晚期内体的分选转运参与Ⅱ型腺相关病毒在IB3人支气管上皮转化细胞中的转导激活过程

Late endosome sorting contributed to the transduction activation of type 2 adeno-associated virus in IB3 transformed human bronchial epithelial cells

目的确认Ⅱ型腺相关病毒(adeno-associated virus,AAV2)是否能够在肺上皮细胞中经过囊泡分选过程进入晚期内体,并且进一步检测在内体中分离的腺相关病毒是否具有不同的转导活性,从而探讨内体转运系统在腺相关病毒感染动力学中的作用。方法以RAB7的GFP融合蛋白作为晚期内体的特异性标记,通过对Ⅱ型腺相关病毒进行荧光标记,在人支气管上皮转化细胞IB3中进行共聚焦显微成像的共定位分析。通过密度梯度离心和内体亲和沉淀对进入细胞内体的病毒颗粒进行分离,并对细胞进行二次感染时的活性在有或无RAB7影响因素的条件下进行比较分析。结果相当部分的Ⅱ型腺相关病毒在感染IB3细胞后能够进入晚期内体且进入晚期内体的病毒比未经细胞分选的病毒表现出更好的外源基因表达活性。结论提示Ⅱ型腺相关病毒在肺支气管上皮细胞中晚期内体的转运可能促进其转导的活化过程。

Objective To test whether type 2 adeno-associated virus（AAV2） is able to undergo vesicular sorting and traffic into the late endosome,and then evaluate the endosome-contained AAV2 possess different transduction activities,thus to imply the role of endosomal trafficking in the activation of AAV2 transduction in transformed human bronchial epithelial cells.Methods Using the RAB7 GFP fusion protein as the specific late endosome marker,its colocalization with AF568 fluorochrome labeled AAV2 was analyzed by confocal microscopy.The infected AAV2 was retrieved by gradient centrifugation and magnetic beads-based immunoisolation together with the endosome,and then subjected to the reinfection of IB3 cells.The efficiency of the AAV2 transgene expression was compared under the conditions without and with the modulation of RAB7 functions.Results It was found that a significant portion of the endocytosed AAV2 was able to traffic into the late endosome in IB3 cells.The AAV2 recovered from the late endosome exerted better transduction activities than the native viruses.Conclusion The results implied that the vesicular sorting of AAV2 into the late endosome might be an important step for the activation of the AAV mediated transgene expression.