检测破骨细胞内活性氧水平方法的实验探究与应用

Inve stigation and application of the me thod to me asure intrace llular ROS in oste oclasts

目的探究可以有效、准确且直观地检测破骨细胞内活性氧(ROS)的方法,并应用其对破骨细胞分化过程中活性氧的变化进行检测。方法培养RAW264.7细胞系作为破骨前体细胞。控制细胞密度铺板后装载检测活性氧的2,7-双乙酸二氯荧光素(DCFH-DA)荧光探针,分别检测阴性对照组(只加α-MEM培养基)、阳性对照组(ROSup)、核因子κB受体活化因子配体(RANKL)组(100ng/mlRANKL)和N-乙酰左旋半胱氨酸(NAC)组(100ng/mlRANKL+1m MNAC)破骨细胞的活性氧水平。检测方法包括使用荧光显微镜观察不同组的荧光强度并对阳性细胞计数统计和使用流式细胞仪检测不同组的荧光强度并对Mean FL1-A定量分析。结果阴性对照组与阳性对照组比较,定性的荧光照片和阳性细胞计数统计,与定量的流式细胞术统计图和Mean FL1-A统计分析,都显示阳性对照组的细胞内活性氧水平远高于阴性对照组(P<0.001和P<0.001)。同时,RANKL组的活性氧水平高于阴性对照组(P<0.05和P<0.01),而NAC组的水平较RANKL组为低(P<0.001和P<0.001)。结论定性观察方法与定量检测方法结果均有效并且高度一致。两种方法的综合使用可以直观准确地检测破骨细胞内的活性氧。使用该方法可验证破骨细胞分化过程中活性氧的增加,并且NAC可抑制此变化。

Objective To explore an effective and accurate ROS measurement method in osteoclasts, and to apply it in the study of ROS alteration during osteoclastogenesis.Methods RAW264.7 cell line was cultured as osteoclast precursors.ROS probe with DCFH-CA was utilized to examine the intracellular ROS in the negative control group （containing α-MEM only）, positive control group （ROSup）, RANKL group （100 ng/ml RANKL）, and NAC group （100 ng/ml RANKL ＋1 mM NAC）.ROS positive cells were observed with fluorescence microscope and counted.The level of intracellular ROS was measured with flow cytometry and the mean FL1-A was analyzed.Results The level of ROS in positive control group was significantly higher than that in negative control group （P〈0.001, P〈0.001）.Meanwhile, ROS in RANKL group was significantly higher than that in the negative control group （P〈0.05, P〈0.01）, and ROS in NAC group was significantly lower than that in RANKL group （P〈0.001, P〈0.001）.Conclusion Both qualitative and quantitative measurements of ROS are effective and accordant.ROS may be examined accurately and intuitively using combined methods.The application of the methods verify that ROS increases during osteoclastogenesis, and the increase can be inhibited by NAC.