外周淋巴细胞冻存技术在复发性流产主动免疫治疗中应用研究.docVIP

外周淋巴细胞冻存技术在复发性流产主动免疫治疗中应用研究 　　【摘要】 目的：探?外周淋巴细胞冻存技术用于复发性流产（RSA）主动免疫治疗的临床效果。方法：对2014年11月-2016年1月经本院中医科门诊确诊的77例RSA患者的临床资料进行回顾性分析，所有浓缩白细胞通过无偿献血方式获得，进一步采用Ficoll密度梯度离心法分离淋巴细胞，淋巴细胞经自体血浆冻存，深低温保存，分次复苏后用于RSA患者主动免疫治疗。通过台盼蓝拒染实验和流式细胞技术比较冻存不同时期淋巴细胞存活率和CD3+T、CD3+CD8+T和CD3+CD4+T细胞免疫表型变化，评价冻存淋巴细胞的活性和稳定性；通过比较治疗前后RSA患者HLA-Ⅰ、Ⅱ类特异性封闭抗体转阳率和再次妊娠成功率，评价冻存外周淋巴细胞用于RSA治疗的效果。结果：与新鲜分离的淋巴细胞比较，存活7、15、30、60、90 d的淋巴细胞存活率和细胞表型稳定性比较，差异均无统计学意义（P0.05）；经冻存淋巴细胞主动免疫治疗后，封闭抗体累计转阳率为93.5%。封闭抗体阳性者再次妊娠成功率为90.3%，显著高于阴性者的20.0%，差异有统计学意义（P0.05）。结论：外周血淋巴细胞经自体血浆冻存，深低温保存后，不影响细胞存活率和稳定性，且对RSA患者治疗效果明确。此外，此法获得的淋巴细胞来源安全、可靠，患者依从性高，且可一定程度缓解临床血液制品来源困难的处境，具有进一步临床推广使用的价值。 　　【关键词】 外周淋巴细胞； 冷冻保存； 复发性流产； 主动免疫治疗 　　【Abstract】 Objective：To explore the clinical efficacy of peripheral lymphocyte cryopreservation technique for lymphocyte active immunotherapy（LIT） in the treatment of recurrent spontaneous abortion（RSA）.Method：The clinical data of 77 patients with RSA who had been diagnosed with traditional Chinese medicine from November 2014 to January 2016 were retrospectively analyzed.All the concentrated white blood cells were obtained by means of blood donation，further using ficoll density gradient centrifugation to separate lymphocytes.Trypan blue exclusion and flow cytometry assay were used to detect the cell viability and the changes of CD3+T，CD3+CD8+T and CD3+CD4+T lymphocytes at different time points respectively.The positive rates of HLA-Ⅰ，Ⅱ specific blocking antibodies and the outcome of re-pregnancy were adopted to evaluate the efficacy of cryopreserved lymphocyte active immunotherapy in treatment of RSA.Result：There were no significant differences in lymphocyte survival rate and and cell phenotype stability between fresh day and 7，15，30，60，90 days compared with isolated lymphocytes（P0.05）.Furthermore，after treatment，the positive rate of blocking antibodies was up to 93.5%.In addition，the probability of successful pregnancy rate was 90.3% in patients with positive blocking antibodies，which was significantly higher than 20.0