钙化纳米微粒诱导人肾小管上皮细胞自噬的研究  

The research on autophagy of human renal tubular epithelial cells induced by calcifying nanoparticles （CNP）

作　　者：刘建河[1] 潘骏[1] 齐隽[1] 

机构地区：[1]上海交通大学医学院附属新华医院泌尿外科,200092

出　　处：《中华泌尿外科杂志》2012年第1期37-40,共4页Chinese Journal of Urology

基　　金：上海交通大学科技基金资助（2007XJ021）志谢：中国科学院药物研究所余强教授对实验设计的热心指导,中国科学院药物研究所戚春婷博士在实验研究过程中的热心帮助.

摘　　要：目的观察不同浓度钙化纳米微粒（calcifyingnaniparticles，CNP）对人肾小管上皮细胞HK-2生长的影响，探讨CNP是否可以诱发体外培养HK-2细胞的自噬作用。方法将0．0125、0．025、0．05、0．1mg／ml等4种浓度CNP加入对数生长期的HK-2细胞，培养12、24、48、72h后，MTT法检测HK-2细胞生长、增殖情况。EGFP—LC3表达质粒转染体外培养HK-2细胞，培养24h后，加入CNP共同孵育，作用3、6、24、48h后收集细胞，透射电镜下检测CNP作用后HK-2细胞自噬体的形成，荧光显微镜检测CNP作用前后HK．2细胞LC-3斑点的形成情况。结果CNP对HK-2细胞的生长抑制作用随着CNP浓度的升高和作用时间的延长而增强，0．05nlg／mlCNP作用48h后-细胞增殖抑制率为14．5％，0．1mg／mlCNP作用72h后抑制率达21．5％。加入CNP6h后透射电镜下可见CNP聚集形成结晶样物并黏附于HK-2细胞表面，HK-2细胞质内见散在的自噬体形成。荧光显微镜下可见转染EGFP—LC3的HK-2细胞质内散在分布的LC-3荧光斑点。结论CNP可以诱发HK-2细胞的自噬作用，可能在肾结石等生物矿化相关疾病发生中起重要的作用。Objective To observe the effects of different concertrations of calcifying nanoparticles （CNP） on growth and proliferation of human renal tubular epithelial cells （HK-2） , and to explore the effects of autophagy of human renal tubular epithelial cells induced by CNP. Methods Normal growth HK-2 cells were cultured in vitro and the culture medium was changed to serum-free medium after cell growth to confluence, CNP with different concentrations were then added and the cell proliferation inhibition was measured by MTT assay after 12, 24, 48 and 72 h concubation. EGFP-LC3 express plasmid was transfected into HK-2 cells, CNP with certain concentration was then added after 24h incubation. The formation of autophagosomes and their adherence to cells were observed by electron microscopy, and EGFP-LC3 staining was used to detect the formation of autophagosome by flourescence microscopy after 3, 6, 24 and 48 h concubation. Results CNP presented a concentration-dependent cell proliferation inhibition on HK-2 cells, the inhibitory ratio in cell proliferation was 14.5% after 48 h co-incubation when CNP density was 0.05 mg/ml, and the inhibitory ratio was 21.5% after 72 h co-incubation when CNP density was 0.1 mg/ml. Autophagosome formation and CNP adherence to cell surface could be observed by electron microscopy 6 h after CNP was added to the DMEM medium. The LC3-positive dots were observed in HK-2 cells incubated with CNP. Conclusions Autophagy of HK-2 could be activated by co-incubation with CNP, which may play an important role in the formation of nephrolithiasis.

关 键 词：钙化纳米微粒 人肾小管上皮细胞 自噬 

分 类 号：R691.4[医药卫生—泌尿科学]