人CD14转基因细胞系的建立.docVIP

人CD14转基因细胞系的建立 目录 TOC \o 1-9 \h \z \u 目录 1 正文 1 文1：人CD14转基因细胞系的建立 1 文2：人心房肌Kv15钾通道稳定表达细胞系的建立 8 1材料和方法 9 参考文摘引言： 12 原创性声明（模板） 13 文章致谢（模板） 13 正文 人CD14转基因细胞系的建立 文1：人CD14转基因细胞系的建立 Establishment of Murine Cell Line Trafected with Human CD14 Gene Abstract This study was aimed to cotruct the CD14 eukaryotic expression vector，establish the trageneic CD14 positive cell line in order to facilitate the establishment of a mouse model of antibody targeting therapy for human acute monocytic leukemia (AML-M5). Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase，the human CD14 gene was cloned and sequenced through the RT-PCR and T-A clone expressional vector (+)/CD14 was cotructed by cleaving with double restriction endonuleases and ligating with T4 murine melanoma cell line B16 was trafected with the (+)/CD14 recombinant with Superfect trafection clones were selected by G418 and the expression of human CD14 on the trafectant was confirmed by flow cytometry (FCM).The results indicated that the sequence of the human CD14 cDNA cloned was exact to be same as the one from GenBank recombinant (+)/CD14 was identified with double-enzyme expression of the human CD14 on the trafectant (B16/CD14) was confirmed by conclusion，the murine cell line B16/CD14 frafected with human CD14 gene has been established which can be used for the study of human AML-M5 antibody targeting therapy with mouse model. Key words CD14；trageneic cell line；acute monocytic leukemia; mouse model 急性单核细胞白血病（AML-M5）是难治性白血病之一，CD14是M5细胞膜上的特异性标志，它在其它细胞成分包括T、B、 自然 杀伤细胞(NK)、红细胞、血小板、造血干/祖细胞以及非造血系统细胞上不表达［1］，并且单核细胞能通过CD14将与CD14抗原分子结合的分子内吞入细胞内［2］，因此CD14是导向治疗AML-M5的良好靶点。本研究组已经将自制鼠源性抗人CD14单克隆抗体（ZCH-7-2F9）研制成基因工程抗体，拟进一步进行动物试验。为了建立良好的动物模型，一个稳定表达人CD14抗原分子的肿瘤细胞系是重要的环节。然而，除病人新鲜M5细胞外，国际上很少有CD14表达的单核细胞系存在，从而对该研究的深入进行带来困难。目前，大多数研究室仍然通过VitD3诱导单核细胞来源的细胞株U937 来获得CD14阳性表达的细胞系［3］，这不仅不能保证CD14表达的均一性，也不利于人M5白血病动物模型的建立及其体内导向治疗的研究，通过转基因技术建立表达人CD14抗原的鼠肿瘤细胞系是解决这一问题的可行途径。为此，我们利用C57B