成纤维细胞介导的α干扰素基因疗法的建立

ESTABLISHMENT OF THE FIBROBLAST-MEDIATEDINTERFERON-ALPHA GENE THERAPY

以成纤维细胞为基因载体细胞，建立。干扰素（ＩＦＮ－α）基因疗法的实验模型，并动态观察实验动物体内ＩＦＮ－α的分泌水平。将６８２ｂｐ的全序列入ＩＦＮ－αｃＤＮＡ插入到携有Ｎｅｏ￣Ｒ基因的表达载体ＢＭＧＮｅｏ的ＸｈｏＩ位点上，对此重组表达载体（ＢＭＧＮｅｏ－ＩＦＮ－α）进行了限制性酶切鉴定。用磷酸钙共沉淀法将ＢＭＧＮｅｏ－ＩＦＮ－α转入ＮＩＨ３Ｔ３成纤维细胞中，通过Ｇ４１８抗性筛选、有限稀释和检测上清中ＩＦＮ－α生物活性，从１４株阳性克隆中筛选到一株高分泌ＩＦＮ－α达１０２４Ｕ／ｍｌ的克隆株（ＮＩＨ３Ｔ３－ＩＦＮ－α￣＋）。将此＊阳性克隆细胞体外大量扩增，包裹入胶原，然后埋植入小鼠腹腔内，于１２小时到１５天可从小鼠血清中检测出ＩＦＮ－α，以７２小时的分泌水平最高，表明成纤维细胞能成功地将人ＩＦＮ－α基因导入体内并稳定表达。

he fibroblasts were used as carrier cells to estab-lish an experimental model of human IFN-α genetherapy, and the IFN-α level in mice at different timeafter implantation was observed . The 682-bp full-length of human IFN-α cDNA was inserted into theXhoI site of the BMGNeo vector containing aneomycin resistance gene. The recornbinant vectorBMGNeo-IFN-α DNA was transfected into NIH3T3fibroblasts by the calcium phosphate coprecipitationmethod. A fibroblast cell clone (NIH3T3-IFN-α￣+ ) se-creting 1024 U/ml of IFN-α in supernatant was ob-tained from 14 positive clones by G418 resistance selec-tion, limiting dilution and IFN-α activity assay. Thisclone was expanded in vitro, encapsulated into collagenand implanted intraperitoneally into mice. After im-plantation, serum IFN-α activity could be detectedfrom 12h to day 15 with a peak at 72h. These resultsdemonstrated that IFN-α gene transfected fibroblastscould successfully express the IFN-α gene in vivo afterimplantation.