Preanalytical aspects of quantitative TaqMan real-time RT-PCR: applications for TF and VEGF mRNA quantification

OBJECTIVES: The present paper focuses on preanalytical aspects of tissue factor (TF) and vascular endothelial growth factor (VEGF) mRNA quantification: the choice of blood collection tubes and defining the time frame allowed before processing the sample. DESIGN AND METHODS: Blood was collected from healthy volunteers in K(3) EDTA tubes, CPT, endotoxin-free EndoTube tubes and in PAXgene tubes. Total RNA concentration was determined by absorbance readings at 260 nm with a GeneQuantII UV spectrophotometer. RNA quantity and quality were also determined by the Lab on a Chip technique (Agilent 2100 Bioanalyzer). Real-time RT-PCR assays were performed by the TaqMan technology. RESULTS: The more expensive PAXgene and CPT tubes and the Endo tubes did not give superior results from those obtained in inexpensive routine K(3) EDTA tubes. The PAXgene tubes preserved high molecular mass rRNA better than the other tubes. CONCLUSION: Both the PAXgene system and routine EDTA tubes are suitable for clinical purposes aimed at quantitation of mRNA for TF and VEGF. PAXgene yielded rRNA that was less degraded but had lower mRNA per microg extracted RNA. A time frame up to 24 h until sample processing is acceptable for TF and VEGF mRNA.     

PDGF-BB enhances monocyte chemoattractant protein-1 mRNA stability in smooth muscle cells by downregulating ribonuclease activity

Platelet-derived growth factor (PDGF) has protean manifestations, including the regulation of growth and migration, in many cell types. We have previously reported that PDGF-BB induces the accumulation of monocyte chemoattractant protein (MCP)-1 mRNA in smooth muscle cells (SMC), in large part due to an increase in mRNA stability. To elucidate the mechanism by which PDGF-BB stabilizes MCP-1 mRNA, we have employed in vitro RNA gel mobility shift and decay assays. Cytoplasmic extracts from PDGF-BB-treated SMC increased the half-life of in vitro transcribed MCP-1 mRNA from approximately 45 min to >2 h. PDGF-BB-inhibitable degradation was not dependent on specific regions of the MCP-1 mRNA and was equally effective on a variety of in vitro transcribed mRNAs. Angiotensin II had a similar effect on MCP-1 mRNA stability, whereas tumor necrosis factor-alpha and basic fibroblast growth factor did not. The PDGF-BB-inhibitable RNAse activity was active at pH 6.6 and heat stable, but was sensitive to proteinase K. Extracts from PDGF-BB- or angiotensin II-treated cells inhibited the RNAse activity of control extracts, suggesting that the effect of PDGF-BB and angiotensin II are due to activation of a soluble inhibitor of the RNAse. The effect of PDGF-BB was blocked by inhibitors of tyrosine phosphorylation, but not by inhibitors of phosphatidylinositol 3-kinase or mitogen-activated protein kinases. These studies provide new insights into the mechanisms by which PDGF-BB enhances mRNA accumulation.     

Gene expression profile of transgenic mouse kidney reveals pathogenesis of hepatitis B virus associated nephropathy

Hepatitis B virus (HBV)-associated nephritis has been reported worldwide. Immune complex deposition has been accepted as its pathogenesis, although the association between the presence of local HBV DNA and viral antigen and the development of nephritis remains controversial. To understand better the roles played by HBV protein expression in the kidney, the global gene expression profile was studied in the kidney tissue of a lineage of HBV transgenic mouse (#59). The mice expressed HBsAg in serum, and HBsAg and HBcAg in liver and kidney, but without virus replication. Full-length HBV genome (adr subtype, C genotype) isolated from a chronic HBV carrier was used to establish the transgenic mice #59. Similarly manipulated mice that did not express HBV viral antigens served as controls. Southern blotting, hybridization with HBV probe, and immuno-histochemical staining were used to study HBV gene expression. mRNA extracted from the kidney tissue was analyzed using Affymetrix microarrays. HBsAg and HBcAg were located mainly in the cytoplasm of tubular epithelium. Altogether 520 genes were "up-regulated" more than twofold and 76 genes "down-regulated" more than twofold in the kidney. The complement activation, blood coagulation, and acute-phase response genes were markedly "up-regulated". Compared to the controls, the level of serum C3 protein was decreased in #59 mice, while the level of C3 protein from kidney extract was increased. Results indicate that expression of HBsAg and HBcAg in tubular epithelial cells of the kidney per se can up-regulate complement-mediated inflammatory gene pathways, in addition to immune complex formation.     

逍遥丸对抑郁模型大鼠海马突触素mRNA表达的影响

目的研究逍遥丸(舒肝解郁中药方剂)抗抑郁作用的分子机制。方法以原位杂交和RT-PCR方法,研究慢性应激抑郁模型大鼠的海马突触素mRNA表达以及逍遥丸对其影响。结果模型组大鼠海马突触素mRNA表达量明显上升,与正常对照组比较差异有统计学意义(P<0.05);氟西汀组、逍遥丸组大鼠海马突触素mRNA表达量显著下降,与模型组比较差异有统计学意义(P<0.05)。结论模型大鼠海马神经元内突触素表达上调,逍遥丸能下调其表达。     

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC₅₀ of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.     

左旋多巴甲酯对斜视性弱视猫模型的作用

目的观察左旋多巴甲酯对斜视性弱视猫视皮质c-fos基因表达的影响,探讨盐酸左旋多巴甲酯对弱视的治疗作用及其作用机制。方法正常幼猫30只随机分成6组:左旋多巴甲酯低剂量、中剂量和高剂量组、模型对照组、阳性对照组及正常对照组,每组5只,于4wk时行眼外直肌切除术造成人工斜视(正常对照组除外),经图形视觉诱发电位(P-VEP)确定形成弱视后,灌胃给予左旋多巴甲酯20,40,80mg·kg-1,阳性对照组给予左旋多巴40mg·kg-1,正常对照组及模型组均给予等量生理盐水。观察P-VEP的变化并采用原位杂交技术检测各组猫视皮质的c-fosmRNA表达情况。结果左旋多巴甲酯明显的缩短猫斜视性弱视眼的P100波峰潜时及提高P100波幅值。斜视猫视皮质的阳性染色细胞较正常猫减少,差异有显著性(P<0.01),用药后各组阳性染色细胞较模型对照组明显增加(P<0.01)。结论左旋多巴甲酯明显地改善斜视性弱视猫模型弱视眼的传导和感觉功能,其机制可能与左旋多巴甲酯进入脑内的量增加及介导视皮质c-fos mRNA的表达调控有关。     

HLA-E siRNA沉默肝癌细胞HLA-E基因的表达

目的针对HLA-EmRNA靶序列不同位点,设计合成多个siRNA链,定量分析其对HLA-E(+)肝癌BEL-7402细胞的基因沉默效率,筛选最佳抑制效果的siRNA。方法用IFN-γ(5×105IU·L-1)诱导BEL-7402细胞表达HLA-E基因,经流式细胞技术纯化后作为靶细胞。设计并合成3条HLA-E siRNA链(A、B、C),将各siRNA(0.1mmol·L-1)经脂质体Lipofectamin 2000转染至靶细胞。采用细胞免疫荧光、流式细胞技术、Western杂交、实时PCR等定量方法,比较48h后各siRNA的基因沉默效果,并观察HLA-E基因沉默对NK肿瘤细胞杀伤的影响。结果与空白对照、非特异组相比,3组(A、B、C组)HLA-E siRNA均明显抑制细胞HLA-E抗原、蛋白产物、mRNA、细胞表面HLA-E分子的表达(P<0.01),B、C组抑制效果接近90%,高于A组(P<0.01)。A、B、C组NK肿瘤杀伤率升高(P<0.01),而B、C组肿瘤杀伤效果高于A组(P<0.01)。结论经筛选的HLA-EsiRNA能特异、高效沉默肝癌细胞HLA-E基因表达,可能抑制其非经典HLA-Ⅰ途径免疫逃避,为肝癌"基因-免疫"治疗提供新的治疗策略。