论文部分内容阅读
目的观察甲状旁腺激素(PTH)基因和蛋白体外表达情况,并评价其基因治疗甲状旁腺功能低下模型鼠的作用。方法(1)以脂质体将质粒pcDPG分别1次和多次转染293细胞,观察绿色荧光蛋白(GFP)的表达并计算转染率;(2)转染24、48、72和96h后real-ti me定量PCR和Western blot法检测PTH基因与蛋白表达,并活性鉴定;(3)建立甲状旁腺功能低下症模型,将pcDPG质粒多次肌肉注射治疗,监测血钙和PTH值、存活时间及各器官病理变化。结果转染后24h即见GFP表达,72h达高峰,96h开始减少;多次转染后GFP表达率可达90%以上;PTHcD-NA拷贝数转染24h为5×103,72h达最高为8×104,多次转染显著增高(P<0.01);Western blot见48h和72h有PTH蛋白表达,其可对抗甲状旁腺切除小鼠抽搐症状;术后第2天血钙与PTH明显低于术前(P<0.05),pcDPG质粒大、中剂量组连续治疗48h后血钙与PTH值均恢复正常。结论重组PTH基因治疗甲状旁腺功能低下模型鼠有较好的疗效。
Objective To observe the expression of PTH gene and protein in vitro and to evaluate the role of gene therapy in hypoparathyroidism model rats. Methods (1) The plasmid pcDPG was transfected into 293 cells once and several times by liposome respectively. The expression of green fluorescent protein (GFP) was observed and the transfection efficiency was calculated. (2) After transfection for 24, 48, 72 and 96 h Real-ti me quantitative PCR and Western blot assay PTH gene and protein expression, and activity identification; (3) the establishment of hypoparathyroidism model, the pcDPG plasmid multiple intramuscular injection therapy, monitoring of serum calcium and PTH value, survival Time and pathological changes of various organs. Results The expression of GFP was observed at 24h after transfection, reached the peak at 72h, and decreased at 96h. The expression of GFP was up to 90% after multiple transfection. The transfection of PTHcD-NA was up to 5 × 103 24h × 104, multiple transfection was significantly increased (P <0.01); Western blot see 48h and 72h PTH protein expression, which can be anti parathyroid mice convulsive symptoms; 2 days after the serum calcium and PTH was significantly lower than Preoperative (P <0.05), PCDPG plasmid large and medium doses of 48h continuous treatment of serum calcium and PTH values returned to normal. Conclusion Recombinant PTH gene therapy for hypoparathyroidism model rats have a good effect.