CAJ | 학술논문

目的定量比较分析不同黏膜上皮细胞中人巨噬细胞炎性蛋白-3α(MIP-3α)的转录水平.方法体外转录制备MIP-3α RNA标准品,人工合成MIP-3α mRNA序列特异的引物及TaqMan探针.利用TaqMan EZ RT-PCR试剂盒的反应体系和ABI实时荧光定量PCR仪进行实时荧光定量RT-PCR.通过对RT-PCR产物测序、使用标准品和质控品进行多次独立测试等评估实时荧光定量RT-PCR方法的特异性、灵敏度和可重复性.随后对不同黏膜上皮细胞系Caco-2、T-84、HeLa和淋巴细胞系PM1中MIP-3α mRNA水平进行了定量检测.结果建立了可用于MIP-3α mRNA水平定量检测的实时荧光定量RT-PCR方法,该方法特异性好(扩增片段测序结果与参考序列完全一致)、灵敏度高(25 μl反应体系中有5个拷贝就可以检出)、检测样品浓度范围广(103～1010拷贝/ml).对Caco-2、T-84、HeLa和PM1细胞中MIP-3α mRNA水平的定量分析表明,肠黏膜上皮细胞Caco-2和T-84的MIP-3α mRNA水平比HeLa和PM1细胞高.结论黏膜上皮细胞能表达丰富的MIP-3α,不同黏膜上皮细胞MIP-3α的表达水平可能不同.
목적 정량비교분석불동점막상피세포중인거서세포염성단백-3α(MIP-3α)적전록수평.방법 체외전록제비MIP-3α RNA표준품,인공합성MIP-3α mRNA서렬특이적인물급TaqMan탐침.이용TaqMan EZ RT-PCR시제합적반응체계화ABI실시형광정량PCR의진행실시형광정량RT-PCR.통과대RT-PCR산물측서、사용표준품화질공품진행다차독립측시등평고실시형광정량RT-PCR방법적특이성、령민도화가중복성.수후대불동점막상피세포계Caco-2、T-84、HeLa화림파세포계PM1중MIP-3α mRNA수평진행료정량검측.결과 건립료가용우MIP-3α mRNA수평정량검측적실시형광정량RT-PCR방법,해방법특이성호(확증편단측서결과여삼고서렬완전일치)、령민도고(25 μl반응체계중유5개고패취가이검출)、검측양품농도범위엄(103～1010고패/ml).대Caco-2、T-84、HeLa화PM1세포중MIP-3α mRNA수평적정량분석표명,장점막상피세포Caco-2화T-84적MIP-3α mRNA수평비HeLa화PM1세포고.결론 점막상피세포능표체봉부적MIP-3α,불동점막상피세포MIP-3α적표체수평가능불동.
Objective To compare the mRNA level of macrophage inflammatory protein-3α(MIP-3α) in 3 different mucosal epithelial cell lines. Methods RNA standards were prepared by in vitro transcription. One-step real-time RT-PCR was established and optimized using TaqMan EZ RT-PCR Core Reagents with TaqMan probes and primers specific to human MIP-3α mRNA sequence. The specificity of one-step real-time RT-PCR method was confirmed by sequencing the PCR products. The sensitivity and reproducibility of the method was examined by repeating the test 8 times with the same sample. Results The one-step real-time RT-PCR with a wide detection range is sensitive, reproducible. It was found that MIP-3α mRNA level in Caco-2 and T-84 cells was much higher than that in the HeLa cells. Conclusion High level of MIP-3α mRNA could be found in mucosal epithelial cells and difference in transcription level of MIP-3α may exist in epithelial cells from different mucosa.