IP-10作为牛结核病诊断标志物的初步探究

Preliminary Research on the Potential of IP-10 as Biomarker of Bovine Tuberculosis

关键词:  牛结核病  诊断  IP-10  IFN-γ  Real-Time PCR

Key words:  bovine tuberculosis  diagnosis  IP-10  IFN-γ  Real-Time PCR

摘要:

(目的)为评价细胞因子IL-12 p40、IP-10和TNF-α转录水平与牛分枝杆菌感染之间的关系，及其在牛结核病诊断中的应用潜力，（方法）采集田间筛选的结核病阳性牛、结核病阴性牛以及牛分枝杆菌68002人工感染牛的外周血淋巴细胞，经牛结核菌素（PPDB）、重组蛋白CFP-10-ESAT-6（CE）、MPT63、PET和PBS分别刺激6 h，提取细胞总RNA，用荧光定量PCR检测IL-12 p40、IP-10、IFN-γ和TNF-α的转录水平。（结果）结果显示结核病阳性牛的外周血淋巴细胞经PPDB和CE刺激后，其IP-10 的 mRNA转录水平显著高于结核病阴性牛，且与IFN-γ 的 mRNA 转录水平具有良好的相关性；初步建立牛结核病IFN-γ和IP-10的 Real- time PCR检测方法，其对临床阳性样本的检出率分别为71.4%1和85.7%。（结论）因此， IP-10的mRNA转录水平与牛分枝杆菌的感染相关，有作为牛结核病诊断标志物的潜力。

Abstract:

(Objective) To evaluate the relationship between the mRNA transcription levels of IL-12 p40, IP-10 and TNF-α and the infection of Mycobacterium bovis 68002 (M. bovis 68002), and the potential of these cytokines as biomarkers for the diagnosis of bovine tuberculosis(bTB), (Methods)the peripheral blood lymphocyte (PBLC) of bTB positive cattle, bTB negative cattle and M. bovis experimentally infected cattle were isolated respectively. The PBLC were stimulated by using PPDB, recombinant protein CFP-10-ESAT-6(CE), MPT63, PET and PBS for 6 h, respectively. And then the total RNA were extracted and the mRNA transcription levels of IL-12 p40, IP-10, IFN-γ and TNF-α were detected by real-time PCR. (Results)The results showed the mRNA transcription levels of IP-10 of PBLC stimulated by PPDB or CE were significantly higher in bTB positive cattle than that in bTB negative cattle, and the mRNA transcription levels of IP-10 showed good correlation with IFN-γ. The bTB IFN-γ and IP-10 real-time PCR detection methods were preliminary established, and the detection rate on clinical bTB positive samples were 71.45% and 85.7% respectively.(Conclusion) Therefore, the mRNA transcription levels of IP-10 were related with M. bovis infection，and potential as biomarker for the diagnosis of bTB.