Yang Yi-ning

GW24-e0989 No significant difference of adeno-associated virus serotype 9 transfection efficiency comparison in different cells

Objectives To make a comparison on the efficiency of two kinds of cells for transfecting adeno-associated virus serotype 9 carried enhanced green fluorescence protein, and to demonstrate that adeno-associated virus serotype 9 transfection is feasibility and safety. Methods Human Umbilical Vein Endothelial Cells (HUVECs) and Human Aortic Smooth Muscle Cells (HASMCs) respectively infected with AAV9-eGFP were observed and analysed with fluorescence microscopy and flow cytometry to assess transfection efficiency. Multiplicity of Infection was set to three groups:1*105, 1*106 and 1*107. Non-transfected cells were set as control. Results HUVECs and HASMCs infected with AAV9-eGFP were begin to expressed after transfected 24 hours, and the expression peak appeared in the fourth day in HUVECs, and the fifth day in HASMCs. Cell growth was not affected and cell form was normal in the whole observation period. Multiplicity of infection of 1*105, 1*106 and 1*107 in the fourth day after transfection in HUVECs was 1.4%, 12.3% and 52.8%. And the fifth day in HASMCs was 5.3%, 18.3% and 52.4%. Conclusions There was no obvious differences between HUVECs and HASMCs after transfection in multiplicity of infection of 1*107. This study demonstrates that adeno-associated virus serotype 9 transfection of HUVECs and HASMCs is feasibility and safety.

ASSA13-03-50 Transfection of Rats H9C2 Cells with Recombinant Adeno-Associated Virus Serotype 9 Mediated Anti-NF-kB Ribozyme in Vitro and Effect of Nuclear Factor-kB Activity

Objective To evaluate the transfection efficiency using recombinant adeno-associated virus Serotype 9 mediated Anti-NF-κB ribozyme and enhanced green fluorescent protein (rAAV9-EGFP-R65) to rats H9C2 cells and the effect of Nuclear Factor-κB (NF-κB) activity. Methods rAAV9-EGFP-R65 was transfected into H9C2 cells at multiplicities of infection (MOI = 1 × 106 v.g./cell). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage was determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. H9C2 cells were treated with TNF-α, rAAV9-EGFP- R65 and PDTC. The DNA binding activity of NF-κB was examined by electrophoretic mobility shift assay (EMSA). Results The cells with rAAV9-EGFP-R65 transfection at MOI of 1 × 106 v.g./cell began to exhibit EGFP expression 1 day after transfection. The fluorescence intensity increased with the time of transfection. EGFP expression reached the maximum on day 5, at the point of which the transduction efficiency of rAAV9-EGFP-R65 inH9C2 cells was (32.27 ± 3.19)%. Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells. TNF-a could active NF-κB, rAAV9- EGFP-R65 and PDCT can efficiently decrease NF-κB activation in rats H9C2 cells. Conclusions rAAV9-EGFP-R65 can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition. rAAV9-EGFP-R65 can availably inhibit NF-κB activation in rats H9C2 cells in vitro. This study played foundation for further research.

Relationship between a novel polymorphism of the C5L2 gene and coronary artery disease

Background C5L2 has been demonstrated to be a functional receptor of acylation-stimulating protein (ASP), which is a stimulator of triglyceride synthesis or glucose transport. However, little is known about the variations in the coding region of the C5L2 gene and their association with coronary artery disease (CAD). Methodology/Principal findings The authors identified a novel single nucleotide polymorphism (SNP), 698C>T (P233L), in exon 2 using a PCR direct-sequencing method. This nucleotide change causes the amino-acid order from proline to leucine at codon 233. We examined the role of this SNP for CAD using two independent case–control studies: one was in the Han population (492 CAD patients and 577 control subjects) and the other was in the Uygur population (319 CAD patients and 554 control subjects). Heterozygote carriers of the 698CT genotype were more frequent among CAD patients than among controls in the Han population (7.3% vs 1.7%) and in the Uygur population (4.7% vs 1.6%). The odds ratio (OR) for carriers of the 698CT genotype for CAD was 4.484 (95% CI: 2.197 to 9.174) in the Han group and 2.989 (95% CI: 1.292 to 6.909) in the Uygur population. After adjustment of confounding factors such as sex, age, smoking, alcohol consumption, hypertension, diabetes, as well as serum levels of triglyceride, total cholesterol, high-density lipoprotein, the difference remained significant in the Han group (p<0.001, OR=6.604, 95% CI: 2.776 to 15.711) and in the Uygur group (p=0.047, OR=2.602, 95% CI: 1.015 to 6.671). Conclusion/Significance The 698CT genotype of C5L2 may be a genetic marker of CAD in the Han and Uygur population in western China.

e0054 Recombinant adeno-associated virus serotype 9 transfection of rats H9C2 cells in vitro

Objective To evaluate del transfection efficiency using recombinant adeno-associated virus Serotype 9 mediated enhanced green fluorescent protein (rAAV9- EGFP) to rats H9C2 cells and the impact on growth of H9C2 cells. Methods rAAV9-EGFP was transfected into H9C2 cells at different multiplicities of infection (MOI=1×105, 1×106, 1×107). EGFP expression in the cells was observed under inverted fluorescence microscope and the EGFP-positive cell percentage determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. Results The cells with rAAV9-EGFP transfection at MOI of 1×106 and 1×107 began to exhibit EGFP expression 1u2005days del after transfection and the cells transfection at MOI of 1×105 began to exhibit EGFP expression 2u2005days after transfection. The fluorescence intensity increased with the MOI used for transfection. EGFP expression reached the maximum on day 4, at the point of which the transduction efficiency of rAAV9-EGFP in H9C2 cells was (14.1±0.2)%, (35.1±4.8)% and (56.8±0.1)%. Corresponding to MOIs of 1×105, 1×106 and 1×107, respectively. Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells after transfection. Conclusions rAAV9-EGFP gene can be del transfected in a stable manner and efficiently expressed in H9C2 cells without causing cell growth inhibition. This del The results of this study played foundation (del) (provides a platform) for further research.