Objective To investigate the effect of extracellular signal-regulated kinase(ERK)pathway OH nitriC oxide(NO)release by human umbilical vein endothelial cell(HUVEC)induced by placental growth factor-1(PLGF-1).Methods During January to April 2006,50 samples of umbilical vein blood were collected from newborns delivered by cesarean section due to intrauterine distress and abnormal fetal position.HUVEC were primarily cultured by trypsin digestion.Then the following procedures were performed:(1)Cells were identified using the morphology andⅧfactor immunohistochemistry methods if the culture WaS satisfactory.(2)Cells were collected,and fms.1ike tyrosin kinase(Fit-1)protein and its mRNA expression were detected with immunoprints and RT-PCR methods.(3)The protein wag extractedafter cells were treated with PLGF-1(cells were collected before the treatment and 2.5,5.10,20 min after the treatment).The protein levels of ERK were determined by immunoprints.(4)The cells were cultured witll serum-free culture medim containing PLGF-1 only(culture media were collected 20,40,160,360.480.720 and 1440 rain after the treatment).The quantity of NO was detected with nitrate reductase metllod(5)The ceHs were cultured with serum.free culture medium containing PI)98059,the inhibitor of mitogen-activated protein kinase(MAPK)/MEK for 60 min Then the cells were cultured continuously with the serum-free culture medium containing PLGF-1 for 60 mira The culture media were coilected.The quantity of NO was detected by nitrate reductase method.The samples were divided into treatment group and control group.Control group was exactly the satne in treatment time,culture condition,and time to colleet the cells as the treatment group.except that it WaS not treated with PLGF-l or PD 98059.Resuits (1)By morphology and Ⅷ factor inununohistochemistry the cultured cells were identified to be HUVEC.(2)Fit-1mRNA and protein were expressed in HUVEC.(3)Expression of ERK protein started to increaSe at 2.5 min after treatment of HUVEC with PLGF-1,reaching the peak at 5 min,and decreased at 10 min.(4)Incomparison with the control group.NO started to increase at 20 rain after treatment of HUVEC with PLGF-lat 480 min(15.82±0.69)μmol/L Comparison between the two groups showed a significant difference (P<0.05).(5)ReleaSe of NO from the cells treated with PD98059 for 1 hour and PLGF WaS significantly inhibited,compared with the ceils treated with PLGF-1 only.Conclusion ERK pathways play an important role in N0 release bv HUVEC induced by PLGF.

Objective To investigate the influence of humanistic care on patients in the operation room.Methods 80 cases of surgery patients from November 2010 to October 2011 were given humanistic care,another 80 patients before implementation of humanistic care were chosen,and the satisfaction degree of doctors and patients and recovery of patients before and after the implementation of humanistic care were compared.Results After the implementation of humanistic care,the recovery of patients improved.The total satisfaction degree of patients and doctors reached 93.2％ and 98.1％,significantly higher than those before the impIementation,which were 82.1％ and 90.2％.Conclusions Implementation of humanistic care can improve the psychological and physical state of surgery patients,and help them gain better recovery.

Objective To study the correlation between the drug-resistance variation and the genotypes of hepatitis B virus (HBV)detected by the PCR-reverse dot blot and the relation between the HBV variation loci with the liver function indexes and HBV DNA viral loading.Methods The serum samples from 462 patients with chronic hepatitis B treated by oral nucleoside drugs were screened.The PCR-reverse dot blot was adopted to detect the drug-resistance gene mutation loci and genotypes.The correla-tion between the HBV drug-resistance mutant with the genotypes,liver function indexes and HBV DNA viral loads was performed. Results Among 462 patients taking nucleoside drugs for treating chronic hepatitis B,45 drug-resistance mutants were detected with the mutation rate of 9.74%;in which,16 cases (35.5%)were 180M and 204I/V mutant,6 cases(13.3%)were 204V,13 ca-ses(28.9%)were 204I mutant,3 cases (6.7%)were 180V mutant and 3 cases(6.7%)were 236T mutant.The HBV genotyping showed 105 cases of genotype B,337 cases of genotype C,0 case of genotype D and 2 cases of other genotypes.Conclusion (1)The HBVgenotypes in Maoming area may be different from the genotypes in other southern regions and is dominated by HBV-C geno-type.(2)The PCR-reverse dot blot method is a detection method for fastly and accurately finding the drug-resistance loci after nu-cleosides therapy.(3)The clinical analysis demonstrates that the drug-resistance mutation loci has no correlation with the liver func-tion index ALT(P >0.05),but there was certain correlation between the drug-resistance mutation loci in hepatitis B and HBV DNA viral load(P <0.05).

Objective To investigate the effect of hyperurieemia on cardiovascular and cerebrovaseular complications in type 2 diabetes mellitus(T2DM). Methods According to the level of blood uric acid,430 patients with T2DM were divided into hyperuricemia group and non-hyperurieemia group, the differences of uric acid(UA) ,creatine(Cr) ,total cholesterol(TC) ,triglyeride(TG) ,fasting blood glucose(FBG) ,glycosylated hemoglobin(HbA1c) and body mass index(BMI) were compared,and the occurrences of coronary heart disease, hypertension and acute cerebrovascular accidence were observed. Results The FBG and HbAIc were not significantly different between the two groups(P＞0.05). The BMI ,TG,TC and Cr in the hyperurieemia group were significantly higher than those in the non-hyperuricemia group(P＜0.05 ). The incidence rates of coronary heart disease, hypertension and acute cerebrovascular diseases in the hyperuricemia group were also significantly higher than those in the non-hyperuricemia group (P＜0.05 ). Conclusion Hyperuricemia is obviously correlated with obesity and blood lipid abnormalities. Hyperurieemia can aggravate the metabolic disturbances,and can accelerate the occurrence and development of cardiovascular and eerebrovascular diseases in the patients with T2DM.

Objective To investigate the clinical effects of gliclazide sustained-release tablet for recently diag-nosed type 2 diabetes mellitus. Methods 118 subjects with recently diagnosed type 2 diabetes and normal figure were divided into three groups. The three group subjects used gliclazide sustained-release tablet, repaglinide(import-ed) ,novolin 30R for 12 weeks,respectively. For all pretherapy and post-treatment cases, blood glucose and glycosy-lated hemoglobin were tested before meal. After two hours of meal, blood glucose was tested again. Meanwhile, hypo-glycaemia event was inspected. The test results were analyzed by "mean ± standard deviation" method. Results There are no significant difference(P>0.05) in fasting blood sugar and postprandial blood sugar(2h) level between the group using gliclazide sustained-release tablet and the group using novolin 30R. The clinical effects of both gli-clazide sustained-release tablet and novolin 30R to fasting blood sugar are all better than that of repaglinide(import-ed) (P < 0.01). But there are no significant difference in postprandial blood sugar(2h) level between the group of us-ing gliclazide sustained-release tablet and the following two group: repaglinide(imported) and novolin 30R. Conclu-sion The investigation results show that gliclazide sustained-release tablet is better than repaglinide(imported) in fasting blood sugar. But for the effects of postprandial blood sugar(2h), there is no significant difference between gli-clazide sustained-release tablet, repaglinide(imported) and novolin 30R.

The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) in the placenta of the patients with pregnancy induced hypertension (PIH) was detected and its role in the pathogenesis of PIH was studied. The pathological changes in placental vessels were observed by HE staining. NO2-/NO3-, the stable metabolic end products of NO, was measured with nitrate reductase. The eNOS activity in placental tissues was assayed by spectrophotometry. Western blot analysis was applied to detect NOSTRIN expression. The incidence of thickening and fibronoid necrosis of placental vessels was significantly higher in women with PIH than in the normal group (P < 0.01). The levels of placental NO2-/NO3- in PIH patients (27.53 +/- 7.48 micromol/mg) were significantly lower than in normal group (54.27 +/- 9.53 micromol/mg, P < 0.01). The activity of eNOS was significantly decreased in PIH group (12.826 +/- 3.61 U/mg) as compared with that in normal group (21.72 +/- 3.83 U/mg, P < 0.01). Western blot analysis revealed that both groups expressed 58 kD NOSTRIN, but the protein level was significantly higher in women with PIH than in the normal group (P < 0.01). A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in placental tissue of women with PIH (r = -0.57, P < 0.01). It was concluded that the level of NOSTRIN expression in placenta of women with PIH was increased, which may play an important role in the pathogenesis of PIH.

Objective To investigates factors affecting the positive rate of blocking antibody treated by paternal lymphocyte immunotherapy in patients with recurrent spontaneous abortion (RSA).Methods From January 2008 to August 2012,326 RSA cases undergoing treatment in Infertility Center of Qilu Hospital were studied retrospectively.Those patients were divided into 2 groups randomly:260 cases in intradermal injection group were administered via bilateral forearm intradermal injections for immunotherapy once 21 days,then the blocking antibody was determined after 2 (23 cases),3 (73 cases),4 (74 cases),5(90 cases) times respectively,while in subcutaneous injection group,the 66 cases were administered via subcutaneous injection once 21 days,the blocking antibody measured after 3 times; In both cases,the blocking antibody was all determined 2 weeks later.The positive rate of blocking antibodies and the rate of successful pregnancy was recorded,and then followed up after the blocking antibody turning positive.Results (1)Positive rate of blocking antibodies:the positive rate of blocking antibodies were 17％ (4/23),58％ (42/73),72％ (53/74) and 84％ (76/90) in the 2,3,4,and 5 times of intradermal injection group,respectively (P ＜ 0.05).In subcutaneous injection group,the positive rate of blocking antibodies was 38 ％ (25/66),which was significantly lower than that in group intradermal injection receiving 3 times immunotherapy (P ＜0.05).(2) The rate of pregnancy:the 176 patients out of 200 patients were pregnant when antibody was positive after immunotherapy,with 71.6％ (126/176)of patients gained successful pregnancy(the length of pregnancy more than 5 months).Conclusions The route and frequency of administration of immunotherapy could influence the positive rate of blocking antibody.The rate of successful pregnancy will be increased after blocking antibody turning positive.

Objective To investigate the clinical value of the peripheral blood monocyte human leukocyte antigen-DR (mHLA-DR) for assessment of degree of severity and the diagnosis of acute pancreatitis (AP). Methods A case-control study was conducted. Eighty-six AP patients admitted to Shandong Liaocheng People's Hospital from June 2014 to May 2015 were enrolled. Patients were classified into four groups [mild (n = 33), moderate (n = 25), severe (n = 16), critical (n = 12)] according to the disease classification. Eighty healthy persons subjected to physical examination center of our hospital at the same time were served as controls. Acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) scores in patients were estimated. Flow cytometry was used to measure the expression of the peripheral blood mHLA-DR, and the Pearson method was used to analyze the relationship between the level of mHLA-DR and the APACHE Ⅱ score. The receiver-operating characteristic curve (ROC) was plotted, and then the clinical value of the peripheral blood mHLA-DR was analyzed for the diagnostic value in AP patients. Results The expression of the mHLA-DR in patients with AP was significantly lower than that of healthy control group [(63.7±18.6)% vs. (86.4±8.3)%, t = 5.319, P < 0.001]. The expression levels of the mHLA-DR in mild group, moderate group, severe group, and critical group were (79.6±6.5)%, (66.4±9.4)%, (49.9±8.1)%, (32.5±12.0)%, respectively, and the APACHE Ⅱ score were 4.67±1.99, 5.88±2.05, 9.06±2.62, 12.33±3.96, respectively. Pair wise comparisons were statistically significant (all P < 0.05). The HLA-DR expression level in the peripheral blood of patients with AP was negatively correlated with the APACHE Ⅱ score (r = -0.695, P < 0.001). The area under the ROC curve (AUC) of mHLA-DR expression in peripheral blood for AP was 0.894 [95% confidence interval (95%CI) = 0.847-0.941, P < 0.001], and the cut-off point was 84.40%, with the sensitivity of 75.0%, the specificity of 90.7%, and the accuracy rate of 83.1%. The AUC of mHLA-DR expression for mild AP was 0.938 (95%CI = 0.889-0.987, P < 0.001), and the cut-off point was 72.70%, with the sensitivity of 87.9%, the specificity of 88.7%, and the accuracy rate of 88.4%. The AUC of mHLA-DR expression for severe and critical AP was 0.943 (95%CI = 0.881-1.005, P < 0.001), and the cut-off point was 57.85%, with the sensitivity of 84.0%, the specificity of 96.4%, and the accuracy rate of 90.6%. Conclusions The expression levels of the peripheral blood mHLA-DR in AP patients can reflect the degree of disease, and contribute to the diagnosis of AP. The value of mHLA-DR may be used as a new biological indicator in the diagnosis and assessment for the severity of AP.

Objective To observe the influence of specific short hairpin siRNA targeting EGFR gene on apoptosis of human ovarian cancer Skov-3 cells in vitro. Methods A plasmid of a short hairpin siRNA targeting EGFR was constructed, and it was transfeeted into Skov-3 cell line by lipofectamine 2000. Human ovarian carcinoma cells of the line Skov-3 were cultured and divided into 3 groups: control group; non-specific group, transfected with non-specific plasmid vector; and specific group, transfected with specific small hairpin RNA expression vector. The expression of EGFR mRNA and protein were examined by RT-PCR and immunocytochemistry, Flow cytometry (FCM) was adopted to analyze quantitatively apoptotic cells in each group. Results After transfection of pshRNA-EGFR, mRNA and protein levels of EGFR gene in Skov-3 cells were obviously reduced. Flow cytometry analysis revealed that apoptosis could be induced in Skov-3 cells line transfected with pshRNA-EGFR in a time-dependent manner, no obvious apoptosis were detected in control group and non-specific group. Conclusion The plasmid expressive vector target at EGFR in our study is capable of suppressing EGFR expression of human ovarian cancer Skov-3 cells and inducing apoptosis, which provide a new way for the gene therapy of human ovarian cancer.

Objective To establish a method for the determination of aristolochic acid A inPaishi granule.Methods The HPLC system consisted of the Phenomenex Luna C18(4.6 mm×250 mm, 5μm)column, the mobile phase consisted of acetonitrile and 0.01% HAc, gradient elution flow rate was 1.0 ml/min, the column temperature was 35℃, The UV detector was set at 250 nm.Results The linear response range was 0.029-0.580μg/ml (r=0.999 9). The detection limit and quantitation limit of aristolochic acid A inPaishi granule were 0.9 and 3.0 ng/ml. The average recovery of aristolochic acid A was 96.4%.Conclusion The method is high sensitivity, accurate, repeatable and high specificity,and can be used as an inspection method for safe use of Paishi granule.