骨髓基质干细胞(BMSCs)细胞毒最佳实验条件的研究

On the Optimal Experiment Conditions in Cytotoxicity Tests of BMSCs

目的探讨CCK-8法和MTT法在骨髓基质干细胞(Bone marrow stromal cells,BMSCs)细胞毒实验中的最佳实验条件.方法取对数生长期的P3大鼠BMSCs,制成不同浓度的细胞悬液,于96孔培养板中培养24 h,分别用CCK-8法、MTT法检测其吸光度(A)值,根据细胞浓度与A值关系曲线确定两种方法各自的最适细胞浓度;取最适浓度的细胞于0.5,1,2,3,4和5 h 6个不同时间点,分别于450,490,595 nm处测定吸光度A值,确定两种方法最佳的检测时间和最适检测波长;采用CCK-8和MTT法检测5个浓度的鹿茸Ⅰ型胶原对BMSCs的增殖的影响.结果 CCK-8法测得的A值与细胞数目之间的线性相关性优于MTT法;CCK-8法、MTT法最佳检测波长分别为450,490 nm,最佳检测时间分别为1,4 h;在检测鹿茸Ⅰ型胶原蛋白对BMSCs的增殖的影响时,CCK-8法测得数据的线性相关性优于MTT法,并且数据偏差较MTT法小.结论 CCK-8法与MTT法检测结果相比线性相关性好,灵敏度高,准确性好.在BMSCs细胞毒检测中CCK-8法优于MTT法.

Objective To investigate the optimal experiment conditions for MTT and CCK-8 method in cytotoxicity tests of bone marrow stromal cells（ BMSCs）. Methods The P3 rat bone marrow stromal cells in lograrithm growth stages were prepared to cell suspensions in different cell concentrations,the BMSCs were planted in 96-well plates to be cultured for 24 h,CCK-8 and MTT methods were applied to test A value,respectively. The appropriate cell concentration ranges were determined according to the curves of cell number vs A;A values of the BMSCs in the appropriate cell concentrations at 450,490,595 nm were measured at the different time points,namely,0. 5h,1h,2h,3h,4h and 5h,respectively,to determine the optimal test time and wavelength of CCK-8 and MTT methods. CCK-8 and MTT methods were used to detect the influence of five velvet antler type I collagen at the five concentrations on the proliferation of rat BMSCs. Results The linearity of cell number vs A values detected by CCK-8 was better than that by MTT;The best test wavelength of CCK-8 and MTT method was450 and 490 nm,respectively,and the best test time was 1 and 4 h,respectively;the linearity of measured data byCCK-8 was better than that by MTT,and the data deviation was smaller than that by MTT,when the influence of velvet antler type I collagen on the proliferation of rat BMSCs. Conclusion The linearity of CCK-8 between A and cell viability was better than that of MTT,and CCK-8 assay was better than MTT assay in the detection sensitivity and accuracy. CCK-8 method was better than MTT method in the cytotoxicity test of BMSCs.