sacrificed by overdose anesthesia, and heart tissue sections were made.MAIN OUTCOME MEASURES: ①After haematoxylin-eosin staining, myocardial structure changes were observed under an optical microscope. ②Cells positive for factor Ⅷ were numbered and then to calculate the total number of cells in each visual field for evaluating microvessel density with a German ZEISS Axiotron image analyzer.RESULTS: All the 60 SD rats were involved in the final analysis, without deletion. ①Myocardial structure was relatively disarrayed in the control groups, with collagens and fibroblasts substituted for cardiomyocytes. In the marginal infarct areas, cardiomyocytes were irregular and partial cells were significantly hypertrophied. As compared with control group, myocardial collagen fibers fused less, and tissue structure got more disarranged in the experimental group. Microvessel density in the implanted area was obviously increased. ②In the 2nd, 4th and 8th weeks after EPC infusion, microvessel density at the ischemic myocardium was significantly higher than that at the corresponding time point in the control group (P ＜ 0.01). In the EPCs groups, microvessel density tended to statistically increase with time going (P ＜ 0.05). No prominent difference was observed in the three control groups (P＞ 0.05).CONCLUSION: Relatively purified EPCs can be obtained by certain procedure of isolation and culture from rat peripheral blood. Intramyocardial implantation of autologous EPCs promotes microangiogenesis and has a protective effect on ischemic myocardial tissue.

To study the mechanism of human chronic renal allograft rejection, kidney tissues were taken from 16 patients with chronic renal allograft rejection and from 5 healthy subjects, and underwent the frazed section staining for ICAM-1 and VCAM-1 to anti-ICAM-1 and anti-VCAM-1 respectively by using immunohistochemistry(ABC).The results showed that there were differ-ent distribution of ICAM-1 and VCAM-1 expression in nomal kidney and renal allograft during chronic rejection.It was suggested that ICAM-1 and VCAM-1 might play an important role in the pathogenesis of human chronic renal allograft rejection.

Objective To assess the effect of levothyroxine , lipoic acid and their combination therapy on improving the cognitive deficit and on the level of oxidative stress in the serum and hippocampus tissue of adult hypothyroid rast .Methods Fifty male SD rats were randomly divided into five groups:normal group , hypothyroidism group [ propylthioaracil ( PTU ) group], the levothyroxine sodium (L-T4) treatment group (PTU +L-T4 group), the lipoid acid(LA) treatment group (PTU+LA group), and the two-drug combination group (PTU+LT-4+LA group).After 8 weeks of treatment, the learn-ing and memory abilities of the rats were evaluated by Morris Water Maze ( MWM ) .The content of malondialdehyde ( MDA) and the activities of superoxide dismutase ( SOD) in the serum and hippocampus were respectively measured using thiobarbituric acid ,and xanthine-xanthine oxidase methods .Results Compared with the normal group , the escape latency was longer(P<0.01), the MDA level was increased and the SOD activity was decreased in the serum and hippocampus in the PTU group (P<0.001).Compared with PTU group, the SOD activity was increased and the MDA level was decreased in the PTU +L-T4 group, PTU +LA group and PTU +L-T4 +LA group(P<0.05).There was significant difference in MWM test score .Conclusion The L-T4 and LA combination therapy can significantly improve the cognitive function of hypothyroid rats.

Objective To explore the procedures of isolation,cultivation of endothelial progenitor cells(EPC) from peripheral bloods and the feasibility of local EPC transplantation to promote neovascularization in ischemic myocardium. Methods Peripheral blood was obtained from the femoral artery of each Sprague-Dawley(SD)rat.Myocardial ischemia was induced by ligation of murine left anterior descending coronary artery.Immmdiate timepoint of ligation procedure, autologous EPC from peripheral blood,positive for CD31,D34,Flk-1 and vWF,isolated from the peripheral blood of each animal were injected to ischemic myocardium.In the control groups,cell culture media was injected.Rats were euthanized and neovascularization in local ischemia areas was evaluated.Results Hematoxylin-eosin staining indicated better myocardial arrangement in the EPC group.Capillary density in ischemic myocardium weeks after transplantation was significantly greater in the group of EPC injection than the control groups.Conclusion Relatively purified EPC can be obtained by certain procedure of isolation and culture from rat peripheral blood.Intramyocardial transplantation of autologous EPCs will promote microangiogenesis and has a protective effect on ischemic myocardial tissue.

Objective To observe the therapeutic effect of exenatide combined with metformin therapy in type 2 diabetes with nonalcoholic fatty liver disease.Methods 50 patients with type 2 diabetes and nonalcoholic fatty liver disease were randomly divided into experimental group and control group with 25 cases in each.The control group were treated with metformin, the experimental group were treated with exenatide on the basis of metformin.Before treatment and at the end of 12-week treatment, patients height, weight, body mass index(BMI), waistline(WC), glycated hemoglobin(HbA1c), fasting blood glucose(FBG),fasting C-peptide(FCP), triglyceride(TG), cholesterol(TCH), high density lipoprotein(HDL), low density lipoprotein(LDL),systolic pressure(SBP), diastolic pressure(DBP), alanine aminotransferase(ALT), superoxide dismutase(SOD), liver lipid contents(LFC) were detected.Results Baseline data were not significantly different between the two groups.Compared to baseline, WC, HbA1c, FBG and LFC in experimental group and control group were obviously improved after treatment(P<0.05), and the experimental group improved more significantly than control group(P<0.05).Conclusion Exenatide combined with metformin has an obvious therapeutic effect on BMI, WC, HbA1c, FBG, FCP, SBP, TG, TCH, HDL, LDL, LFC than mere metformin treatment in treatment of type 2 diabetes with nonalcoholic fatty liver disease.

Objective To explore the clinic values of intraoperative fluid overload in evaluating the perioperative prognosis of patients after cardiopulmonary bypass cardiac operation. Methods A prospective observational study was conducted. The adult patients admitted to the Third People's Hospital of Chengdu from April 2014 to March 2016 for selective cardiopulmonary bypass cardiac operation monitored by pulmonary artery catheter or pulse-indicated continuous cardiac output (PiCCO) were selected. All patients received therapy with restrictive fluid management strategy after admission to the intensive care unit (ICU) and were divided into two groups based on the value of intraoperative fluid accumulation ratio at the time of admission to the ICU: group A with intraoperative fluid accumulation ratio of less than 10% and group B with equal to or more than 10%. Then the changes and different prognosis of the patients in groups were observed. Risk factors affecting the prognosis were analyzed using logistic regression, and the predictive values of various parameters on prognosis were analyzed using receiver operating characteristic curve (ROC). Results 224 cases were included, with 172 in group A and 52 in group B. No significant differences were found between both groups in gender, age, pre-operative scores by European system for cardiac operative risk evaluation (EuroScore), acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) and sequential organ failure assessment (SOFA), operation ways, operation time, cardiopulmonary bypass time and blood loss (all P > 0.05). Both APACHE Ⅱ score and SOFA score in group B were significantly higher than those in group A at admission and 24, 48 and 72 hours after ICU admission (APACHE Ⅱ: 24.5±4.1 vs. 21.8±3.5, 14.8±6.5 vs. 9.9±3.9, 12.3±5.4 vs. 9.4±3.7, 10.9±5.0 vs. 8.9±3.3, SOFA: 12.3±2.9 vs. 10.6±2.1, 8.8±2.8 vs. 5.7±1.7, 7.2±3.0 vs. 5.0±2.0, 6.4±3.6 vs. 5.2±1.7, all P < 0.05). Compared with group A, incidence of combination with acute kidney injury (AKI) was significantly increased in group B (92.3% vs. 68.6%, P < 0.01), the level of post operation cardiac index (CI) was significantly lower in group B (mL·s-1·m-2: 40.67±4.00 vs. 49.84±7.50, P < 0.01). Both the duration of mechanical ventilation and the length of stay in the ICU in group B were significant longer than those in group A (days: 3.2±2.1 vs. 1.8±1.3, 5.0±1.7 vs. 3.6±1.2, both P < 0.01). The post-operation complications, 7-day and 28-day mortality in group B were all significantly higher than those in group A (65.4% vs 30.2%, 19.2% vs. 1.7%, 26.9% vs. 3.5%, all P < 0.01). Logistic regressive analysis showed that after controlling the influence of postoperative AKI and CI on mortality, the intraoperative fluid accumulation ratio at ICU admission was still an independent risk factor [odds ratio (OR) of 7-day mortality = 1.380, 95% confidence interval (95%CI) = 1.019-1.869, P = 0.037; OR of 28-day mortality = 1.302, 95%CI = 1.026-1.654, P = 0.030]. The area under the curve of ROC (AUC) in predicting the 28-day mortality of patients after operation using intraoperative fluid accumulation ratio was 0.874 (P = 0.000), with a sensitivity of 95.0 % and a specificity of 78.4% at the optimal threshold value of 7.5%. Conclusions Intraoperative fluid overload in patients admitted to the ICU would aggravate their condition, prolong the duration of mechanical ventilation and the length of ICU stay, and increase post-operative complications morbidity and mortality. After controlling the influence of AKI and cardiac insufficiency on mortality, the fluid overload was still an independent risk factor for the death of patients after cardiopulmonary bypass cardiac operation.

Objective To explore the relationship between neutrophil‐to‐lymphocyte ratio (NLR) and early‐stage diabetic nephropathy in patients with newly diagnosed type 2 diabetes(T2DM ). Methods A total of 160 patients with newly diagnosed T2DM were enrolled in this study and divided into two groups:early‐stage diabetic nephropathy (EDN ) group and non‐DN group (T2DM group). The clinical and biochemical data were collected. Results NLR ,neutrophia cell count ,2 hPG ,HbA1 c ,age were significantly higher in EDN group than in T2DM group[NLR (2.27 ± 0.92)vs(1.81 ± 0.56) ,neutrophia cellcount(4.35±1.47)vs(3.78±1.08)109/L,2hPG(21.98±4.30)vs(20.37±4.40)mmol/L,HbA1c (11.06±2.02)% vs (10.22 ± 1.89)% ,age(49.22 ± 12.71) vs (44.41 ± 10.81)years] (P< 0.05).Logistic regression analysis showed that NLR (OR= 6.529 ,95% CI:1.946 ~ 21.873 ,P= 0.002)and 2 hPG(OR = 1.213 ,95% CI:1.002 ~ 1.467 ,P= 0.047 ) were independent risk factors for EDN.Conclusion Increased NLR is significantly associated with EDN. High NLR level may be a reliable predictive marker for EDN.