毒力相关基因芯片分析感染单核细胞的贝氏柯克斯体毒力相关基因表达

摘要

目的 建立贝氏柯克斯体感染单核-巨噬细胞的细胞模型,利用建立的贝氏柯克斯体毒力相关基因芯片分析感染不同时期细菌的转录变化,尝试在分子水平上阐述感染早期贝氏柯克斯体的致病机制. 方法 选择贝氏柯克斯体九里株的166个毒力相关基因进行PCR扩增,制备贝氏柯克斯体毒力相关基因芯片.宿主细胞采用单核-巨噬细胞THP-1,用贝氏柯克斯体九里株感染细胞,在感染后的各时间点提取RNA,逆转录成cDNA,时间零点样品设定为参照组.采用双色荧光标记,待测DNA(即各感染后的各时间点得到cDNA)用Cy5标记,参照基因组DNA用Cy3标记.芯片杂交后,采用软件GenePixPro4.1,辅助以软件Excel,进行图片处理和数据分析. 结果 芯片分析发现贝氏柯克斯体16个基因在感染单核细胞的24h内得到显著表达. 结论 感染单核细胞得到显著表达的贝氏柯克斯体毒力相关基因与细胞内入侵、削弱吞噬细胞内杀菌抑菌作用、适应吞噬体/吞噬溶酶体内生存环境、增强菌体复制有关.%Objective To establish the cell model of the human monocytes THP-1 infecting the Coxiella burnetii in vitro, and to explain the pathogenic mechanism of the Coxiella burnetii in the early phase of infection at the molecular level based on the gene transcript expression of C. burnetii. Methods Total 166 virulence-related genes implicated in adhesion, invasion, intracellular trafficking, host modulation, detoxification were selected from the whole genome of Coxiella burnetii Nine Mile and processed by PCR. The PCR products were purified and used to make the microarray. The human monocytes THP-1 were infected with C. burnetii in vitro. RNA was extracted at the indicated time points after the infection, and then converted to cDNA. The samples at the zero-time was set as the reference. The two-fluorescence comparative hybridization was used to label the microarray with the reference DNA (cDNA of Nine Mile) labeled by Cy3 and the tested cDNA labeled by Cy5. After the hybridization,the GenePix Pro4. 1 and Excel software were uaed to analyze the processing image and data. Results Sixteen of the 166 virulence-associated genes were effectively expressed in 24 h. Conclusion These expressed genes are involved in the intracellular invading, trafficking, detoxification, adaption, survival, and replication of C. burnetii.