【作者】 于玲珠；

【导师】 刘娣；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 本实验旨在建立成纤维细胞的培养系统和细胞周期同步化方法从而为体细胞核移植提供良好的供体细胞。 实验中改进了胰蛋白酶消化法，成功地分离和培养了多种鼠成纤维细胞和多种猪成纤维细胞。 在培养猪耳皮肤成纤维细胞时从筛选细胞分离方法、比较供体年龄和改善培养条件这三方面着手提高培养的成功率。实验中发现胰蛋白酶冷热处理结合法培养的细胞存活率明显高于胰蛋白酶热处理法，说明使用胰蛋白酶冷热处理结合法对细胞的不利影响较少，细胞产量较高。实验发现来自幼年个体的细胞比来自老年个体的易于培养。在培养条件方面选取DMEM／F12培养液+20％胎牛血清培养猪耳皮肤成纤维细胞，效果较好。在进行传代培养时，利用了流式细胞仪检测了培养细胞的凋亡情况，发现传至第12代的细胞仍无明显凋亡现象，细胞已传至16代以上，说明应用此方法可稳定地进行猪耳皮肤成纤维细胞的传代培养。 一般认为核移植胚的正常发育取决于供体核处于细胞周期的G0期或G1期。因此，实验中对培养的猪耳皮肤成纤维细胞进行抑制处理，并且对处理前后细胞的周期分布和凋亡情况做进一步研究。 通过流式细胞仪来同时检测细胞的DNA和蛋白质的含量，这样就能够计算出细胞处于G0期，G1期，S期以及G2期+M期的百分率。与正常对照组的细胞相比经过血清饥饿处理、汇合处理及艾菲地可宁处理的细胞包含更高的百分率处于G0期及G0期+G1期。 对于小的，中等的，大的细胞分别检测时发现随着细胞体积的由大到小，正常对照组、饥饿处理组及汇合处理组的细胞处于G0期及G0期+G1期的百分率显著升高，艾菲地可宁处理组细胞处于G0期的百分率明显升高。汇合处理后的小细胞处于G0期的百分率显著升高，且明显高于其他处理。解除这些处理后细胞周期能迅速恢复。实验证明这些处理是可逆的。 通过流式细胞仪检测，发现不同处理的猪耳皮肤成纤维细胞的凋亡率均较低。在几种细胞周期抑制处理中，汇合处理后的细胞凋亡率最低。 猪耳皮肤成纤维细胞能够有效地同步化于G0期及G0期+G1期，而且不会影响扩增能力。更多还原

【Abstract】 The purposes of this experiment were to establish cuture systems of fibroblasts and methods of cell cycle synchronization so as to provide better donor cells for somatic nuclear transfer.In this experiment, the method of trypsin digestion was improved, and that several kinds of mouse fibroblasts and porcine fibroblasts were successfully dissociated and cultured.During the culture of the porcine ear skin fibroblasts, the successful rate was increased by selecting culture methods, contrasting donor age and improving culture condition.The rate of living cells in the method of combining trypsin cold treatment with trypsin heat treatment was much higher than that in the method of trypsin heat treatment.The results showed that the method of combining trypsin cold treatment with trypsin heat treatment had less adverse effects on cells than the method of trypsin heat treatment. The harvest quantity of cell in the method of combining trypsin cold treatment with trypsin heat treatment was higher than that in the method of trypsin heat treatment. The culture of young individual’s cells was easier than that of adult individual’s cells. The culture results of the porcine ear skin fibroblasts was good by using DMEM/F12 containing 20% fetal bovine serum. The apoptosis in the passage cells was detected by using flow cytometry.It was fmded that the apoptosis of passage 12 cells was not obvious. Cells were subcultured to sixteenth passage.It proved that the subculturing of porcine ear skin fibroblasts was stable by using this kind of method combining trypsin cold treatment with trypsin heat treatment.Normal development of nuclear transfer embryos was thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore,the cell cycle characteristics and the apoptosis of porcine ear skin fibroblasts cultured under a variety of cell cycle-arresting treatments were investigated.This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S and G2+M phases of the cell cycle. Compared to values in cycling cells, higher percentages of cells in GO+G1 and GO alone were apparent when cells were serum starved or cultured to confluency or treated with aphidicolin.Separate assessment of small-, medium- and large-sized cells showed that as the cell size decreased from large to small, percentages of cycling cells, starved cells and confluent cells in G0+G1 and G0 alone increased remarkably, percentages of aphidicolin treatment cells in G0 increased obviously. Percentages of small cells after confluency treatment in G0 increasedremarkably and were higher than those of other treatments obviously. The effects of these treatments were reversible, and their removal led to a rapid progression in the cell cycle.It was finded by using flow cytometry that the apoptosis rate of porcine ear skin fibroblasts cultured under a variety of cell cycle-arresting treatments was quite low. The apoptosis rate of confluent cells was lowest among a variety of cell cycle-arresting treatments.These data indicated that porcine ear skin fibroblasts could be effectively synchronized at GO-H31 and GO alone without compromising their proliferation capacity.更多还原