大鼠外周血单个核细胞保存方法研究

Study on Preservation Methods of Peripheral Blood Monouclear Cells of Rat

通过观察大鼠外周血单个核细胞（PBMCs）的形态，了解不同保存方法和不同保存时期对PBMCs活性和细胞周期分布的影响，建立其短期保存方法．用密度梯度离心法获得大鼠外周血单个核细胞，分别在RPMI1640培养液中4℃保存和冷冻保护剂中－80℃保存，并分别在第1－4天和第7天，14天，21天，28天，35天用台盼蓝染色法测定细胞活力，用流式细胞仪检测细胞周期．结果分离获得的PBMCs成活率为97．69％±1．59％，基本处于G0／G1期．PBMCs置4℃保存4d后，存活率在60．84％±2．75％；置－80℃保存35d后，存活率在78．54％±2．97％，PBMCs基本处于G0／G1期．最后得出－80℃低温保存方法对PBMCs活性和细胞周期检测有一定影响，但其活性率接近80％，是一种简便易行短期保存PBMCs的方法的结论．

The gole of study was to explore a simple and effective method of short-term preservation for peripheral blood monouclear cells （PBMCs） of rat by observation morphous of PBMCs. PBMCs got from venous blood of the rat by density gradient centrifugation were preserved in RPMI1640 at 4 ℃ and -80 ℃ refrigerator, respectively. Ttrypan-blue chromophore assay was used to detect the viability and flow cytometry to detect the cell cycle of PBMCs after 1 day, 2 days, 3 days,4 days,and 7 days, 14 days,31 days,28 days, 35 days,respectively. The rate of the viability of PBMCs was 97.69 %±1.59 %,and 60.84 %±2.75 % preserved at 4 ℃ for 4 days,and 78.54 %±2.97 % at -80℃ for 35 days, respectively. Almost all cells gathered in G0/G1 phase while cell debris added when preservation time prolonged. So preservation of PBMCs at -80 ℃ refrigerator was simple and effective method to keep stability and viability （80 %） of PBMCs.