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本文用Wistar大鼠肺巨噬细胞(alveolar macrophage,简称AM)的胞浆标志酶乳酸脱氢酶(lactate dehydrogenase,简称LDH)和溶酶体的标志酶β-葡萄糖醛苷酶 (β-D-glucuronidase,简称β-glu)的漏出为指标,观察了超声、反复冻融和Triton X-100三种处理方法破碎细胞浆膜和溶酶体膜的适宜条件.并对三种方法的破碎效果进行了比较.结果证明:(1)用形态学方法证明,LDH漏出百分数确实可作为细胞膜破碎程度的指标.(2)三种处理方法的适宜条件分别是:超声时间为50s,超声振幅为10;-20℃冰箱反复冻融5次;0.2%的Triton X-100作用30min.(3)三种处理方法对胞膜的破碎效果是相同的.反复冻融对溶酶体膜的破碎效果低于超声和Triton X-100.本文还对三种破碎细胞方法的优缺点以及处理因子对酶活性的影响进行了讨论.
In this study, the cytosolic markers lactate dehydrogenase (LDH) and lysosomal enzyme β-glucuronidase (β-D-glucuronidase) in Wistar rat alveolar macrophage (AM) glucuronidase (abbreviated as β-glu) as an indicator to observe the appropriate conditions for the disruption of cytoplasmic membrane and lysosomal membrane by ultrasonic, repeated freeze-thaw and Triton X-100 treatment methods, and the crushing effect of the three methods The results showed that: (1) Morphology proved that the leakage percentage of LDH could indeed serve as an indicator of the degree of cell membrane disruption. (2) The suitable conditions for the three treatment methods were as follows: ultrasonic time 50s, ultrasonic amplitude 10 ; Freezing and thawing at -20 ℃ for 5 times; 0.2% Triton X-100 for 30min. (3) The three methods of treatment were the same on the cell membrane, the effect of repeated freezing and thawing on lysosomal membrane fragmentation was low Ultrasound and Triton X-100. The advantages and disadvantages of the three methods of cell disruption and the effect of treatment factors on enzyme activity are also discussed.