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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。
Cytokine gene therapy is an important advance in biotherapy in recent years. In this study, human IL-6 gene for the treatment of gene, fibroblast cells as a carrier, the establishment of an experimental model of human IL-6 gene therapy and dynamic observation of IL-6 secretion in vivo levels of 650bp of human IL- 6 cDNA was inserted into the Xho I site of BCMGNeo expression vector carrying Neo ~ R gene. The recombinant expression vector was identified by restriction enzyme digestion. BCMGNeo-IL-6 was transfected into NIH3T3 fibroblasts by calcium phosphate co-precipitation method. One of the high positive clones was screened by screening G418, limiting dilution and determination of IL-6 activity in the supernatant Clonal strains secreting IL-6 (184.6 U / ml). Southern blot analysis was performed on this positive clone. The positive clone was amplified in vitro, encapsulated in collagen and then transplanted into the peritoneal cavity of mice. IL-6 was detected from mouse serum (up to 15 days after transplantation), and fibroblasts successfully transfected IL-6 gene Into the body and effective expression, proves that the gene therapy is feasible.