ABSTRACT
OBJECTIVE: The mitogenic and chemotactic effects of resistin-like molecule alpha (RELMα) are thought to contribute to vascular remodeling in pulmonary arterial hypertension. Here we evaluate the expression of RELMα in atherosclerotic plaque and investigate its effects on the proliferation and migration of vascular smooth muscle cells (VSMCs). METHODS: An atherosclerotic model was established by feeding 4-week-old C57BL/6J ApoE-/- mice (n = 9) with a high-fat diet. Wild-type 4-week-old C57BL/6J (n = 9) were fed the same diet and were used as controls. RELMα expression was evaluated by immunohistochemistry and quantified using real-time PCR (RT-PCR). A (3)H-thymidine incorporation assay and the Boyden chamber assay, respectively, were used to explore the effects of different concentrations of RELMα on the proliferation and migration of VSMCs. RESULTS: Immunohistochemistry identified positively stained granules in atherosclerotic plaques. These results were confirmed by detection of RELMα mRNA using RT-PCR. We also demonstrated that in vitro exposure to RELMα significantly promoted the proliferation and migration of VSMCs in a dose-related manner (p < 0.01). CONCLUSIONS: RELMα expressed in atherosclerotic plaque of ApoE-/- mice appears to enhance the proliferation and migration of aortic VSMCs in a dose-related manner.
Subject(s)
Aorta/cytology , Intercellular Signaling Peptides and Proteins/physiology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Animals , Cell Movement/physiology , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Male , Mice , Mice, Inbred C57BL , Plaque, Atherosclerotic/pathology , RNA, Messenger/metabolismABSTRACT
Objective To develop a sensitive,specific, simple and rapid quantitative real-time PCR (Q-PCR) assay for detection of Staphylococcus aureus with SmartCycler.Methods According to the nuc gene sequences specific to S.aureus, a pair of primers and one TaqMan probe were designed. An internal amplification control (IAC) which is a chimeric double-stranded DNA constructed from a fragment of the Listeria monocytogenes hly gene flanked by the nuc-specific target sequences was added to the reaction system. This IAC was detected using a second TaqMan probe labeled with a different fluorophore. The performance of the nuc-IAC Q-PCR was evaluated using artificially contaminated drinking water and commercial UTH whole milk samples spiked with ATCC 6538.Results The nuc-IAC assay could be used reliably for detection with a sensitivity of 5 copies of linear plasmid DNA per reaction, 10 fg of genomic DNA in 62.5% of the reactions or 50 cfu/ml S.aureus cells with 50% probability. The quantification was linear (r~2≥0.998) over a 6-log dynamic range, with a PCR efficiency over 0.967. The 5×10~2 CFU per 25 ml mimic sample of drinking water or milk could be detected by this assay consistently and quantifiably.Conclusion The nuc-IAC Q-PCR assay for S.aureus is developed. It could not only be applied for the quantitative detection of S.aureus, but also prevent the false negatives and underestimations of contamination loads due to PCR failure.
