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首页> 美国卫生研究院文献>Clinical and Diagnostic Laboratory Immunology >Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha interleukin 6 (IL-6) and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization.
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Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha interleukin 6 (IL-6) and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization.

机译：猪细胞因子特异性cDNA的克隆以及通过逆转录PCR和化学发光杂交检测猪肿瘤坏死因子α白介素6（IL-6）和IL-1β基因的表达。

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A reverse transcription PCR assay with porcine cytokine-specific primers was developed to clone cDNA fragments and generate cDNA probes that were specific for porcine tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-1 beta. The specificities of the cDNA PCR products were confirmed by sequence analysis on the basis of known porcine cytokine gene sequences. The reverse transcription PCR assay was also used to study cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated and control unstimulated porcine alveolar macrophages. The cDNA products were analyzed in ethidium bromide-stained agarose gels, and the transcription level of each cytokine was determined relative to the endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA level of each cytokine by measuring the intensity of the chemiluminescence hybridization signals by densitometric scanning. Various levels of cytokine mRNAs were detected in both LPS-stimulated and control unstimulated cells. Thus, TNF-alpha mRNA levels were enhanced in the cell cultures stimulated for 6 h with LPS compared with those in control cell cultures. No differences in TNF-alpha transcription levels between LPS-stimulated and control cells were observed after incubation for 24 or 55 h. Enhancements of IL-6 and IL-1 beta mRNA levels were also observed in the cultures stimulated with LPS for 6 and 24 h compared with the cytokine mRNA levels in control cell cultures. The presence of cytokine mRNA transcripts in the LPS-stimulated macrophage cultures correlated with the detection of these soluble cytokines by the bioassays. In contrast, no soluble cytokine was detected in control macrophage culture supernatants in the presence of cytokine mRNA transcripts.
机译：开发了一种使用猪细胞因子特异性引物的逆转录PCR检测试剂盒，以克隆cDNA片段并生成对猪肿瘤坏死因子α（TNF-alpha），白介素6（IL-6）和IL-1 beta特异的cDNA探针。根据已知的猪细胞因子基因序列，通过序列分析证实了cDNA PCR产物的特异性。逆转录PCR测定法还用于研究脂多糖（LPS）刺激和对照未刺激的猪肺泡巨噬细胞中细胞因子mRNA的表达。在溴化乙锭染色的琼脂糖凝胶中分析cDNA产物，并通过测量化学发光杂交信号的强度，确定每种细胞因子的转录水平相对于每种细胞因子的内源性甘油醛-3-磷酸脱氢酶（GAPDH）RNA水平。密度扫描。在LPS刺激的细胞和对照未刺激的细胞中均检测到各种水平的细胞因子mRNA。因此，与对照细胞培养相比，LPS刺激6小时的细胞培养物中TNF-αmRNA水平升高。温育24或55小时后，在LPS刺激的细胞和对照细胞之间未观察到TNF-α转录水平的差异。与对照细胞培养物中的细胞因子mRNA水平相比，LPS刺激6和24小时的培养物中也观察到IL-6和IL-1βmRNA水平的增强。 LPS刺激的巨噬细胞培养物中细胞因子mRNA转录物的存在与通过生物测定法检测这些可溶性细胞因子有关。相反，在存在细胞因子mRNA转录物的对照巨噬细胞培养物上清液中未检测到可溶性细胞因子。

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