Sheridan J W, Bishop C J, Simmons R J
J Cell Sci. 1981 Jun;49:119-37. doi: 10.1242/jcs.49.1.119.
An investigation was made of the sequential biophysical and morphological changes that occur as cultured human melanoma cells (MM96) outgrow their supply of nutrient. Simultaneous buoyant-density and velocity-sedimentation fractionation experiments were used to characterize cells from 3 kinetically differing types of culture. Cells from exponential cultures were large, moderately dense and rapidly sedimenting; cells from post-exponential cultures were of intermediate size, less dense and much more slowly sedimenting; and dye-excluding cells from reproductively non-viable late post-exponential cultures were small, of widely variable though generally high density and sedimented moderately rapidly. Although reproductive viability was high in cells from both exponential and post-exponential cultures, depletion of clonogenic cells was seen at the extremes of the distribution profiles of cells fractionated by either method. This was particularly evident at the low-density extreme of the buoyant-density profiles where cells retained viability despite their loss of proliferative potential. As cells became post-exponential, nuclear size diminished in parallel with cell size, the number of microvilli declined, mitochondria condensed, cytoplasm vacuolated, the frequency of osmiophilic vacuolar inclusions rose, chromatin clumped and nucleoli became prominent. Progression to a reproductively non-viable late post-exponential state resulted in a continued parallel fall in nuclear size, increased cytoplasmic blebbing, further mitochondrial condensation, an increased proportion of cytoplasmic vacuoles containing osmiophilic material, the major part of which was melanin, and further clumping and margination of chromatin. Cells progressed rapidly from this newly described pre-apoptotic state to death by apoptosis, a process characterized by the budding and division of cells into a number of ultrastructurally well-preserved membrane-bound fragments.
对培养的人黑色素瘤细胞（MM96）耗尽营养供应时发生的一系列生物物理和形态变化进行了研究。同时进行了浮力密度和速度沉降分级实验，以表征来自3种动力学不同类型培养物的细胞。指数生长期培养物中的细胞体积大、密度适中且沉降迅速；指数生长期后培养物中的细胞大小中等、密度较小且沉降慢得多；而来自增殖性无活力的指数生长期后期培养物的拒染细胞体积小、密度变化很大但通常较高，沉降速度适中。尽管指数生长期和指数生长期后培养物中的细胞增殖活力都很高，但在用任何一种方法分级的细胞分布曲线的两端都观察到克隆形成细胞的减少。这在浮力密度曲线的低密度端尤为明显，在那里细胞尽管失去了增殖潜力但仍保持活力。随着细胞进入指数生长期后期，细胞核大小与细胞大小同步减小，微绒毛数量减少，线粒体浓缩，细胞质空泡化，嗜锇性液泡内含物的频率增加，染色质聚集，核仁突出。进展到增殖性无活力的指数生长期后期状态导致细胞核大小持续同步下降，细胞质泡化增加，线粒体进一步浓缩，含有嗜锇性物质（主要是黑色素）的细胞质空泡比例增加，染色质进一步聚集并边缘化。细胞从这种新描述的凋亡前状态迅速进展到凋亡死亡，这一过程的特征是细胞出芽并分裂成许多超微结构保存完好的膜结合碎片。
