应用荧光细胞分选仪研究白血病的细胞周期

本文用荧光细胞分选仪和秋水仙素结合的方法,体外观察小鼠粒单核细胞白血病(MMoL)、粒细胞白血病(L 833-B)细胞的细胞周期和各时相持续时间。体外培养的白血病细胞于指数增殖阶段加入秋水仙素,持续作用12 h,每隔2 h取1组(两瓶)标本,其中1瓶做涂片,计数核分裂细胞数,计算MI;另1瓶用FACS测细胞DNA相对含量,从组方图中计算出每单位时间G_2M期细胞增加率,以及对照组细胞各时相在细胞群中的比例,以便推算细胞Tc及各时相细胞持续时间。用涂片MI方法得到秋水仙素阻断细胞,不同时间核分裂细胞的累积率,其直线回归方程式,MMoL及L 833-B细胞分别为Y=3.10+0.96 X,Y=1.28+0.86 X;Tc分别为104.2和116.3h。Tc太长,可能主要由于涂片时受秋水仙素细胞毒作用后细胞变性,易于破碎,人为降低单位时间MI值。用FACS测定G_2M细胞在秋水仙素作用后其积累率的直线回归方程式,MMoL及L 833 B细胞的分别为Y=26.13+4.01 X,Y=21.04+3.76 X,由此式计算的Tc分别为24.9和26.6 h。这一结果比以往用ARG法所得Tc分别为16和20 h的长些,可能是由于接种的细胞量不同和细胞开始实验时体外培养时间长短不同而引起的,以及秋水仙素对细胞核分裂期除阻断作用外,还有一定杀伤作用和制备标本时对细胞有一定机械性损伤等因素有关。FACS技术用于研究细胞动力学具有快速、简便和避免用放射性核素等优点。

A STUDY ON THE LEUKEMIC CELL CYCLE WITH FLUORESCENCE ACTIVATED CELL SORTER

By using fluorescence activated cell sorter (FACS) and colchicine, cell cycle and cell-phase durations of mouse myelomonocytic(MMoL) and myelocytic (L833-B) leukemia were studied in vitro. The two kinds of leukemic cells were blocked by colchicine for 12 hours. Two specimens were taken each time with interval of 2 hours following colchicine administration, one of them was prepared for smear, counting the mitotic number, estimating MI, and calculating Tc; the other was used for assay of the DNA content of each cell, estimating the increase rate of G_2M phase, and at least calculating Tc. The following results were obtained: Using the MI method, the cell cycle of MMoL and L_(833_B)cells were 104.2 hrs and 116.3 hrs respectively. The cell cycle were artificially prolonged due to decrease of MI caused by colchicine which not only blocked the cells in metaphase, but also destroyed these cells. Using FACS technique to estimate the increasing rate of G2M phase during the period of colchicine administration, cell cycle of the leukemia of MMoL and L_(833-B) were calculated as 24.9 hrs and 26.6 hrs respectively. These figures were near the real cell cycle or a little longer than them. There might be some loss of metaphase due to cytotoxic action of colchicine and the preparing process of samples. FACS technique in the study on cell kinetics has the advantage of fast, simple, and without using radionuclides.