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ObjectiveTo investigate the effects of pravastation intervention on tumor necrosis factor (TNF)-α-indueed ossifie calcification in human umbilical artery smooth muscle cells (hUASMCs). MethodshUASMCs were cultured by tissue explant in vitro, hUASMC were treated with TNF-α 50 μg/L and pravastatin of three different concentrations. The calcium deposition was determined by O-cresolphthalein eomplexone method. The mRNA expression of BAP and OPN was determined by real time-PCR. The protein expression of BAP, OPN and BMP-2 was determined by Western blotting. ResultsPravastatin inhibited the proliferation of hUASMC (r=-0.946, P＜0.01) and decreased the cell calcium deposition (r=-0.973, P＜0.01) in a dosedependent manner. Pravastatin down-regulated the expression of BAP, OPN and BMP-2 induced by TNF-α in a dose-dependent manner (mRNA, r=-0.972, P＜0.01;BAP protein, r=-0.820, P＜0.01;OPN protein, r=-0.972, P＜0.01;BMP-2 protein, r=-0.928, P＜0.01). ConclusionPravastatin can inhibit the proliferation of hUASMC, decrease the cell calcium deposition and inhibit the ossifie calcification of hUASMC induced by TNF-α.
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Objective To establish a real-time fluorescent quantitative RT-PCR for detecting SARS-Coronavirus (CoV) mRNA in gargling liquid and serum of SARS patients.Methods The assay is based on simplified nested fluorescent RT-PCR. Total RNA was extracted from gargling liquid and serum by using high performance system and reverse-transcription by antisense primer.The specific TaqMan probe was designed according to the published DNA sequence of SARS-CoV polymerase and labeled with FAM and TAMRA. Standard curves for SARS-CoV quantification were prepared with serial dilutions of the recombinant plasmid.Results The method is highly sensitive and specific. The sensitivity of assay was 1?105 copies/L. The positive rate of SARS-CoV mRNA in the gargling liquid of patients was 65.0% (26/40) and 11.5% (6/52) in suspected patients, respectively. SARS-CoV mRNA was not detectable in the gargling liquid from 40 healthy individuals. The positive rate of SARS-CoV mRNA in the serum of SARS patients was 25.6% (10/39). PCR amplification products of 6 suspected patients with SARS-CoV mRNA were confirmed by sequencing and the sequence data were consistent with that of BJ01 AY278488.Conclusions Real-time fluorescent quantitative RT-PCR should be a rapid, specific tool for early diagnosis of SARS-CoV infection.