摘要
细胞因子基因治疗是近年来生物治疗的重要进展。本文以人IL-6基因为治疗目的基因,以成纤维细胞为载体细胞,建立了人IL-6基因疗法的实验模型,并动态观察了其体内IL-6分泌水平.将650bp的人IL-6 cDNA 插入到携有Neo^R 基因的表达载体BCMGNeo 的Xho I 位点上,对此重组表达载体进行限制性酶切鉴定。用磷酸钙共沉淀法将BCMGNeo-IL-6转入NIH3T3成纤维细胞中,通过G418抗性筛选、有限稀释和上清中IL-6活性的测定,从多株阳性克隆中筛选到一株高分泌IL-6(184.6U/ml)的克隆株。对此阳性克隆进行了Southern 杂交分析。将此阳性克隆体外扩增、包裹入胶原中,然后移植入小鼠腹腔内,可从小鼠血清中(直至移植后15天)检测出IL-6,明成纤维细胞能成功地将IL-6基因导入体内并有效表达,证明该基因疗法是可行的。
In the present study,we have established an experimental model of human IL-6 gene thera-py with fibroblasts as cellular delivery and then observed its in vivo IL-6 level at different time.650-bp-long hIL-6 eDNA was inserted into Xho Ⅰ site of expression vector BCMGNeo carryingNeo^R gene and the resulting recombinant expression vector BCMGNeo-IL-6 was identified by re-striction enzyme digestion.BCMGNeo-IL-6 was transfected into NIH3T3 fibroblast cells by cal-cium phosphate coprecipitation method.A fibroblast cell clone secreting IL-6 activity in the cul-ture supernatants,and then analysed by Southern blot.The cell clone was expanded in vitro,en-capsulated into collagen and implanted i.p.into mice.Serum IL-6 could be detected even after15 days implantation.These results demonstrated that fibroblast cells could mediate in vivotransfer and efficient expression of hIL-6 gene,suggesting that this kind of gene therapy is reli-able.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
1993年第6期323-327,共5页
Chinese Journal of Immunology
关键词
白细胞介素6
基因疗法
成纤维细胞
Interleukin-6
Gene therapy
Fibroblast cells
Implantation
Gene transfer
