重组pEGFP-N1-IGF-1质粒体内转染骨质疏松大鼠的可行性

Feasibility of recombinant plasmid pEGFP-N1-IGF-1 transfection in an osteoporosis rat

背景:胰岛素样生长因子1水平的下降与骨质疏松密切相关,目前应用基因技术是骨质疏松等骨代谢疾病治疗的发展方向,但病毒载体可能引起严重免疫反应,质粒重组体pEGFP-N1-IGF-1活体转染骨质疏松大鼠可能成为一种有效的治疗手段。目的:观察质粒重组体pEGFP-N1-IGF-1转染骨质疏松大鼠中胰岛素样生长因子1的表达。方法:雌性SD大鼠随机分为假手术组、pEGFP-N1组和pEGFP-N1-IGF-1组。后2组予双侧卵巢切除。术后12周,3组依次给予生理盐水、pEGFP-N1载体和质粒重组体pEGFP-N1-IGF-1分别复合脂质体转染。转染后48h荧光活体成像及肝脏切片观察,定期测定血清胰岛素样生长因子1质量浓度。结果与结论:转染后pEGFP-N1组和pEGFP-N1-IGF-1组大鼠均可见全身大部出现明显荧光表达,以肝脏及尾部为著,肝脏切片镜下观察可见明显荧光表达。去卵巢大鼠血清胰岛素样生长因子1质量浓度显著降低(P<0.05);pEGFP-N1-IGF-1转染使去卵巢大鼠血清胰岛素样生长因子1质量浓度明显升高(P<0.05),而后随时间延长而逐渐降低(P<0.05)。结果证明质粒重组体pEGFP-N1-IGF-1能够在骨质疏松大鼠体内成功转染并表达胰岛素样生长因子1蛋白,为应用胰岛素样生长因子1基因治疗骨质疏松症提供了理论基础。

BACKGROUND：The decline of insulin-like growth factor-1（IGF-1） levels has been proven to be an important reason leading to osteoporosis.Gene therapy for osteoporosis and other bone metabolic diseases has becoming a direction of the research,but the viral vector may cause serious immune response.Therefore,the recombinant plasmid pEGFP-N1-IGF-1 in vivo transfection of osteoporosis rats may be a more effective treatment way.OBJECTIVE：To study the IGF-1 expression during recombinant plasmid pEGFP-N1-IGF-1 in the osteoporosis rats in vivo.METHODS：Female Sprague Dawley rats were randomly assigned to sham-surgery group,pEGFP-N1 group and pEGFP-N1-IGF-1 group.Bilateral ovariotomy was performed in pEGFP-N1 group and pEGFP-N1-IGF-1 group.At 12 weeks following surgery,the three groups were given saline,pEGFP-N1 vector and recombinant plasmid pEGFP-N1-IGF-1 complex liposomes hydrodynamic injection,respectively.Fluorescence in vivo imaging and liver biopsy were observed at 48 hours following transfection.Serum concentrations of IGF-1 were determined at regular intervals.RESULTS AND CONCLUSION：After transfection,rats of pEGFP-N1 group and pEGFP-N1-IGF-1 group developed noticeable fluorescence expression not only in the whole body but also liver biopsy,especially in the liver and tail.IGF-1 levels were significantly decreased in rats undergoing ovariotomy（P 0.05）.The expression level of serum IGF-1 in rats of pEGFP-N1-IGF-1 group were significantly increased（P 0.05）,and then reduced over time（P 0.05）.Results indicated that recombinant plasmid pEGFP-N1-IGF-1 can be successfully transfected and expressed IGF-1 protein in osteoporosis rats,which can be a substantial foundation for further application of IGF-1 gene therapy to osteoporosis.