AlM: To silent hypoxia inducible factor-1α ( HlF-1α) gene in malignant melanoma of the choroid cell by small interference RNA ( siRNA ) and investigate its effect on the expression of matrix metalloproteinase-2 ( MMP-2 ) in the choroid cell line human uveal melanoma cell (OCM-1) in hypoxia environment.METHODS:OCM-1 cells cultured on culture flask were divided into normal group and hypoxia group. Hypoxia group were divided into five groups: simple hypoxic group, and interference group, and negative control group, and positive control group, and liposome group. Normal group cells were cultured on DMEM culture flask with 10% FBS, 100U/mL penicillin, 100μg/mL streptomycin as well as high concentration of glucose. The cells were maintained at 37℃ in a humidified 5% CO2 incubator. Cells in good condition were selected for experiment. For hypoxia group, chemical hypoxia inducer CoCl2 was added into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment. We designed and synthesised siRNA ( siRNA + negative control+positive control ) , the target sequences of the HlF-1α to transfect hypoxic malignant melanoma of the choroid cell. SiRNA including HlF-1α siRNA, β-actin siRNA and negative control group synthesized in vitro transfected hypoxic OCM - 1 cell through Lipofectamine2000. The expression of HlF-1α, MMP-2 gene and the protein were detected by RT-PCR and Western blot. RESULTS: Compared with the normal group, the expression of HlF-1α mRNA was not obviously changed (P>0. 05), but the expression of HlF-1α protein and MMP- 2 mRNA protein was significantly higher ( P<0. 05) . Compared with the other hypoxia groups,β-actin mRNA expression of positive control group decreased ( P< 0. 05 ) , which proved successful transfection. The expression of HlF-1α mRNA and the expression of its protein and both MMP-2 mRNA and its protein was significantly lower ( P < 0. 05 ). The negative control group, liposome control group had no significant difference in the detection of factors (P>0. 05).CONCLUSlON: Hypoxia status may upregulate the HlF-1α in OCM-1 cells by increasing the expression of protein. Hypoxia can also inactivate MMP-2, resulting in upregulation of MMP-2 RNA and the expression of its protein. The expression of HlF-1α and MMP-2 mRNA can be down-upregulated by transfecting OCM-1 with HlF-1α siRNA.

To develop a sensitive and specific high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of mosapride in human plasma, mosapride and internal standard tamsulosin were extracted from plasma with liquid-liquid extraction, then separated on a Waters ACQUITY UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 microm ID) with gradient elution at flow-rate of 0.25 mL x min(-1). The mobile phase was water (containing 0.3% formic acid) and acetonitrile under gradient conditions. Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 422 --> m/z 198 and m/z 409 --> m/z 228 were used to quantify mosapride and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 0.17 - 68.00 ng x mL(-1). The lower limit of quantification was 0.17 ng x mL(-1). The inter- and intra-day precision (RSD) was less than 13%, and the accuracy (RE) was within +/- 6.3% calculated from QC samples. The method was used to determine the concentration of mosapride in plasma after a single oral dose of 5 mg mosapride citrate to 20 healthy male Chinese volunteers. The method has been proved to be selective, sensitive, rapid and suitable for pharmacokinetic study of mosapride.
Administration, Oral ; Area Under Curve ; Benzamides ; administration & dosage ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; methods ; Gastrointestinal Agents ; administration & dosage ; blood ; pharmacokinetics ; Humans ; Male ; Morpholines ; administration & dosage ; blood ; pharmacokinetics ; Sensitivity and Specificity ; Serotonin Receptor Agonists ; administration & dosage ; blood ; pharmacokinetics ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry ; methods

OBJECTIVETo investigate the effect of Salvia miltiorrhiza on glomerulosclerosis induced by Ang II.

METHODRat mesangial cells were exposed to 100 nmol L(-1) Ang II. Meanwhile, we added S. miltiorrhiza injection of different concentrations to Mcs. PAI-1 mRNA and protein, TGF-beta1 in serum free MEM medium, and the level of cellular reactive oxygen species (ROS) were measured.

RESULTS. miltiorrhiza notably attenuated Ang II induced expression of PAI-1 in a concentration-dependent manner. Meanwhile, S. miltiorrhiza suppressed the production of TGF-beta1 and cellular ROS in mesangial cells.

CONCLUSIONS. miltiorrhiza can alleviate glomerular sclerosis. The renoprotective effects of S. miltiorrhiza may be due to its ability to decrease Ang II -induced PAI-1 and TGF-beta1 secretion and cellular ROS level.

Angiotensin II ; pharmacology ; Animals ; Blotting, Western ; Cells, Cultured ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Gene Expression ; drug effects ; Injections ; Mesangial Cells ; drug effects ; metabolism ; Plants, Medicinal ; chemistry ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Salvia miltiorrhiza ; chemistry ; Transforming Growth Factor beta1 ; blood ; secretion

No abstract available.
Pulmonary Embolism

OBJECTIVETo evaluate the relationship between circulating levels of insulin-like growth factor-1 (IGF-1), IGF-binding protein-3 (IGFBP-3) and colorectal cancer.

METHODSA meta-analysis of 6 epidemiological studies on insulin-like growth factors and risk of colorectal cancer were performed.

RESULTSThe pooled odds ratio (OR) of IGF-1 and IGFBP-3 were 1.56 (95% CI: 1.14-2.13) and 0.78 (95% CI: 0.43-1.44) respectively. According to the results from different measurements (enzyme-linked immunoabsorbent assay and immunoradiometric assay), the pooled OR were 1.92 and 1.23 for IGF-1, 0.46 and 1.44 for IGFBP-3 respectively.

CONCLUSIONHigh serum levels of IGF-1 were independent risk factors of colorectal cancer but the OR of IGFBP-3 was not statistically significant. The heterogeneity between studies on IGFBP-3 and colorectal cancer was caused by different measurements used, but there was still a need to conduct simultaneous large size study under 2 different measurements for further conclusion.

China ; epidemiology ; Colorectal Neoplasms ; blood ; epidemiology ; Enzyme-Linked Immunosorbent Assay ; methods ; Insulin-Like Growth Factor Binding Protein 3 ; blood ; Insulin-Like Growth Factor I ; metabolism ; Radioimmunoassay ; Risk Factors

This study was aimed to investigate the aggregation of rehydrated-lyophilized platelets. The aggregation rate of fresh and rehydrated-lyophilized platelets were measured by using thrombin, ristocetin, ADP and collagen as inductors and APACT2 aggregameter; the effects of intra- and extra-cellular trehalose on maximum aggregation rate of rehydrated-lyophilized platelets were detected by using ADP as an inductor. The results showed that the aggregation rate of fresh platelets was all about 100%, while aggregation rate of rehydrated lyophilized platelets was (70.17 +/- 7.36)%, (15.3 +/- 2.81)%, (68.67 +/- 6.86)%, (64.67 +/- 11.6)% respectively, when the concentration of thrombin, ristocetin, ADP and collagen was 1 U/ml, 1.6 mg/ml, 20 micromol/L and 2 microg/ml. The maximum aggregation rates of rehydrated-lyophilized platelets in intra- and extra-cellular trehalose, extracellular trehalose and blank control groups were (66.0 +/- 4.69)%, (25.3 +/- 2.42)% and (11.5 +/- 1.87)% (P < 0.01), meanwhile there was significant difference of rehydrated-lyophilized platelet aggregation rate between intra- and extra-cellular trehalose and extracellular trehalose groups (P < 0.01). It is concluded that the concentrations of thrombin (1 U/ml), ristocetin (1.6 mg/ml), ADP (20 micromol/L) and collagen (2 microg/ml) are optimal for platelets aggregation tests, the internal and extracellular trehalose significantly enhance the aggregation of rehydrated-lyophilized platelets.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Freeze Drying ; methods ; Humans ; Platelet Aggregation ; physiology

The aim of this research was to study the technology and methods of loading lyoprotectant-trehalose into cytoplasm of human platelets before lyophilization, to optimize experimental conditions of loading trehalose, to investigate the changes of platelets response to agonists and activation after incubation of platelets for 4 hours at 37 degrees C in the presence of lyoprotectant-trehalose, to protract the figures of loading efficiency and intracellular trehalose concentration versus incubation time, temperature and external trehalose concentration, to optimize loading parameters. The response of platelets to different agonists--thrombin, ADP, collagen and ristocetin were measured respectively by APACT2 aggregometer before and after loading trehalose into platelets; the expressions of CD62p and PAC-1 on platelet membranes in the presence and absence of reversible platelets activation inhibitors were measured by flow cytometry respectively before and after loading trehalose into cytoplasm of platelets. The results showed that the loading efficiency was linear to incubation time (2 hours later) and incubation temperature (rang from 30 degrees C to 40 degrees C), respectively. The loading efficiency almost reached 60% when the platelets were incubated at 37 degrees C for 4 hours. The intracellular trehalose concentration was higher with the increase of the extracellular trehalose concentration (< 50 mmol/L). Compared to untreated groups, the values of MPV and aggregation to different agonists in treated groups showed no significant difference, respectively (P > 0.01). After incubation of platelets for 4 hours, the expression of CD62p increased to some extent, however, the expression of CD62p decreased again when the reversible platelets activation inhibitor PGE-1 and adenosine were added to the incubation buffer. It is concluded that 37 degrees C, 4 hours and the extracellular trehalose concentration < 50 mmol/L are the optimal conditions for loading with trehalose. The processing of loading with trehalose before platelet lyophilization has no significant effects on response of platelets to agonists and activation.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cell Survival ; Cryopreservation ; methods ; Freeze Drying ; Humans ; Trehalose ; blood ; pharmacology

OBJECTIVETo evaluate the 5-item version of the international index of erectile function (IIEF-5) as a method to differentiate the causes of vasculogenic erectile dysfunction (ED).

METHODSIn all, 103 ED patients (mean age 46.8 +/- 18.7) were reviewed by IIEF-5. Penile blood flow was also assessed in each patient after an intracavernosal injection (ICI) and audio-visual sex stimulation by duplex Doppler ultrasonography. The 99mTc-(113m)In dual radioisotope test was performed to confirm specific vascular causes in the vasculogenic ED cases. Kruskal-Wallis TEST was employed to compare the scores of IIEF-5 with the results of ICI, duplex Doppler ultrasonography and the 99mTC-(113m)In dual radioisotope test.

RESULTSOf the total number of ED cases, 37 (37/103, 35.9%) were nonvasculogenic, 18 (18/103, 17.5%) arteriogenic, 35 (35/103, 34.0%) venogenic and 13 (13/103, 12.6%) combined vasculogenic. There was no significant difference in the IIEF-5 scores either between the vasculogenic group and the non-vasculogenic one (P = 0.253) or among different groups of the vasculogenic ED patients.

CONCLUSIONIIEF-5 cannot be used as a tool for differential diagnosis of vasculogenic ED, or to compare its specific vascular causes, nor can the scores of IIEF-5 reflect penile vascular conditions.

Adult ; Humans ; Impotence, Vasculogenic ; diagnosis ; diagnostic imaging ; Male ; Middle Aged ; Penis ; diagnostic imaging ; Smoking ; Surveys and Questionnaires ; Ultrasonography

Objective To understand the major genotype-resistant mutation sites and change trends of HIV/AIDS patients with failure of antiviral therapy (ART) in Lincang City, Yunnan Province. Methods The In-House method was used to amplify the Pol gene region in the plasma samples of HIV/AIDS patients with failure of ART in Lincang City from 2011 to 2018. The target sequence was spliced and submitted to the HIV resistance database to identify and analyze the HIV-1 subtypes and resistant mutation sites. Results The 950 strains of HIV/AIDS patients with antiviral failure were mainly CRF08_BC, accounting for 75.5% (717/950), and the total gene mutation rate was 67.1% (637/950), which was dominated by non-nucleoside reverse transcriptase inhibitors (NNRTIs), accounting for 62.4% (593/950); followed by nucleoside reverse transcriptase inhibitors (NRTIs), accounting for 34.7% (330/950); protease inhibitors (PIs) was 7.5% (71/950). A total of 15 NRTIs of resistance-related mutation sites were detected, mainly M184V (29.3%) which was detected mostly in AZT/D4T+3TC+NVP programs; including 17 kinds of NNRTIs, mainly was K103N/S (25.1%)，the most detected in AZT/TDF+3TC+EFV programs. There were 22 kinds of PIs，mainly secondary sites were L10F/V/I (2.2%) and L33F (2.1%). The top three NRTIs mutation sites in the area were changed from T69D/N/G，M184I/V and D67N/G/S to M184I/V, K70R/Q/E/T and T215Y/F/V/I/N/A/D. NNRTIs mutation sites were changed from V179D/T/E/F, E138A/K/G/R and Y181C/F/G/V to K103N/S, E138A/K/G/R and V179D/T/E/F. The mutation sites of the first three PIs did not change much. Conclusions The second-line regimen based on PIs is a better choice in free antiviral treatments. Mastering the drug resistance of different gene mutations is beneficial to the compatibility of first-line drugs, thus delaying the use of second-line drugs.

Objective To investigate the effect and its mechanism of targeted inhibition of the expression of chemokine receptor 4(CXCR4) gene by small interfering RNA (siRNA) on the invasion and proliferation of nasopharyngeal carcinoma cells.Methods Forty-two nasopharyngeal carcinoma tissue and its adjacent tissues in the First Affiliated Hospital of Xinxiang Medical University from January 2014 to December 2016 were collected.The expression of CXCR4 mRNA and protein in nasopharyngeal carcinoma tissue and its adjacent tissues were detected by real time-polymerase chain reaction and Western blot.The human nasopharyngeal carcinoma cell line CNE-2Z were divided into blank control group,negative control group and CX-CR4 transfection group.The cells in blank control group were not given any treatment;the cells in negative control group were transfected nonsense siRNA sequence;the cells in CXCR4 transfection group were transfected CXCR4 targeting siRNA sequence.The protein expression of CXCR4,matrix metalloproteinases-2 (MMP-2),MMP-9,β-catenin,Cyclin D1 were detected by Western bloting after 48 h of transfection.The proliferation and invasion ability of the cells were detected by cell counting kit and Transwell chamber.Results The expression of CXCR4 mRNA in nasopharyngeal carcinoma tissue and adjacent tissues was 5.526 ±0.143,0.953 ±0.091 respectively;the expression of CXCR4 protein in nasopharyngeal carcinoma tissue and adjacent tissues was 0.522 ± 0.047,0.053 ± 0.011 respectively.The expression of CXCR4 mRNA and protein in nasopharyngeal carcinoma tissue were significantly higher than those in adjacent tissues (P ＜ 0.05).The protein expression of CXCR4,MMP-2,MMP-9,β-catenin,Cyclin D1 in cells,cell survival rate and the number of cell invasion in CXCR4 transfection group were significantly lower than those in the blank control group and negative control group (P ＜ 0.05);however,there was no significant difference in above indexes between the blank control group and negative control group (P ＞ 0.05).Conclusion Inhibiting of CXCR4 gene expression in nasopharyngeal carcinoma CNE-2Z cells can significantly decrease the proliferation and invasion ability of cancer cells,and the mechanism may be related to down regulation of Wnt/β-catenin signaling pathway.