胰岛素样生长因子结合蛋白3通过线粒体途径诱导软骨细胞凋亡

Exogenous exogenous insulin-like growth factor binding protein -3 induces chondrocyte apoptosis through Nur77 nucleus -mitochondria trauslocation

目的观察外源性胰岛素样生长因子结合蛋白-3（IGFBP-3）对诱导软骨细胞凋亡的效应，探讨其是否通过“细胞核内蛋白Nur77-线粒体易位途径”诱导凋亡。方法原代培养大鼠软骨细胞并传代2—3次后，转入无血清培养培养环境，添加不同浓度的外源性IGFBP-3以及设置对照组，同时处理24h后，分别通过普通及荧光显微镜观察IGFBP-3对软骨细胞的形态学影响、细胞计数试剂盒（CCK-8）吸光度法和流式细胞仪检测软骨细胞凋亡比例、激光共聚交显微镜观察IGFBP-3诱导细胞核内受体Nur77。结果（1）原代培养的单个软骨细胞贴壁后呈不规则多边形，生长至融合时呈“铺路石样”，经过数次传代，细胞由多边形逐渐伸展为成纤维细胞样；（2）普通明场和荧光显微镜下，均可见凋亡小体，表明外源性IGFBP-3软骨细胞能诱导软骨细胞凋亡；（3）CCK-8数值：空白组为1．43±0．03；阴性对照组为1．44±0.05；IGFBP-3组（0．5、1．0μg／ml与2．0μg／m1）分别为1．31±0．03、1．06±0．07、0．82±0．10；空白组与阴性对照组差异无统计学意义（P=0．845），IGFBP-3组内差异有统计学意义（P=0．000、0．000），IGFBP-3组与空白组和阴性对照组分别差异有统计学意义（P=0．000）；（4）流式细胞仪结果：空白组为97．57％；阴性对照组为97．63％；IGFBP-3组（0．5-2．0μg／m1）分别为90．03％、80．03％和67．33％，统计学分析结果同CCK-8；（5）激光共聚交显微镜观察表明，IGFBP-3处理组中，细胞核内受体Nur77从细胞核易位至线粒体，而空白组和阴性对照组均未见此现象。结论IGFBP-3可通过诱导Nur77从细胞核易位至线粒体，从而诱导软骨细胞凋亡。

Objective Observing whether exogenous insulin -like growth factor binding protein -3 （IGFBP- 3） could induce chondroeyte become apoptotic, and examining if the nucleus - mitochondria transloeation of Nu77 （ orphan nuclear receptor） was concerned. Methods Primary cultured rat chondro- cyte was harvested and underwent 2 - 3 passages, then they were transferred to serum - free culture. We exposed these chondrocytes in the IGFBP - 3 with gradient concentrations and the controls, following another 24 hrs incubation. Then the morphological observations by bright field or fluorescence microscope, the cell vitality by cell counting kit - 8 （ CCK - 8） assays, the apoptotic proportion of chondrocytes by flow cy- tometry and the Nur77 signal analysis by laser confocal microscope, were respectively done by us. Results （ 1 ） The cultivated and confluent chondrocytes, which were adherent and irregular polygonal after the isola- tion and the primary culture, were ＂stone - road like＂ then extended as fibroblast - like when underwent several passages; （2） The apoptotic bodies were observed under both bright filed and fluorescence microscope, suggesting a apoptotic effect of IGFBP - 3 on chondrocytes ; （3） CCK - 8 tests showed blank control （ 1.43 ± 0.03 ）, negative control （ 1.44 ±0. 05 ） had no statistical difference （ P = 0. 845 ） ; IGFBP - 3 groups （0. 5, 1.0 μg/ml and 2. 0 μg/ml）, which were 1.31 ±0. 03, 1.06 ±0. 07, 0. 82 ±0. 10 respectively, had significant statistical differences between each two of them （ P = 0. 000, 0. 000 ） ; any IGFBP - 3 group also had statistical differences with blank control or negative control （ P = 0. 000） ; （4） flow cytometry results showed as the follows： 97.57% （ blank control）, 97.63% （ negative control）, 90. 03% , 80. 03% and 67. 33% （0. 5, 1.0 μg/ml and 2. 0μg/ml respectively） , and the statistical analysis were the same as the CCK - 8 shows ; 5, A translocation of Nur77 from ehondrocyte nucleus to mitochondria was captured under laser confocal microscope merely in IGFBP - 3 group but the other controls. Conclusion The exogenous IGFBP - 3 could induce rat chondrocytes apoptosis under the nucleus - mitochondria translocation of Nur77.