Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells
Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells
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DOI：
10.1095/biolreprod60.4.1013
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发表时间：
1999-04-01
影响因子：
3.6
通讯作者：
Prather, RS
中科院分区：
文献类型：
--
作者：
Boquest, AC;Day, BN;Prather, RS
核移植胚胎的正常发育被认为依赖于细胞周期的G 0或G1期的核转移。因此，我们研究了在各种细胞周期阻滞处理下培养的猪胎儿成纤维细胞的细胞周期特征。这是通过使用面粉细胞仪同时测量细胞DNA和蛋白质含量来实现的，从而能够计算细胞周期的CO、G1、S和G2+M期的细胞百分比。与快速循环培养物（G 0 +G1：74.1 +/- 3.0; G 0：2.8 +/- 1.2）相比，血清饥饿5天的培养物含有更高（p < 0.05）百分比的G 0 + G1（87.5 +/- 0.7）和单独的G 0细胞（48.3 +/- 9.7）。与循环培养物相比，生长至汇合增加了（p < 0.05）G 0 +G1百分比（85.1 +/- 2.8），但没有增加GO百分比（6.0 +/- 5.3）。对小、中、大尺寸细胞的单独评估显示，随着细胞尺寸从大到小减小，G 0 +G1和单独G 0中的细胞百分比增加（p < 0.05）。我们在单独的G 0 +G1和GO中分别发现95.2 +/-0.3%和72.2 +/-12.0%的小血清饥饿细胞。还用细胞周期抑制剂处理培养物。用二甲基亚砜（1%）或秋水仙素（0.5 μ M）处理分别增加了CO（24.8 +/- 20.0）或G2+M（37.4 +/- 4.6）中的细胞百分比。然而，细胞仅对含羞草碱处理有轻微反应。更全面地了解供体细胞的细胞周期应能提高核移植程序的效率。
Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in GO or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flour cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in CO, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0.7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase GO percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and GO alone increased (p < 0.05). we found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and GO alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 mu M) increased percentages of cells in CO (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.