小鼠胚胎干细胞转基因治疗甲状旁腺功能低下症的实验研究

Experimental study on gene therapy of hypoparathyroidism with murine embryonic stem cells

目的 探讨小鼠胚胎干细胞 (TC 1)转基因治疗甲状旁腺功能低下症 (HPT)。方法包装出重组人甲状旁腺素 (PTH )基因的小鼠干细胞病毒 (MSCV) ,以其感染小鼠ESCs ,检测基因转导效率 ,PTH分泌情况 ;观察体内外分化情况 ,以及注入模型鼠体内各组小鼠血PTH和血钙变化情况。结果 获得滴度为 2× 10 7集落形成单位 (CFU ) /ml的重组逆转录病毒 ,经聚合酶链反应(PCR)扩增未检测到有野生型病毒存在 ,可以安全应用。感染TC 1的效率为 70 % ,每 10 6未分化TC 1每 48h分泌PTH约 10ng。重组有PTH基因的TC 1在体内外均可分化出三胚层组织 ,注入模型鼠体内 ,在观察期间实验组动物未再出现甲旁低表现 ,而且血PTH和血钙均保持在接近正常值范围内。结论 MSCV介导外源PTH基因可高效转导TC 1并持续分泌PTH ;体内外分化实验证明TC 1具有全能性 ,而且内环境并不是决定TC 1分化的唯一因素。经基因转导的TC 1可较好的改善模型鼠的症状 ,是未来细胞移植的一种潜在来源。

Obejective To investigate the gene therapy of hypoparathyroidism with murine embryonic stem cells (TC-1).Methods The murine stem cell viruses which recombinated with parathyroid hormone (PTH) gene were harvested.The recombinant retroviruses were titred and used to infect TC-1.The gene transduction efficiency in TC-1 was assayed and the differentiation in vivo and in vitro observed.The chang in symptoms and serum PTH and Calcium levels in the models after TC-1 injection were observed.Results We acquired the condensed recombinant retroviruses whose titre was 2×10 7 colony forming unit (CFU)/ml.Wild type of viruses were not found through PCR amplification and the recombinant viruses proved to be applied safely.The gene transduction efficiency could reach 70% in TC-1.The secretory amount reached 10 ng per 10 6 undifferentiated TC-1 per 48 h.The tissues of all three embryonal layers could be seen in the differentiation test in vivo and in vitro.During the observing time no manifestations of HPT in experimental group models occurred any more after recombinant TC-1 injection.The serum PTH and calcium level maintained in the normal range.Conclusion MSCV could transduct the PTH gene into TC-1 with high efficiency and secreted PTH persistently.The in vivo differentiation test proved that TC-1 were totipotent and the environmental condition was not the single factor to determine TC-1 differentiation.TC-1 transducted with PTH gene could relieve the symptoms in animal models and were a potential source for future cell transplantation.