目的: 采用CFSE法与CCK-8法评价同种异体骨对人外周血淋巴细胞的活性与增殖效应。方法: 通过人新鲜外周血淋巴细胞与供试品(脱钙骨基质与冻干型组织工程骨)浸提液共培养后,采用CCK-8法检测淋巴细胞的活性水平。通过CFSE标记的人新鲜外周血淋巴细胞与供试品浸提液共培养后,采用流式细胞术检测淋巴细胞增殖能力。结果: CCK-8法试验结果表明,脱钙骨基质组的吸光度值与培养液对照组相比无显著性差异,而冻干型组织工程骨组的吸光度值与培养液对照组相比显著增高(P<0.05),表明冻干型组织工程骨提高了淋巴细胞的活性。CFSE法试验结果表明,与培养液对照组相比,冻干型组织工程骨组的淋巴细胞增殖相关指标(Dil、PF、EI、RI)以及淋巴细胞亚群增殖指标Dil均无显著性差异(P>0.05),表明冻干型组织工程骨未对人外周血淋巴细胞的增殖产生显著性影响。结论: 与培养液对照组相比,冻干型组织工程骨提高了人外周血淋巴细胞的活性,但是未对人外周血淋巴细胞的增殖产生显著影响。CCK-8法主要反映的是细胞活性,而CFSE法可以更客观地反映细胞有无增殖。
Objective: To investigate the effect of allogeneic bone on viability and proliferation of human peripheral blood lymphocyte by CCK-8 method and CFSE method. Methods: The lymphocyte viability was detected by CCK-8 method after co-culture of human fresh peripheral blood lymphocytes and the sample extraction (decalcified bone matrix and freeze-dried tissue engineered bone). Also, the lymphocyte proliferation was determined by flow cytometry after co-culture of fresh peripheral blood lymphocytes labeled with CFSE and the sample extraction. Results: The result of CCK-8 method showed that there was no significant difference in absorbance value between decalcified bone matrix and medium control. But there was a significant increase of absorbance value in freeze-dried tissue engineered bone compared with that in medium control (P<0.05), suggesting that freeze- dried tissue engineered bone improved lymphocyte viability. The result of CFSE method showed that there was no significant difference in Dil, PF, EI and RI of lymphocyte, and Dil of lymphocyte subtype between freeze-dried tissue engineering bone and medium control (P>0.05). The results suggested that freeze-dried tissue engineered bone had no significant effect on the proliferation of human peripheral blood lymphocyte. Conclusion: Compared with medium control, decalcified bone matrix has no significant effect on human peripheral blood lymphocyte viability. Freeze-dried tissue engineered bone improves the human peripheral blood lymphocyte viability, but dose not significantly affect the proliferation of human peripheral blood lymphocyte. CCK-8 method mainly reflects the cell viability, but CFSE method could reflect the cell proliferation more reasonable.
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