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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。
Objective To construct a recombinant eukaryotic expression plasmid of parathyroid hormone (PTH) gene and evaluate the expression and biological activity of PTH gene after transfection in vitro. At the same time, observe its gene therapy effect on hypoparathyroidism animal. Methods (1) PTH gene was cloned from human parathyroid tissue and the recombinant eukaryotic expression vector pcDNA3.1-PTH-GFP (pcDPG) was constructed by topological method. The recombinant plasmid was digested by restriction endonucleases, polymerase chain reaction (2) Lipofectamine 2000 was transfected into 293 cells by lipofectamine 2000. The expression of green fluorescent protein (GFP) was observed and transfection efficiency was calculated. The expression of PTH gene was verified by reverse transcription-polymerase chain reaction (RT-PCR) (3) Purification of PTH protein in the supernatant of transfected cells, and identification of biological activity; (4) Establishing a rabbit model of hypoparathyroidism by intramuscular injection of pcDPG plasmids to monitor serum calcium, phosphorus and PTH value and survival time, through the morphological observation of the pathological changes in various organs. Results (1) The results of enzyme digestion and PCR were the same as expected. The sequencing results were 99.3% homologous to the published sequences. (2) The expression of GFP was observed 24 h after transfection and the expression was enhanced with time. The transfection efficiency at 48 h Up to 38.9% and 62.5%, respectively, while PTH gene expression was detected by RT-PCR. (3) Purified PTH protein was able to counteract seizure symptoms in parathyroid mice. (4) L ± 0.09mmol / L, 1.73mmol / L ± 0.03mmol / L, 1.75mmol / L ± 0.03mmol / L, 1.65mmol / L ± 0.04mmol / L) were significantly lower than the preoperative (2.82mmol / L ± 0.21mmol /L,P<0.05), postoperative PTH values (5.03pg / ml ± 0.05pg / ml, 5.04pg / ml ± 0.05pg / ml, 5.03pg / ml ± 0.07pg / ml, 5.29pg / ml ± 0.03pg / ml) was also significantly lower than preoperative (11.63pg / ml ± 1.60pg / ml), but after 2d postoperative serum phosphorus increased (P <0.05) Phosphorus and PTH values returned to normal. Conclusion The liposome-mediated plasmid pcDPG transfection in vitro in high rate, can express active PTH protein, while hypoparathyroidism rabbits have a good effect, which gene therapy for hypoparathyroidism Further research laid the foundation.