转基因造血干细胞移植治疗小鼠甲状旁腺功能低下症的研究

Treatment of murine hypoparathyroidism with transgenetic hemopoietic stem cell transplantation

目的 探讨输注整合有甲状旁腺素(PTH)基因的造血干细胞对甲状旁腺功能低下症的治疗效果。方法 将以pcDNA 3.1 PTH 为模板扩增出的PTH 基因插入到逆转录病毒载体MSCV中,得到含PTH基因的重组质粒,并转染PA317 包装细胞,以抗生素Geneticin筛选阳性克隆,获得重组有PTH基因的浓缩病毒悬液,以其感染人脐血造血干细胞,然后注入甲状旁腺功能低下症模型小鼠血中,术后观察小鼠症状的改善情况、血PTH及血钙浓度变化情况。结果 所获得的重组有PTH基因的浓缩病毒悬液,其病毒滴度为2×107 CFU/ml,PTH的分泌量为15 ng/48 h(106个细胞),未检测到有野生型病毒存在。实验组小鼠接受转染有PTH基因的血干细胞后,症状改善,血PTH及血钙浓度逐渐上升,并维持于接近正常水平;仅接受重组有PTH基因的浓缩病毒悬液的小鼠,短期内血PTH及血钙浓度明显升高,以后则呈缓慢下降趋势,并逐渐出现甲状旁腺功能低下症的表现;只接受造血干细胞移植的小鼠术后20 d左右全部死亡。结论 甲状旁腺功能低下症小鼠接受整合有PTH基因的造血干细胞静脉输注可获得较长期的治疗效果。

Objective To investigate the gene therapy of hypoparathyroidism (HPT) with hemopoietic stem cell s(HSCs) integrated with parathyroid hormone (PTH) gene.Methods Human PTH gene was amplified by polymerase chain reaction from pcDNA 3.1 -PTH templates and inserted into murine stem cell virus (MSCV) vectors. The plasmids recombinated with PTH gene were transfected into PA317 packaging cells and screened for positive clones with Geneticin. The condensed recombinant retroviruses were obtained and used to infect HSCs coming from umbilical cord blood. The infected HSCs were injected into the mice suffering from HPT. The change of symptoms and serum level of PTH and calcium in each group of mice was observed.Results Human PTH gene was proved to be inserted into MSCV vectors and the titres were up to 2×10 7 colony forming unit (CFU)/ml in condensed retroviral solution. The secretion of PTH reached 15 ng per 10 6 cells per 48 h. The wild type viruses were not detected by PCR amplification. The mice suffering from HPT recovered quickly and the serum level of calcium and PTH remained normal for about 3 months after the HSCs recombinated with PTH gene were injected into them. In the groups of mice subject to the injection of the recombinant retroviruses containing PTH gene, the serum level of PTH and Calcium was significantly increased in a short time, and then declined slowly. And the mice took on the manifestation of hypoparathyroidism again. The mice only subject to the implantation of HSCs were all dead within 20 days.Conclusion A long term therapeutic effect could be obtained by infusing the HSCs which were integrated with PTH gene into the mice suffered from hypoparathyroidism.