中文摘要：

目的探讨环孢菌素A（CsA）对肾小管上皮细胞（TEC）自噬．溶酶体通路的影响。方法不同浓度（3、5、10μmol／L）CsA刺激人近端肾小管上皮（HK-2）细胞24h。用MTT法检测细胞活力；AnnexinV—PI双染流式细胞术检测细胞凋亡；免疫荧光法检测细胞自噬相关蛋白微管相关蛋白1轻链3（LC3）Ⅱ和p62表达；共聚焦显微镜下观察mRFP—GFP串联荧光标记LC3（tfLC3）质粒转染HK．2细胞后自噬体与溶酶体融合及降解情况；用流式细胞术检测HK-2细胞溶酶体对DQ-卵清蛋白的消化能力。结果与对照组相比，5、10μmol／LCsA组细胞活力下降（均P〈0．01）；细胞凋亡数增多（均P〈0．05）。与对照组相比，3、5、10μmol／LCsA组细胞LC3-II和p62表达增加（均P〈0．05）；自噬体降解受阻；细胞溶酶体消化DQ-卵清蛋白能力下降（均P〈0．01）。结论CsA通过损害溶酶体功能而阻滞肾小管上皮细胞自噬通路，可能是CsA肾病的发生机制之一。

英文摘要：

Objective To investigate the effect of cyclosporine A （CsA） on autophagylysosomal pathway in tubular epithelial cells. Methods Human renal tubular epithelial cell line （HK-2 cell） was treated with different concentrations （3, 5 and 10 μmol/L） of CsA for 24 h. Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV- PI staining followed by flow cytometry analysis, respectively. Autophagyrelated protein LC3- H and p62 were detected by immunofluorescence assay. Autophagic flux was analyzed in HK- 2 cells transfected with a tandem mRFP- GFP fluorescenttagged LC3 （tfLC3） plasmid by laser confocal microscope. The lysosomal degradation was evaluated by DQ- ovalbumin staining followed by flow cytometry analysis. Results The viability of HK- 2 cells was significantly decreased with CsA stimulation when compared with control group （P 〈 0.01）, but the number of apoptotic cells was markedly increased by CsA treatment （P 〈 0.05）. Compared with the control group, different doses of CsA dramatically increased the expressions of LC3- II （P 〈 0.01） and p62 （P 〈 0.05） in HK-2 ceils. Moreover, HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes. In HK-2 cells, exposured to CsA caused a decrease in lysosomal degradation by DQ- ovalbumin staining when compared with control group （P 〈 0.01）. Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.