pcDPG基因治疗甲状旁腺功能减退症的实验研究

目的构建甲状旁腺激素(PTH)基因的重组真核表达质粒,评价体外转染后PTH基因的表达与生物学活性,同时观察其对甲状旁腺功能减退动物的基因治疗作用。方法(1)从人胚甲状旁腺组织中克隆PTH基因,拓扑法构建其重组真核表达质粒pcDNA3.1-PTH-GFP(pcDPG),并采用酶切、聚合酶链反应(PCR)及DNA测序鉴定;(2)用脂质体转染pcDPG入293细胞,观察绿色荧光蛋白(GFP)表达并计算转染率,同时逆转录-聚合酶链反应(RT-PCR)验证PTH基因的表达;(3)纯化转染细胞上清中PTH蛋白,进行生物学活性鉴定;(4)建立甲状旁腺功能减退症的家兔模型,将pcDPG质粒以肌肉注射进行分组治疗,监测血钙、磷和PTH值及存活时间,通过形态学观察各器官的病理变化。结果(1)酶切与PCR结果与预期相同,测序结果与文献中序列同源性为99.3%;(2)细胞转染后24h即可见GFP表达,随时间延长而表达增强,48h转染率达38.9%和62.5%,同时RT-PCR见PTH基因表达;(3)纯化的PTH蛋白可对抗甲状旁腺切除小鼠的抽搐症状;(4)模型兔术后第2d血钙(1.71mmol/L±0.09mmol/L,1.73mmol/L±0.03mmol/L,1.75mmol/L±0.03mmol/L,1.65mmol/L±0.04mmol/L)明显低于术前(2.82mmol/L±0.21mmol/L,P<0.05),术后第2d PTH值(5.03pg/ml±0.05pg/ml,5.04pg/ml±0.05pg/ml,5.03pg/ml±0.07pg/ml,5.29pg/ml±0.03pg/ml)也明显低于术前(11.63pg/ml±1.60pg/ml),但是术后第2d血磷增高(P<0.05),pcDPG质粒大、中剂量组治疗后48h血钙、磷与PTH值均恢复至正常。结论脂质体介导的质粒pcDPG体外转染率较高,能表达有活性的PTH蛋白,同时对甲状旁腺功能减退家兔有较好的疗效,从而为甲状旁腺功能减退症基因治疗的进一步研究奠定了基础。

pcDPG parathyroid hormone gene therapy of hypoparathyroidism: an experimental study

OBJECTIVE: To construct recombination eukaryote expression plasmid for human parathyroid hormone (PTH) gene, assay PTH expression and biological activity after transfection in vitro and evaluate gene therapy effect on hypoparathyroidism (HPT). METHODS: (1) PTH gene was amplified from human embryonic parathyroid gland tissue, and plasmid pcDNA3.1-PTH-GFP (pcDPG) was constructed by TOPO recombination technique. Digestion, PCR and sequencing were used to identify the positive vectors. (2) pcDPG was transformed into 293 cells by Lipofectamine 2000(TM), fluorescent inverted microscope was used to observe GFP expression, and PTH gene expression was assayed by RT-PCR technique. (3) PTH protein in supernatant was purified and evaluated biological activity. (4) HPT rabbit models were developed and plasmid pcDPG was injected in skeletal muscles, respectively. Serum calcium, phosphonium and PTH were assayed and pathological changes observed. RESULTS: (1) The findings in digestion and PCR were accorded to anticipation and sequences in report were identified to reference at 99.30%. (2) 24 h after transfection GFP expression could be detected and enhanced with time prolonged arriving to 38.91% and 62.45% at 48 h. PTH gene expression could be detected by RT-PCR. (3) Purified PTH protein made the signs of HPT disappear. (4) Serum calcium and PTH levels were lower than those of pre-operation (P < 0.05) and serum phosphonium enhanced to normal standard 48 h after treatment at the plasmid pcDPG doses of 300 microg/kg and 500 microg/kg. CONCLUSION: Recombination plasmid pcDPG was transformed effectively in vitro and the transfected cells produced PTH protein with biological activity. Besides, the satisfactory therapeutic effect of HPT rabbits was attained by pcDPG plasmid, which provided a foundation for further study of HPT gene therapy.