Screening and validation of double allele-specific binding F-primers for the measurement of antihypertensive pharmacogenomics.

Objective: Previous studies have proposed that genetic polymorphisms of CYP2D6*10, ADRB1, NPPA, CYP3A5*3, ACE, CYP2C9*3, and AGTR1 are involved in antihypertensive pharmacogenomics. The purpose of this study is to develop an amplification analysis using double allele-specific (AS) binding primers for accurate measurement of antihypertensive pharmacogenomics. Methods: To establish a quadruplex quantitative PCR (qPCR) analysis for genotyping of CYP2D6*10, ADRB1 (1165 G>C), NPPA (2238 T>C) and CYP3A5*3, and a triplex qPCR analysis for genotyping of ACE (I/D), CYP2C9*3 and AGTR1 (1166 A>C), mismatch AS F-primers were screened by detection of plasmid/gDNA, and were validated by agreement analysis/reproducibility evaluation, in which the ΔCq (differences in threshold cycles between the wild-type F-primer-based amplification assay and the mutant-type F-primer-based amplification assay) was employed to determine genotypes. Results: Seven pairs of primers were successfully selected through three rounds of F-primers screening. Except for ADRB1, the robustness assessment showed the amplification efficiency ranging from 0.9 to 1.1. In agreement analysis, two specimens in the training set (n = 203) were defined by the triplex analysis rather than NGS as heterozygotes for ACE, which was evidenced by gel electrophoresis. Reproducibility evaluation demonstrated that the coefficient of variation (CV) was <5%. Conclusion: Multiplex amplification analysis using screened AS binding primers is a simple, reliable, and accurate tool to guide drug delivery in antihypertensive personalized treatment.

Analytical capability of flow cytometric bead array for lung cancer markers / 药物评价研究

Objective To appraise the analytical capability of flow cytometric bead array for lung cancer markers through the tests of limit of detection,relative standard deviation,specificity,methods comparation and linearity rang.Methods The limit of detection,relative standard deviation,specificity and linearity rang in detection of Carcinoembryonic antigen (CEA),cytokeratin 19 (Cyfra21-1) and neuron specific enolase (NSE) in serum were evaluated by flow cytometer.Western blotting method was ultilized to validate the specificity of antibody-antigen recognization.The interference of hemoglobin,three acyl glycerol and bilirubin on the detection of CEA,Cyfra21-1 and NSE was tested.Compared to electrochemiluminescence immunoassay,the relative error for flow cytometric bead array was assessed.Results Flow cytometric bead array demonstrated that the limit of detection was 1.71 pg/mL for CEA,3.97 pg/mL for cyfra21-1,and 2.27 pg/mL for NSE.The relative standard deviation for intra-assay and inter-assay were below 10％ and 15％,respectively.The pair of antibodies can defferentially recognize antigens.The measurement for CEACAM6,CK18,NSE appeared that there was no significant cross-talking reaction.Three acyl glycerol and bilirubin did not significantly interfere with the detection for serum samples.Hemoglobin of 500 ng/mL can significantly interfere with the detection of Cyfra21-1 (P ＜ 0.05) and NSE (P ＜ 0.05).The correlation coefficient between flow cytometric array and electrochemiluminescence immunoassay was 0.984 2 for serum CEA,0.962 2 for serum cyfra 21-1 and 0.982 0 for serum NSE.The linearity ranged from 355.76 pg/mL to 367.74 ng/mL for CEA,from 87.89 pg/mL to 107.8 ng/mL for cyfra21-1,and from 90.12 pg/mL to 86.07 ng/mL for NSE.Conclusion Flow cytometric array for lung cancer markers may be of use in clinical detection.

Regulative Effects of Hawthorn Leave Flavonoids on Cytotoxicity,NO and Ca2+in Hypoxia-treated Human Umbilical Vein Endothelial Cells / 航天医学与医学工程

Objective To evaluate the potential effect of HLF (Hawthorn leave flavonoids, w/w, 80% flavonoids) against thrombus formation, effect of HLF on hypoxia-treated human umbilical vein endothelial cell (HUVECs) was studied. Method The levels of cytotoxicity and NO upon HUVECs were studied by flow cytometry. Moreover, the level of calcium ion in HUVECs was examined through laser scanning confocal microscopy. Result Data from this study showed that HLF at concentrations of 5 μg/ml and 10 μg/ml decreased the cytotoxicity of hypoxia to HUVECs (P<0.05, P<0.01). The intracellular levels of NO and calcium ion were downregulated by HLF at concentrations of 5 μg/ml (P<0.01; P<0.01) and 10 μg/ml (vs control, P<0.01; P<0.01) too. Conclusion Results observed suggest that HLF protect HUVECs from hypoxia partly through its regulative effect on NO and calcium ion levels.