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用人体肝癌细胞株(7402)和中国地鼠肺细胞系(CH-CL)的细胞建立了同步化方法:7402细胞在指数生长时,加入秋水仙胺处理后,用机械摇晃法收集中期细胞。或者在传代培养的7402细胞中加入TdR的DNA合成抑制剂,培养24小时后更换新的培养液解除细胞的抑制。同上加入秋水仙胺使细胞停止在分裂中期。这两种方法均可获得85～96％的中期细胞。此外,按上述方法加入秋水仙胺处理细胞并结合机械摇晃法,同样可收获70～90％的同步的中期细胞。染色体分离方法:按上述同步化收集的中期细胞,用Hanks液洗涤,TM低渗液处理,离心;去上清液,加入TMS缓冲液与细胞沉淀混匀,转入注射器或研磨器内,吸打或研磨细胞,使细胞膜破裂释放出染色体,再离心,收集上清液,在沉淀中再加入TMS缓冲液,低速离心,再收集上清液。这样反复离心,收集的上清液汇集于大离心管内,再离心,去上清液,加入TMS液混匀,低速离心,收集上清液。如此反复,就可获得纯度高的中期染色体。最后比较了两种同步化方法的优劣,并讨论了分离中期染色体的各种因素。
Synchronization was established using human hepatoma cell line (7402) and Chinese hamster lung cell line (CH-CL) cells: 7402 cells were treated with colcemid and exponentially growing, and midgut cells were harvested by mechanical shaking. Alternatively, TdR DNA synthesis inhibitor was added to subcultured 7402 cells, and after 24 hours of incubation, the medium was replaced with fresh medium to release the cells. Colchicine was added as above to stop the cells in the metaphase. Both methods yield 85-96% of metaphase cells. In addition, colchicine treatment of cells as described above in combination with a mechanical shaking method can also harvest 70-90% of synchronized metaphase cells. Chromosome separation method: according to the synchronization of the collected metaphase cells, washed with Hanks solution, TM hypotonic solution, centrifuged; to the supernatant, add TMS buffer and the cell pellet and mix, transferred to a syringe or grinder, suction Hit or grind the cells, rupture the cell membrane to release the chromosomes, centrifuge again, collect the supernatant, re-add the TMS buffer to the pellet, centrifuge at low speed, and collect the supernatant. This repeated centrifugation, collected supernatant collected in a large centrifuge tube, and then centrifuged to the supernatant, adding TMS liquid mix, low speed centrifugation, the supernatant was collected. So repeated, you can get high purity metaphase chromosomes. Finally, the advantages and disadvantages of the two methods of synchronization were compared, and various factors of metaphase chromosomes were discussed.