CD3AK/iNOS细胞对慢性粒细胞原代细胞 白血病体外净化效应

Purging Effects of CD3AK/iNOS in vitro on Primary Leukemic Cells from Chronic Myeloid Leukemia Patients

本研究旨在探讨免疫细胞性一氧化氮供体CD3AK/iNOS细胞对慢性粒细胞白血病(CML)原代白血病 细胞的体外净化效应。培养并扩增PA317/iNOS细胞并用G418筛选;用NIH3T3细胞测定病毒滴度,分离外周血 单个核细胞并用抗CD3单克隆抗体激活;病毒感染靶细胞CD3AK及用G418筛选,用逆转录-聚合酶链反应 (RT PCR)检测CD3AK/iNOS中iNOScDNA的转录;用硝酸还原酶法检测CD3AK/iNOS细胞培养上清中NO含 量以及iNOS活性;用系列稀释半定量巢式RT PCR检测CML患者白血病原代细胞经CD3AK/iNOS细胞净化后 bcr/abl融合基因的表达水平。结果表明,PA317/iNOS细胞能稳定合成并分泌重组逆转录病毒颗粒;NIH3T3细胞 测定病毒滴度为1.0×105CFU/ml;RT PCR检测发现CD3AK/iNOS细胞中有iNOScDNA的转录;CD3AK/iNOS 细胞培养上清中NO含量和iNOS活性较CD3AK细胞明显升高。上述两项指标,经统计学检验,均有显著性差异 (P<0.001,P<0.001)。系列稀释半定量RT PCR检测发现,经CD3AK/iNOS细胞净化后CML患者白血病细胞 bcr/abl融合基因的表达明显下调。实验结果统计分析表明,CD3AK/Neo细胞组净化后与CD3AK/iNOS细胞组净 化后bcr/ablmRNA表达的比较具有显著性差异(P<0.001)。结论:逆转录病毒可介导iNOS基因转入CD3AK细 胞,成功地构建了CD3AK/iNOS;CD3AK/iNOS能明显地增加NO含量和iNOS活性,应用CD3AK/iNOS可能成 为一个有效的AHSCT体外净化方法。

To investigate the purging effect of CD3AK/iNOS on primary leukemic cells from chronic myeloid leukemia patients in vitro, amphotropic packaging cell line PA317 transfected with the whole length of iNOS gene was cultivated, amplified and screened by G418. The viral titer was determined by the NIH3T3 cells. Human peripheral blood mononuclear cells were isolated and activated by anti-CD3 monoclonal antibody in vitro. CD3AK cells were incubated with viral supernatant and selected by G418. Resistant clones were assayed for iNOS gene expression by RT-RCR. The content of nitric oxide and the activity of iNOS in the culture supernatant of CD3AK/iNOS were evaluated by the method of Griess. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene was detected by serial dilution semi-quantitative net RT-PCR assay. The results showed that anti-G418 positive packaging cell line PA317 transfected with the whole length of iNOS gene clones could stably synthesize and excrete recombinant retroviral vectors. The titer of recombinant retroviral vectors was 1.0 × 10^5 CFU/ml. After being transfected by recombinant retroviral supernatant, the iNOS cDNA was expressed in CD3AK/iNOS. The content of NO and activity of iNOS that synthesized and excreted by CD3AK/iNOS were notably increased, compared with those of CD3AK. There were statistically significant differences in NO content and iNOS activity between two groups. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/ iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene in all of them was downregulated by serial dilution semi-quantitative RT-PCR assay. It is concluded that construction of CD3AK/iNOS can markedly increase the content of NO and the activity of iNOS, which can be more efficient in in vitro purging leukemia cells for autologous hematopoietic stem cell transplantation.