流式细胞术分析比较癌症患者新鲜和冻存复苏后外周血淋巴细胞亚群实验研究.docVIP

流式细胞术分析比较癌症患者新鲜和冻存复苏后外周血淋巴细胞亚群实验研究 　　[摘要] 目的 建立标准化的实验和分析方法，采用流式细胞术比较分析癌症患者新鲜和冻存复苏后的外周血淋巴细胞亚群的差异。 方法 于2014年6～12月收集肺恶性肿瘤患者静脉血样本（2 mL/样本），每个样本取300 μL全血用于淋巴细胞亚群分析；剩余全血经裂解红细胞后冻存 （40%或90%胎牛血清）。30 d后复苏细胞，流式细胞术分析淋巴细胞亚群变化。 结果 复苏并培养24 h后，癌症患者PBMC的存活率分别为（67.70±2.80）%（90% FCS） 和（80.47±3.32）% （40% FCS，P=0.0069）。新鲜和冻存的的外周血CD3+、CD3+CD4+、CD3+CD8+、CD3-CD16+56+、CD3+CD16+56+和CD4+CD25+ T细胞的百分率之间无显著差异（P0.05，n=8，40%FCS）。 结论 短期冻存癌症患者的PBMC可用于后续检测T淋巴细胞亚群，尤其对于晚期癌症患者而言，可避免多次采血造成的负担同时提高实验的一致性。此外，本实验建立的方法也可用于临床抗肿瘤免疫治疗和其他免疫监测。 　　[关键词] PBMC；细胞冻存和复苏；淋巴细胞亚群；流式细胞术 　　[中图分类号] R329.2；R697.3 [文献标识码] A [文章编号] 1673-9701（2015）29-0029-03 　　Comparative analysis of lymphocyte subsets between fresh and frozen cancer patients PBMC by flow cytometry 　　WEI Tianxue YU Hong HE Chunying LIU Yuxia 　　Jilin Tumor Institute， Changchun 130012，China 　　[Abstract] Objective To develop a protocol to comparative analysis of lymphocyte subsets between fresh and cryopreserved cancer patients PBMC for immunological studies by flow cytometry. Methods Blood samples （2 mL/sample） from lung malignant tumor patients were collected by venipuncture from June to December 2014. For each sample， 300 μL of fresh whole blood was immediately used for lymphocyte subsets analysis. While the remaining were lysed and the isolated PBMC were cryopreserved with 40% or 90% FCS. Following storage for 30 days， the samples were removed and quickly thawed in 37℃ water bath. After cultured for 24 h， the cells were stained with antibodys. The analysis was performed using an Epics XL/MCL flow cytometry（Beckman Coulter）. Results After separating， the cell recovery of PBMC was（67.70±2.80）% of 90% FCS vs （80.47±3.32）% of 40% FCS（P=0.0069）. There were no significant differences in the frequencies of CD3+，CD3+CD4+，CD3+CD8+，CD3-CD16+56+，CD3+CD16+56+，and CD4+CD25+ T cells between the fresh and frozen PBMC（P0.05，n=8，40% FCS）. Conclusion The results of the present study suggest that frozen PBMC may be used to evaluate T cell subset frequencies and facilitat