慢病毒介导的 TACO-shRNA 通过促进吞噬体与溶酶体融合提高巨噬细胞清除结核分枝杆菌的能力

Lentivirus-mediated RNA interference targeting TACO gene increases the intracellular killing of Mycobacterium tuberculosis by promoting the fusion of bacteria-containing phagosomes and lysosomes

目的 构建以富含色氨酸天冬氨酸膜蛋白（ TACO）基因为靶点的短发夹状RNA（ shRNA）表达慢病毒载体,鉴定其下调TACO表达的效果,以及对巨噬细胞吞噬和杀伤胞内结核分枝杆菌（ M.tb）的影响及相关机制. 方法 设计3条靶向小鼠TACO基因的shRNA表达片段及1条随机序列对照片段,插入慢病毒表达载体pSicoR 后利用293 T细胞进行病毒包装. 采用包装成功的慢病毒颗粒感染RAW264 .7细胞,分别采用real-time RT-PCR和Western blot方法鉴定干扰效果. 选择沉默效果最佳的重组慢病毒感染RAW264.7细胞,48 h后再采用M.tb对这些巨噬细胞进行感染,倍比稀释培养法进行细胞内荷菌量的检测,免疫荧光结合激光共聚焦显微镜检测巨噬细胞内M.tb与溶酶体的共定位情况,cyto-ID细胞自噬检测试剂盒检测各组细胞的自噬水平,Western blot法检测自噬相关蛋白LC3的表达水平.结果 测序结果证实重组慢病毒表达载体均构建成功. 靶向TACO基因的3种重组慢病毒均可下调RAW264 .7细胞中TACO的表达,其中LV-pSRT1的效果最佳. LV-pSRT1感染组RAW264 .7细胞内的荷菌量在感染后0 h时即显著低于对照慢病毒（LV-pSRTc）感染组（5.50×104 vs 8.1×104, P〈0.05）. 以M.tb感染后0 h时间点的细胞内荷菌量为基准,M.tb感染后48 h和72 h时LV-pSRT1感染组RAW264.7细胞内M.tb 的存活率显著低于对照慢病毒感染组（48 h： 134.54% vs 213.58%, P〈0.05; 72 h：148.18%vs 262.96%, P〈0.05）. 与对照慢病毒感染组比较,LV-pSRT1感染组RAW264.7细胞内M.tb与溶酶体的共定位率显著上调（75.67%vs 10.66%, P〈0.05）,同时细胞的自噬水平显著上调（16.20%vs 8.50%, P〈0.05）,LC3Ⅱ的相对表达水平也显著提高（0.51 vs 0.34, P〈0.05）. 结论 TACO基因特异性RNAi慢病毒表达载体可有效抑制RAW264.7细胞中TACO的表达,抑制巨噬细胞对M.tb的吞噬能力,增强巨噬细胞对胞内M.tb的清除能力. 下调TACO表达对巨噬细胞杀伤胞内M.tb影响的机制与其通过自噬溶酶体途径提高M.tb吞噬体与溶酶体的融合有关.

Objective To construct a lentiviral vector-based short hairpin RNA （shRNA） targeting the gene encoding tryptophan-aspartate containing coat protein （ TACO） and to evaluate its inhibitory effect on the expression of TACO , and to further elucidate its effects on the phagocytosing and intracellular killing of My-cobacterium tuberculosis （M.tb） by macrophages and the possible mechanisms.Methods Three shRNA frag-ments targeting TACO gene and a scrambling control shRNA fragments were designed and cloned into the lentivi -ral vector pSicoR .The recombinant lentiviral vectors were identified by sequencing analysis and then packed in 293T cells.Real-time RT-PCR and Western blot assay were performed to evaluate the gene-silencing efficiency of the recombinant lentiviral vectors among RAW 264.7 cells transfected with the concentrated lentivirus .The most effective lentivirus was screened out to transfect the RAW 264.7 cells for 48 hours, followed by infection those cells with M.tb strains.The entry and intracellular survival of M .tb strains in RAW264.7 cells were de-termined by bacterial culture at indicated time points .The colocalization of M .tb and lysosomes was detected by immunofluorescence staining .The cyto-ID autophagy kit was used to detect the cellular autophagy and the auto-phagy-associated protein LC 3 was determined by Western blot assay .Results The recombinant lentiviral vec-tors were successfully constructed and confirmed by sequencing analysis .Decreased expression of TACO in RAW264 .7 cells was detected after transfecting the cells with the lentiviral vector-based shRNA vectors targeting TACO gene for 48 hours.The most effective lentivirus , LV-pSRT1, decreased the expression of TACO by 85.24%and 69.00%at the mRNA and protein levels, respectively.The bacterial loads in LV-pSRT1 trans-fected RAW264.7 cells were significantly decreased at the time point of 0 h after M.tb infection as compared with those in the control lentivirus treated cells （5.50×104 vs 8.1 ×104, P〈0.05）.Compared with the RAW264 .7 cells transfected with control lentivirus , the survival rate of intracellular M .tb strains in LV-pSRT1 transfected cells was significantly decreased at the time point of 48 h （134.54% vs 213.58%, P〈0.05） and 72 h （148.18%vs 262.96%, P〈0.05） considering the bacterial load at the time point of 0 h as the standard. The immunofluorescence staining demonstrated that the colocalization of M .tb strains with lysosomes was signifi-cantly enhanced in LV-pSRT1 transfected cells as compared with that in control lentivirus treated cells （75.67%vs 10.66%, P〈0.05）.Moreover, significantly enhanced autophagy and relative expression of LC 3Ⅱ protein were observed in RAW264.7 cells with TACO gene knockdown （16.20%vs 8.50%, P〈0.05;0.51 vs 0.34, P〈0.05）.Conclusion The lentiviral vector-based shRNA targeting TACO gene could effectively knockdown the expression of TACO protein , decrease the entry and increase the intracellular killing of M .tb strains in mac-rophages.The enhanced intracellular killing of M .tb strains by macrophages was associated with the increased fusion of M.tb-containing phagosome and lysosome .