Objective To investigate the expressions of phosphatidylinositol 3-kinase (PI3K), Akt and E-cadherin and their clinical significance in thyroid papillary carcinomas.Methods Expressions of PI3K, Akt, and E-cadherin were detected in 62 cases of thyroid papillary carcinomas,30 cases of thyroid goiter and 30 cases of normal thyroid by immunohistochemistry (EnVison),and simultaneously compared with age, sex, tumor size, clinical tumor node metastasis( TNM) stages, and lymph node metastasis in thy-roid papillary carcinomas.Results The expression rate of PI3K, Akt, and E-cadherin was 74.2%(46/62), 66.1%(41/62), 16.1%(10/62),respectively.Expressions of three proteins in thyroid papillary carcinomas were significantly different from those in thyroid goiter and normal thyroid tissues ( P <0.05). The lower positive rates of PI3K and Akt proteins were obtained in the group of stageⅠ~Ⅱthan that in the group of stageⅢ~Ⅳ(χ2 =4.976, P =0.026;χ2 =6.233, P =0.013).Higher positive rates of PI3K and Akt proteins were obtained in the group of lymph-node metastasis than that in group of non-lymph-node metastasis (χ2 =6.675, P =0.010;χ2 =7.511, P =0.006).Higher positive rate of E-cadherin protein was obtained in the group of stage Ⅰ~Ⅱ than that in the group of stage Ⅲ ~Ⅳ (χ2 =6.558, P =0.010 ) .Higher positive rate of E-cadherin proteins was obtained in the group of non-lymph-node metastasis than that in the group of lymph node metastasis(χ2 =5.678, P =0.017).There was significant positive correlation between expressions of PI3K and Akt through Spearman correlation analysis ( r =0.423, P <0.05).PI3K was negatively correlated with E-cadherin with Spearman correlation analysis ( r =-0.527, P <0.05).Akt was also negatively correlated with E-cadherin ( r =-0.417, P <0.05).Conclusions PI3K/Akt pathway might regulate thyroid papillary carcinoma cells proliferation, invasion and metastasis.

Objective To investigate the expressions of cancerous inhibitor of protein phosphatase 2A (CIP2A) and vascular endothelial growth factor (VEGF) in hepatocellular carcinomas and their clinical significance.Methods The expressions of CIP2A and VEGF proteins were tested with immunohistochemistry in 60 cases of hepatocellular carcinomas and adjacent paracancerous tissues.Results The positive rate of CIP2A in hepatocellular carcinomas was significantly higher than adjacent paracancerous tissues (83.3％ vs 9.5％,P ＜ 0.05).Significant differences were also observed in the expression rate of VEGF between the patients with hepatocellular carcinomas and paracancerous tissues (75.0％ vs 14.3％,P ＜0.05).The expression of CIP2A in hepatocellular carcinomas was significantly positively correlated with its pathological grading,differentiation,and tumor node metastasis (TNM) stage (P ＜ 0.05).The expression of VEGF in hepatocellular carcinomas was significantly positively correlated with its pathological grading,differentiation,and TNM stage (P ＜ 0.05).Significantly positive correlation was found between expressions of CIP2A and VEGF with Spearman correlation analysis (rs =0.465,P ＜ 0.01).Conclusions The abnormal expressions of CIP2A and VEGF gene may promote tumor angiogenesis and progression of a hepatocellular carcinoma.The study supports positive regulation between expressions of CIP2A and VEGF in a hepatocellular carcinoma.

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.
Amino Acid Sequence ; Enterovirus D, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; metabolism

Hypertension is an important risk factor for cardiovascular and cerebrovascular diseases,which can damage structure and function of vital organs such as the heart,brain and kidney.Pseudo hypertension (PHT) refers to the blood pressure measured by ordinary cuffmanometry is higher than that of the arterial puncture,which is an essential factor of refractory hypertension.Recent findings have suggested that the elderly patients with concomitant history of atherosclerotic disease,renal insufficiency,and diabetes mellitus have the highest risk of pseudohypertension.The incidence rate ofpseudohypertension is about 1.7％-50％ in domestic and international studies.In the clinical treatment,the misdiagnosis of hypertension of patients with excessive blood pressure will lead to severe perfusion defects.In this review,we will focus on the diagnosis and pathogenesis of pseudohypertension.

AIM To observe whether acute stimulation of BRL 37344, a β3-adrenergic receptor (β3-AR) agonist, has the same effects of exacerbating hemodynamics and increasing in β3-AR expression on rats with failing heart as chronic administration. METHODSRats received two doses of isoprenaline (Iso, 340 mg·kg-1, sc, with a 24-h interval) to prepare heart failure model. After 8 weeks, rats were given iv BRL 37344 0.4 nmol·kg-1·min-1 for 10 min. Hemodynamics were measured at 0, 10, 30 min, 1, 2, 3 h, 1, 2 and 7 d after BRL 37344. Levels of β-AR mRNA in myocardium were measured at 0, 1, 2 and 7 d after BRL 37344 by reverse transcription-polymerase chain reaction. RESULTSCompared with Iso group, heart rate, left ventricular end systolic pressure and +dp/dtmax were significantly higher, and left ventricular end diastolic pressure was significantly lower during 1-3 h after BRL 37344 injection in Iso+BRL group. Then, they were restored to the same level as that prior to BRL 37344 injection. Compared with normal control, the levels of β1-, β2- and β3-AR mRNA displayed no significant change in BRL group;the level of β1-AR mRNA was lower and the level of β3-AR mRNA was higher in Iso group. In Iso+BRL group, much more lower β1-AR mRNA level and much higher β3-AR mRNA level were shown on d 2 and d 7 than Iso group. CONCLUSION Acute administration of β3-AR agonist has a shorter improved hemodynamics. But it caused the same result as chronic administration in reduction of β1-AR mRNA and increment of β3-AR mRNA in failure hearts, which may aggravate the cardiac functions.

Objective To explore the effects of the β3 adrenergic receptor (β3-AR) agonist BRL37344 on cardiac fibroblast proliferation and collagen fiber hyperplasia in Wistar rats by promoting the phosphaticlylinositol 3 kinase-protein kinase B(PI3K-Akt) signal transduction pathway. Method Cardiac fibroblasts(CFbs) were isolated from 1 - 3 day-old Wistar rats under the sterile environment in the laboratory of the First Affiliated Hospital of Hasbin Medical University, by using enzyme digestion and an modified technique of differential anchoring velocity.The cultured myocardial cells were randomly (random number) divided into five groups. ① In blank group, rats were not treated with drug; ②in BRL group, rats were treated with BRL37344; ③in LY group, rats were treated with LY294002(PI3K antagonist) for one hour before treated with BRL37344;④in Akt-Ⅰ group,rats were treated with Akt-Ⅰ (Akt antagonist) for one hour before treated with BRL37344;⑤in L-A group, rats were treated with LY294002 and Akt-Ⅰ for one hour before treated with BRL37344. MTT colorimetric method and RT-PCR were used to observe the role of β3-AR agonist with or without PI3K antagonist and/or Akt antagonist in cardiac fibroblast proliferation (CFP) and collagen fiber hyperplasia (CFH). Comparisons between groups were made by one-way ANOVA and Tukey test. Results ①β3-AR was present in the CFbs. ②Compared with BRL group, the CFP and CFH in LY and Akt-Ⅰ groups were lower (P ＜0.01) and those in L-A group were lowest (P ＜ 0.01). ③Compared with blank group,the expressions of type Ⅰ and type Ⅲ fiber mRNA obliviously increased in BRL group (P ＜ 0.01),and at 48 hours,the expressionrs reached peak. At 48 hours,compared with BRL group,the expressions in LY and Akt-Ⅰ groups were lower, and were lowest in L-A group ( P ＜0.01). Conclusions BRL37344 promotes cardiac fibroblast proliferation and expressions of type Ⅰ and Ⅲ collagen fiber mRNA by activating the PI3K-Akt signal transduction pathway.

Objective To investigate the influence of proline-rich tyrosine kinase 2 (Pyk2)gene RNA interference on proliferation,invasion and migration of Hep3B hepatocellular carcinoma cells.Methods The Pyk2 gene RNA interference vector was transfected in Hep3B hepatocellular carcinoma cells by lipofectamine. The Hep3B cells divided into three groups:siRNA group (the vector with Pyk2 RNAi gene was transfected), negative control group (the vector without Pyk2 RNAi gene was transfected),and blank control group (no vectors was transfected).Pyk2 mRNA and protein were detected using reverse transcription reverse transcription-poly-merase chain reaction (RT-PCR)and Western blotting.The biological behavior including cell proliferation,inva-sion and migration were detected by 3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl-2H-tetrazolium bromide (MTT), transwell and wound healing assay,respectively.Results The expression of Pyk2 mRNA of Hep3B cell line in siRNA group (0.1 6 ±0.03)was significantly decreased than those in negative group (0.74 ±0.1 3)and blank control group (0.77 ±0.1 6),with statistically significant differences (t=51 .46,P=0.000;t=53.21 ,P=0.000).The expression of Pyk2 protein of Hep3B cell line in siRNA group (0.24 ±0.06)was significantly decreased than those in negative group (0.83 ±0.05)and blank control group (0.91 ±0.06),with statisti-cally significant differences (t=57.29,P=0.000;t=68.53,P=0.000).The cell proliferation inhibition rate at 48 hours in siRNA group (26.1 7%±0.28%)was significantly raised than those in negative group (9.28%± 0.22%)and blank control group (6.47%±0.31%),with statistically significant differences (t=31 .45,P=0.004;t=34.64,P=0.002).The number of transmembrane cells in siRNA group (32.5 ±8.5)/1 0 HP was significantly declined than those in negative group (98.4 ±1 2.3 )/1 0 HP and blank control group (1 1 2.6 ± 1 1 .3)/1 0 HP,with statistically significant differences (t=95.64,P=0.000;t=1 05.1 7,P=0.000).The wound healing assay in siRNA group (28.1 7%±1 .46%)was significantly lower than those in negative group (77.38%±2.24%)and blank control group (79.41%±3.1 7%),with statistically significant (t=85.86,P=0.000;t=89.37,P=0.000).Conclusion Pyk2 gene involves the proliferation,invasion and migration of Hep3B cells,which has close correction with development and metastasis of hepatocellular carcinoma.Pyk2 gene is very helpful to become a molecular target for the diagnosis and treatment of hepatocellular carcinoma.

ABSTRACT:Objective To observe the effects of late Na current (INa‐L ) and rapidly activating delayed rectifier K current (IKr ) on ventricular heterogeneity and frequency dependency by using high resolution voltage sensitive optical mapping technology .Methods The model of 12 isolated hearts was constructed in rabbits . Voltage sensitive dye Di‐4‐ANEPPS were perfused into the isolated hearts by Langendorff method .LED source with the wave length of 532 nm was used to record APD80 and APD50 of the left and right ventricles .Experimental groups were divided into 3 groups by perfusion drugs dofetillide (30 nmol/L) ,dofetillide+ATX‐Ⅱ(1 nmol/L) ,and dofetillide +ATX‐Ⅱ +mexiletine (10μmol/L) .The subjects were intervened by the above drugs in order ,and they were self‐compared before dosing .After each drug administration ,the hearts were stimulated respectively with the BCL of 2 000 ms ,1 000 ms ,500 ms ,and 300 ms .Then we observed the changes of APD80 and APD50 in the left and right ventricles before and after the interventions .Results ① In the control group ,APD80 and APD50 of the right ventricle were longer than those of the left ventricle in response to different stimulation , and the differences increased with the decrease of stimulating frequency .② When BCL was 1000 ms ,APD80 and APD50 of the left and right ventricles were prolonged respectively after administration of dofetillide , but the differences in APD80 and APD50 were insignificant between the left and right ventricles (P>0 .05) .ΔAPD80 of the two ventricles increased significantly with the decrease of stimulating frequency . ③ After administration of ATX‐Ⅱ , when BCL was 1000 ms ,APD80 and APD50 of the left and right ventricles increased significantly compared with those in the control group and dofetillide intervention group (P<0 .05) .And the increase of APD in the left ventricle was greater than that of the right ventricle .ΔAPD80 of the two ventricles increased significantly with the decrease of stimulating frequency .④ After administration of mexiletine ,when BCL was 1000 ms ,APD80 and APD50 of the left and right ventricles reduced significantly compared with those of the primary state (P<0 .05) .APD80 and APD50 of the left and right ventricles reduced significantly compared with those of the control group (P< 0 .05) and ATX‐Ⅱ group (P>0 .05) .The increase of ΔAPD80 of the two ventricles became milder when the stimulating frequency decreased . Conclusion ① IKr blocked by dofetillide did not affect the heterogeneity between the two ventricles , which showed reverse‐frequency dependence . ② In the context of blocking IKr , ATX‐Ⅱ increased the heterogeneity between the left and right ventricles and enhanced the reverse‐frequency dependence .In contrast ,mexiletine ,the blocker of INa‐L ,decreased the heterogeneity between the two ventricles and reverse‐frequency dependence .

OBJECTIVETo probe into an effective method for treatment of poststroke depression.

METHODSTwo hundred and fifty-six cases of poststroke depression were randomly divided into a control group (n = 76) treated by oral administration of amitilin and an acupuncture group (n = 180) treated by activating brain and regaining consciousness needling method. Their clinical therapeutic effects and changes of relative factors of poststroke depression and plasma contents of monoamine neurotransmitters were observed.

RESULTSIn the acupuncture group, the clinical symptoms and the relative indexes in the depression scales improved and plasma contents of the monoamine neurotransmitters increased significanty as compared with the control group (P < 0.05).

CONCLUSIONThe needling method for activating brain and regaining consciousness can improve relative factors to cure poststroke depression.

AIM:To investigate the roles of feeder cells ln stratification of murine corneal epithelial cells and build an ideal method to engineer stratified epithelial sheet.METHODS:Using contact feeder culture,separated feeder culture,compound feeder culture and culture without feeder cells by Air-lifting method in Transwell chamber culture system,tissue engineered corneal epithelium was reconstructed.Corneal sheets were stained with hematoxylin and eosin(HE)for histological observation.The expression of p63 and keratin 19(K19)and involucrin(IVL)was investigated by immunocytochemistry analysis.RESULTS:Stratification was limited to three to four layers in the contact feeder group,whereas separate feeder sheets were slightly more stratified.The compound feeder group produced a stratified epithelium with five to seven layers of cells.The group without 3T3 feeder cells formed only two to three layers of cells.Immunostaining images in the compound feeder group showed expression of progenitor markers p63 and K19 in the basal and suprabasal layer,as well as differentiation marker involucrin in all layers.CONCLUSION:The remarkable stratification as well as the Iimbal phenotype makes the compound feeder system a candidate tool for cultivating transplantable epithelial sheets.