Objective To establish a real-time fluorescence quantitative methylation assay to investigate the methylation status of GSH-sulphur-transferase P1(GSTP1) gene promoter region in hepatocellular carcinoma(HCC) and to investigate whether which can be used as the early diagnostic indicator of HCC .Methods Ninety-five serum samples were collected from 40 patients with HCC ,30 patients with liver cirrhosis and 25 individuals with healthy physical examination as controls .The methylation level of GSTP1 gene in these serum samples were quantitatively determined by using the real-time fluorescence quantitative methylated spe-cific PCR technique .The receiver-operation characteristic(ROC) curves were adopted to evaluate its diagnostic value for HCC .Re-sults The methylation quantitative level of GSTP1 gene in HCC serum was significantly higher than that in the healthy controls (P<0 .05) .The ROC curve analysis demonstrated that the methylation quantitative analysis of GSTP1 gene could efficiently distin-guish HCC and cirrhosis from healthy controls (AUC=0 .8641) .With the methylation rate of 2% as the critical value for diagno-sing HCC ,its diagnostic specificity was 87 .5% ,the sensitivity was 69 .6% ;the combination detection of serum GSTP1 gene methy-lation and serum AFP could increase the detection rate of HCC to 75% .Conclusion The real-time fluorescence quantitative methyl-ation assay can accurately quantify the methylation level of serum GSTP1 gene ,which has certain application value for the early di-agnosis of HCC .

Objective To evaluate the vestibular function in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Methods Seventeen CADASIL patients were recruited in the present study and 17 healthy volunteers served as control subjects. Electronystagmogram examinations including gaze nystagmus test, spontaneous nystagmus test, saccade test, pursuit test, optokinetic nystagmus test and caloric test were performed in the subjects. Results Neither patients nor controls had gaze nystagmus or spontaneous nystagmus. There was no difference in the latency and velocity of saccade movement between patients and controls. The accuracy of the saccade movement, the accuracy in leftward saccade, was significantly lower in CADASIL group compared with controls. The pursuit movement gains was also significantly lower in CADASIL group than in control group(G_L:0.79±0.08, G_R:0.76±0.12)(t=-3.739、-2.911,P <0.05) compared with controls(G_L:0.87±0.04, G_R:0.86±0.06).The optokinetic nystagmus gains were significantly decreased in CADASIL group(G_L:0.79±0.17,G_R:0.78±0.18)(t=-2.342、-2.335,P<0.05) compared with controls(G_L:0.90±0.08,G_R:0.89±0.09). The caloric test was performed in one CADASIL patient and the result revealed an incomplete fixing inhibition. CADASIL group was further divided into normal subgroup and abnormal subgroup based on the pursuit curve. The comparison between those two subgroups demonstrated a significant correlation between the pursuit movement and the symptoms of vertigo or dizziness(P<0.05). Conclusions The central vestibular function is impaired in CADASIL patients and the abnormal vestibular function is related to the symptom of vertigo or dizziness in CADASIL patients.

Aim To investigate the effects of DGK in-hibitor R59022 on ET-1-induced myocardial hypertro-phy and autophagy, and explore the possible mecha-nisms. Methods Myocardial hypertrophy was in-duced by ET-1 in cultured rat neonatal cardiomyo-cytes. Western blot was used to detect the expression of microtubule-associate protein 1 light chain 3 ( LC3 ) , beclin-1, p62, p-Akt and Akt. mRNA expression of brain natriuretic peptide ( BNP) and beta mysion heav-y chain (β-MHC) and the cell size of cardiomyocytes were detected by RT-PCR and immunofluorescence, respectively. Results Treatment cardiomyocytes with ET-1(10 -7 mol·L-1 ) for 24 h induced the myocardi-al hypertrophy in cultured neonatal rat cardiomyocytes with the activation of autophagy as evidenced by the in-creased expression of autophagy-related proteins LC3-II/I and beclin-1 , as well as the increased p62 degra-dation. While, myocardial hypertrophy induced by ET-1 , including the increased myocardial cell size and the mRNA expression of fetal gene BNP and β-MHC, could be reversed by autophagy inhibitor 3-methyl ade-nine (3-MA) and chloroquine ( CQ) ,but promoted by autophagy agonist rapamycin ( RAPA ) . Pretreatment cardiomyocytes with R59022, an inhibitor of DGK, en-hanced ET-1-induced myocardial hypertrophy by en-hancing autophagy in cardiomyocytes. Furthermore,ET-1 treatment inhibited the activation of Akt by the down-regulation of the Akt phosphorylation, and R59022 en-hanced the effect of ET-1 on the activation of Akt. Conclusions Enhanced autophagy contributes to car-diomyocyte hypertrophy. R59022 deteriorate ET-1-in-duced myocardial hypertrophy by activating autophagy. The possible mechanism may be related to the inhibi-tion of activation of mTOR signaling pathway by inhibi-ting the activation of Akt.

Aim To develop King cobra antivenom from the egg yolks of immunized hens,and study dynamic expression of IgY in egg yolks.Methods chickens(white Leghorn) were immunized with detoxicated King cobra venom by formaldehyde ,Egg yolk antibody (IgY) was isotated by thiophilic interaction chromatography and identified by SDS-PAGE. Activity of IgY was evaluated by enzyme-linked immunoserbent assay(ELISA) and Double immuodiffusion. Protein was measured using folin-lowry method. Results Specific antivenom could be detected in the yolk laid by the hens 9 d after immunization, At the 60th day after primary immunization ,ELISA titers reached 1∶ 100 000,and 97.5 mg IgY?ml -1 yolk was obtained from thiophilic interaction chromatography. IgY was highly specific, No cross reactivity was observed among IgY and agkistrodon acutus venom and vipera venom ;Little cross reactivity was shown with cobra genus venoms.Conclusion King cobra IgY was obtained and purified from the egg yolks of immunized hens,The dynamic expression of IgY was manitered during the course of immunization,Further investigation is needed.

Aim To study the effect of activating Sonic hedgehog( Shh) pathway on the function of endothelial progenitor cells ( EPCs ) in type 1 diabetic mice. Methods EPCs were isolated and cultured by density gradient method from diabetic mice. The effects of Shh N-terminal peptide and agonist SAG on EPCs prolifera-tion were evaluated by using the MTT colorimetric as-say. EPCs migration was measured by Transwell meth-od. EPCs tube formation ability was estimated by Matrigel . EPCs senescence activity was determined by β-galactosidase staining. Results Compared with control mice, the function of EPCs in type 1 diabetic mice was impaired. The proliferation, migration and tube formation of diabetic EPCs could be promoted by Shh peptide and agonist SAG. The senescence of dia-betic EPCs could be decreased by Shh peptide and ag-onist SAG. Conclusion Activating Shh signaling pathway can improve the impared function of diabetic EPCs in type 1 diabetic mice.

Purpose To study the effect of China cobra venom active factor(CCVAF) from China cobra venom on endothelial cells and its mechanism.Methods MTT experiment was adopted to evaluate the effect of CCVAF on bovine arteria pulmonalis vascular endothelial cells(BAVEC).The Eosin-Coomassie brillient blue and rhodamine-phalloidin method was used for actin cytoskeleton.Flow cytometry for [Ca~(2+)]_i and spectrophotometry were used for lactate dehydrogenase(LDH) and nitrogen oxide(NO) levels in cell culture supernatant.Results CCVAF(0.625-20 μg/mL) inhibited the proliferation of BAVEC in dose-dependent manner,and IC50 of CCVAF on BAVEC was 2.45 μg/mL. After CCVAF and BAVEC coincubation, it was showed that regression of intercellular conjunctions and disorder of F-actin distribution occurred. The content of [Ca~(2+)]_i, [LDH] and [NO] increased respectively.Conclusion CCVAF can inhibit BAVEC proliferation and it maybe associated with the change of cytoskeleton and increasing of [Ca~(2+)]_i,[LDH] aod [NO].

This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on type 2 diabetic mice model and to provide mechanistic insights into its therapeutic effect. Type 2 diabetic animal model was established with high calorie fat diet and low dose streptozotocin (STZ) injection. Mice were then randomized into 5 groups: model control, FGF21 0.25 and 0.05 μmol x kg(-1) x d(-1) groups, insulin treatment group. Ten age-matched normal KM mouse administered with saline were used as normal controls. Serum glucose, insulin, lipid products and the change of serum and liver tissue inflammation factor levels between five groups of mouse were determined. The results showed that blood glucose, insulin, free fatty acids (FFAs), triglycerides, and inflammatory factor average FGF-21 of type 2 diabetes model group and normal control group were significantly higher (P < 0.01), while compared with insulin group, no difference was significant. Average blood glucose, insulin, blood lipid and inflammatory factor of FGF-21 treatment group compared with type 2 diabetes group was significantly lower (P < 0.01) and insulin group has no difference with the model control group. The results of OGTT and HOMA-IR showed that insulin resistance state was significantly relieved in a dose-dependent manner. Thus, this study demonstrates that FGF-21 significantly remits type 2 diabetic mice model's insulin resistance state and participates in the regulation of inflammatory factor levels and type 2 diabetes metabolic disorders.

In order to obtain the serotype distribution of E.coli from duck and to screen the vaccine bacterial strains,the serotype identifications and biological characteristics of E.coli were analyzed in recent years from Shandong,Hebei and other areas of commercial duck field;selections of vaccine strains were detected by the virulence and immunogenicity.Totally 44 isolated bacterial strains of E.coli from duck were identified to a total of six serotypes:O78,O93,O76,O2,O92 and O32.The O78 serotype was the dominant serotype,accounting for 56.8％ (25/44);O93 serotype for 15.9％ (7/44) according to bacterial Oantigen typing.The strain SD (O78 serotype) was confirmed to have strong virulence and good immunogenicity.The O78,O93 and O76 are the dominant serotypes of duck E.coli in the study areas.The SD strain could be used as the candidate for the next development of inactivated vaccine.

OBJECTIVETo evaluate the correlation of liver hardness testing

RESULTSobtained by FibroTouch and FibroScan and the liver pathological stage.

METHODSSeventy-five patients with chronic hepatitis B who presented to our clinic between January 2011 and April 2013 were examined with FibroTouch and FibroScan to evaluate the degree of liver fibrosis. Forty-six of those patients also underwent liver biopsy examination.

THE RESULTSfrom technology-based testing and histopathological evaluation of the biopsy were compared by statistical analysis to determine the consistency of FibroTouch and FibroScan in regard to histological stage.

RESULTSAnalysis by paired t-test showed that the

RESULTSfrom FibroTouch and FibroScan were not significantly different (t = -0.17, P =0.8616), and the correlation coefficient from Pearson's correlation analysis was 0.9949 (P less than 0.05), suggesting that the two technologies'

RESULTSare correlated. Based on the histopathology

RESULTSfor liver fibrosis stage, the FibroTouch diagnosis of liver fibrosis more than or equal to S 1 had a receiver operating characteristic (ROC) area under the curve (AUC) of 0.889, diagnosis of liver fibrosis more than or equal to S2 had a ROC AUC of 0.941, diagnosis of liver fibrosis more than or equal to S3 had a ROC AUC of 0.908, and diagnosis of liver fibrosis more than or equal to S4 had a ROC AUC of 0.911.

CONCLUSIONCompared to FibroScan, FibroTouch has a better ability for detecting liver fibrosis and a better consistency with liver pathological stage determined by histopathological analysis.

Adult ; Aged ; Area Under Curve ; Biopsy ; Case-Control Studies ; Elasticity Imaging Techniques ; instrumentation ; Female ; Hepatitis B, Chronic ; pathology ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Prospective Studies ; Young Adult

To enhance the penetration of P53 into tumor cells by fusion it with the cell penetrating peptide 9R. The fusion gene of 9R-p53 was cloned into the expression vector. The fusion protein, CPPs-P53, was expressed and purified. We detected the rate of cell growth inhibition and apoptosis by MTT and Annexin-V-FITC/PI double stained method respectively for measuring its effect on tumor cells. CPPs-P53 and P53 were successfully expressed and purified, the purity of both proteins reached up to 90%. MTT assay showed that the cell growth inhibition by CPPs-P53 was more efficient than P53, and the rate of cell growth inhibition is dose-dependent. The apoptosis experiment showed that P53 could induce apoptosis of tumor cells. Compared with the P53, CPPs-P53 had a more significant effect in inducing cell apoptosis (**P < 0.01). The CPPs-P53 shows more significant effects than P53 in inhibiting cell growth and inducing apoptosis on tumor cells.
Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Humans ; Tumor Suppressor Protein p53 ; pharmacology