人外周血淋巴细胞高分辨染色体制备技术的研究

The preparation of high-resolution chromosome in human peripheral blood lymphocytes

目的：建立一种具有分裂指数高和染色体分散好等优点的550～850条带纹高分辨染色体的制备方法。方法：取10例健康人外周血为样本制备淋巴细胞高分辨染色体。固定5-氟尿嘧啶核苷、尿嘧啶核苷、胸腺嘧啶核苷、溴化乙锭、秋水仙胺的剂量，进行5个因素3个水平的正交设计15种实验方案。5个因素分别为培养时间、胸腺嘧啶核苷、溴化乙锭、秋水仙胺作用时间以及低渗时间。3个水平分别为培养时间64、72、80h；胸腺嘧啶核苷作用时间16、17、18h；溴化乙锭作用时间3、4、5h；秋水仙胺作用时间10、15、20min以及低渗时间30、40、50min。每例样本同时采用15种方案进行实验，比较各方案带纹在550条以上时染色体分裂指数和分裂相分散的情况。结果：在15种方案中培养时间以及秋水仙胺作用时间对分裂指数有显著影响俨〈0．01），其中培养72h后加5-氟尿嘧啶核苷和尿嘧啶核苷进行同步化和秋水仙胺作用15min高分辨染色体分裂指数最大。37℃低渗40min高分辨染色体分散效果最佳。结论：本实验方案的高分辨染色体制备方法，具有分裂指数高和染色体分散好等优点，具有较好的推广价值。

OBJECTIVE： To develop a method for human 550-850 band high-resolution chromosome preparation with advantages such as high mitotic division index and good dispersion rate. METHODS： High-resolution chromosomes were prepared from 10 periphearal blood samples. Doses of 5-fluorouridine, uridine, thymidine, ethidium bromide and demecolcine were constant among 15 experimental designs through arranging 5 factors and 3 standards. 5 factors were adding time of 5-fluorouridine and uridine, action time of thymidine, ethidium bromide and demeeolcine. 3 standards referred to addition of 5-fluorouridine and uridine at 64, 72 and 80 h, action time of thymidine for 16, 17 and 18 h, of ethidium bromide for 3, 4 and 5 h, of demecolcine for 10, 15 and 20 min, and hypo-osmotic time for 30, 40 and 50 min. Each specimen underwent all 15 kinds of treatment designs at the same time. And high-resolution chromosome division index and good dispersion rate were compared among 15 designs. RESULTS： Great significant differences on high-resolution chromosome division index were found from the action time of 5-fluorouridine, uridine and demecolcine （P 〈 0.01）. The highest division index of high-resolution chromosome was obtained when cells had been cultured for 72 h before adding 5-fluorouridine and uridine and havested 15 min after adding demecolcine. The best dispersion high- resolution chromosomes could be made when cells had been diluted for 40 min at 37 ℃. CONCLUSION： The method of human high-resolution chromosome preparation from the first experimental design, with advantages such as high cell division index and good dispersion rate, showed great value to be extended to G-banding preparation.