摘要
目的研究人胰岛素基因表达质粒在体外转染鼠成纤维细胞(NIH3T3)后对糖尿病大鼠血糖的影响。方法采用壳聚糖转染法将重组的人胰岛素基因表达质粒转染鼠成纤维细胞(NIH3T3),转染后72h,经G418抗性筛选出阳性克隆并培养到第24d,用免疫组织化学方法检测胰岛素的表达,同时用ELASA法监测转染后24d的细胞培养液的胰岛素水平。并将阳性克隆大量扩增,注射至糖尿病大鼠腹腔内,并与转染空载体的对照细胞比较,观察在糖尿病大鼠体内的胰岛素表达及对血糖等变化的影响。结果转染PCMV.INS的NIH3T3细胞培养液的胰岛素水平明显高于未转染组(P<0.01)及PCMV转染组(P<0.01),未转染组与PCMV转染组细胞培养液的胰岛素水平无明显变化(P>0.05)。接受胰岛素表达载体细胞的糖尿病大鼠,血糖明显下降,体重逐渐恢复。结论人胰岛素基因能成功转染非胰岛β细胞,并表达目的基因使血糖水平下降说明基因治疗有望成为1型糖尿病的重要治疗手段,壳聚糖是一种很有前途的胰岛素基因载体。
Objective To study the effect of human insulin gene modified fibroblasts in diabetic rats.~Methods An expression vector for human insulin gene was constructed by recombinant gene technique and introduced into NIH3T3 cells by chitosan-mediated DNA transfection. The transfection cells were grown in DMEM medium containing G418 at 72 hours after transfection, and the clones of cells were selected and continued to grow in G418 medium until the 24th day. The transfected cells were examined by immunohistochemical stain. And insulin levels were monitored by ELASA. Thereafter, these cells were transplanted into Streptozotocin-induced diabetic rats.~Results Extracellular insulin levels in PCMV. INS transfected group increased significantly as compared with that in untransfected group (P<0.01) and PCMV transfected group (P<0.01), while there were no changes of extracellular insulin levels in untransfected group and PCMV group (P>0.05). After the modified fibroblasts were injected into STZ-induced diabetic rats, they could decrease blood glucose significantly (P<0.01) and obviously increased the body weight (P<0.05).Conclusion Human insulin gene can be transfected successfully by chitosan-mediated and expressed efficiently in cells ,indicating chitosan is a promising non-viral vector. The cell lines gene can execute metabolic effect on diabetic rats. And the gene therapy of type 1 diabetes is possible.
出处
《公共卫生与预防医学》
2005年第3期17-19,共3页
Journal of Public Health and Preventive Medicine
关键词
人胰岛素基因
糖尿病
基因治疗
壳聚糖
Human insulin gene
Diabetes mellitus
Gene therapy
Chitosan
