Campbell J H, Kocher O, Skalli O, Gabbiani G, Campbell G R
Cell Biology Laboratory, Baker Medical Research Institute, Prahran, Melbourne, Australia.
Arteriosclerosis. 1989 Sep-Oct;9(5):633-43. doi: 10.1161/01.atv.9.5.633.
To investigate a possible correlation between cytodifferentiation, proliferation, and actin expression, smooth muscle cells from the 9-week-old rabbit aortic media were enzyme-dispersed into single cells and were plated in primary culture at different initial seeding densities. The volume fraction of myofilaments (Vv myo) in cells seeded moderately densely fell from 39.5% +/- 1.2% in the intact aortic media to 11.5% +/- 1.6% on Day 5, one day before the onset of logarithmic growth. The Vv myo remained low over the next 3 days, then began to rise as the density of cells increased, returning almost to the original levels after confluency and 1.84 cumulative population doublings (CPD). The expression of alpha-smooth muscle actin mRNA followed a similar time course of change, falling from 84.7% +/- 1.2% of total actin mRNA in freshly isolated cells to 54.0% +/- 6.5% on Day 5, returning to 87.5% +/- 0.5% after confluency. In these cultures, the alpha-smooth muscle actin protein content was 93.7% +/- 2.9% of total actin in freshly isolated cells, 68.7% +/- 3.1% on Day 5, and 73.3% +/- 2.5% 3 days after confluency. In densely seeded cultures, the Vv myo and expression of alpha-smooth muscle actin mRNA fell only slightly on Day 5 and rose to original levels upon confluency after 0.33 CPD. However, at the protein level, alpha-smooth muscle actin decreased on Day 5 and remained low on Day 12. The Vv myo, alpha-smooth muscle actin mRNA, and actin protein of sparsely seeded cells fell on Day 5 and then remained low throughout the culture period, including 5 days after confluency (Day 24), when the cells had undergone 5.37 CPD. Cells that were maintained subconfluent but quiescent on Day 7 in culture had the same low Vv myo, low alpha-actin mRNA expression, and low alpha-actin protein content as actively proliferating cells. The results show that Vv myo and alpha-smooth muscle actin mRNA undergo parallel changes during primary culture according to seeding density, but not to replication, and that alpha-smooth muscle actin protein decreases in culture then remains low irrespective of culture conditions.
为了研究细胞分化、增殖与肌动蛋白表达之间的可能相关性，将9周龄兔主动脉中膜的平滑肌细胞用酶分散成单细胞，并以不同的初始接种密度接种于原代培养。中等密度接种的细胞中肌丝的体积分数（Vv myo）在对数生长期开始前一天，即第5天，从完整主动脉中膜的39.5%±1.2%降至11.5%±1.6%。在接下来的3天里，Vv myo保持在较低水平，然后随着细胞密度的增加而开始上升，在汇合和1.84次累积群体倍增（CPD）后几乎恢复到原始水平。α-平滑肌肌动蛋白mRNA的表达遵循类似的时间变化过程，从新鲜分离细胞中总肌动蛋白mRNA的84.7%±1.2%降至第5天的54.0%±6.5%，汇合后恢复到87.5%±0.5%。在这些培养物中，α-平滑肌肌动蛋白的蛋白质含量在新鲜分离细胞中占总肌动蛋白的93.7%±2.9%，在第5天为68.7%±3.1%，汇合后3天为73.3%±2.5%。在高密度接种的培养物中，Vv myo和α-平滑肌肌动蛋白mRNA在第5天仅略有下降，并在0.33次CPD汇合后恢复到原始水平。然而，在蛋白质水平上，α-平滑肌肌动蛋白在第5天下降，并在第12天保持在低水平。低密度接种细胞的Vv myo、α-平滑肌肌动蛋白mRNA和肌动蛋白在第5天下降，然后在整个培养期（包括汇合后5天，即第24天，此时细胞已经历5.37次CPD）保持在低水平。在培养第7天维持亚汇合但静止的细胞，其Vv myo、α-肌动蛋白mRNA表达和α-肌动蛋白蛋白质含量与活跃增殖细胞相同，均较低。结果表明，在原代培养过程中，Vv myo和α-平滑肌肌动蛋白mRNA根据接种密度而非复制情况发生平行变化，并且α-平滑肌肌动蛋白蛋白质在培养中减少，然后无论培养条件如何都保持在低水平。
