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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。
The purpose of this study was to investigate the in vitro purification of CD3AK / iNOS cells, an immunocyte-derived nitric oxide donor, on primary leukemic cells of chronic myeloid leukemia (CML). PA317 / iNOS cells were cultured and expanded and screened with G418; virus titers were determined with NIH3T3 cells, peripheral blood mononuclear cells were isolated and activated with anti-CD3 monoclonal antibody; the virus was infected with target cells CD3AK and screened with G418 using reverse transcription-polymerase The transcription of iNOS cDNA in CD3AK / iNOS was detected by reverse transcription-polymerase chain reaction (RT-PCR). The content of NO and the activity of iNOS in CD3AK / iNOS cell culture supernatant were detected by nitrate reductase method. The leukemic primary The expression level of bcr / abl fusion gene after purified by CD3AK / iNOS cells. The results showed that PA317 / iNOS cells could stably synthesize and secrete recombinant retrovirus particles. The titer of NIH3T3 cells was 1.0 × 105CFU / ml. The transcription of iNOS cDNA in CD3AK / iNOS cells was detected by RT-PCR. CD3AK / iNOS cell culture The content of NO and iNOS in supernatant were significantly higher than that of CD3AK cells. The above two indicators, the statistical test, there was a significant difference (P <0.001, P <0.001). A series of semi-quantitative RT-PCR assays revealed that the expression of bcr / abl fusion gene was significantly down-regulated in leukemic cells of CML patients after being purified by CD3AK / iNOS cells. Statistical analysis of the experimental results showed that there was a significant difference (P <0.001) in the expression of bcr / abl mRNA after purification in CD3AK / Neo cells compared with CD3AK / iNOS cells. CONCLUSION: The retrovirus can transduce iNOS gene into CD3AK cells and construct CD3AK / iNOS successfully. CD3AK / iNOS can significantly increase NO content and iNOS activity. Using CD3AK / iNOS may be an effective AHSCT in vitro purification method.