一种定量分析小鼠单核细胞体外吞噬诱导凋亡卵泡颗粒细胞能力的方法

Quantitative measurement of in vitro phagocytosis of apoptotic granulosa cells by monocytes in mice

为了建立一种简单有效的定量分析吞噬能力变化的研究方法,本实验采用体外培养和大蒜诱导凋亡的方式,模拟体内单核细胞吞噬凋亡的卵泡颗粒细胞的过程,在不同时点(1h,2h,3h,4h,5h)对共培养物进行瑞氏染色后,用显微镜观察并照相,对吞噬程度随时间变化的规律进行回归分析。结果显示,在颗粒细胞凋亡形变的初期,即可被单核细胞识别,形成吞噬泡,与此同时,颗粒细胞的凋亡程度也越来越大,在吞噬泡内可见凋亡细胞崩解的碎片。对照片进行分类计数发现,单核细胞与颗粒细胞共同培养约3h的时候,外接凋亡细胞的单核细胞个数与内吞凋亡细胞的单核细胞个数的比值接近1,说明此时单核细胞中半数呈现吞入状态,半数呈现外接(识别阶段)状态,笔者建议将此状态的时间点称为"半吞期"。SPSS13.0回归分析,可得线性回归方程y=–0.247x+1.644(y,外接与内吞的单核细胞个数比值;x,共培养时间),R2=0.912,回归方程显著性检验F=31.095,P=0.011(<0.05),回归系数显著性检验T=–5.576,P=0.011(<0.05)。以上结果表明,这种对早期凋亡细胞吞噬过程的体外模拟可以作为一种定量分析的方法,判断吞噬能力的变化,从而可用于对一些影响因素,如促进或抑制吞噬的药物,进行定量分析。

To establish a method for quantitative measurement of phagocytosis, the phagocytic process of apoptotic granulosa cells by monocytes was imitated in vitro. Monocytes and granulosa cells were isolated from Kunming mice and cultured. Granulosa cells were induced to apoptosis by garlic, and then co-cultured with monocytes. At different time points （1 h, 2 h, 3 h, 4 h, 5 h）, co-cultured cells were observed by microscope after Wright＇s staining. The results showed that at the beginning of morphological changes in apoptotic granulosa cells, monocytes captured the apoptotic cells. Meanwhile, the apoptosis of granulosa cells were progressing. Debris was found in phagocytic vacuole. At the point of 3 h after co-culture, the ratio of monocytes which attached to apoptotic granulosa cells to those which engulfed the apoptotic cells was close to one. Namely, half of monocytes were in the state of recognition and half were in the state of engulfment, and this time point was named as ＇half phagocytic period＇. Regression analysis showed that the equation of linear regression was y=-0.247x ＋ 1.644 （y represents Attachment/Engulfment ratio, x represents co-culture time）, R^2=0.912, F= 31.095, P=0.011 （〈0.05）, T=-5.576, P=0.011 （〈0.05）. In conclusion, the present mode of phagocytosis in vitro can be used as a method to quantitatively assay some effective factors such as medicines which could enhance or restrain phagocytosis.