甲状旁腺激素基因治疗甲状旁腺功能低下症  

Gene therapy of parathyroid hormone for hypoparathyroidism

作　　者：温济民[1] 钟宇华[2] 孙芸[3] 于春梅[4] 吴全理[1] 

机构地区：[1]广西北海市人民医院普外科,536000 [2]广西北海市人民医院内分泌科,536000 [3]广西北海市人民医院妇产科,536000 [4]北京大学医学部附属肿瘤医院外科

出　　处：《中华实验外科杂志》2006年第1期49-51,共3页Chinese Journal of Experimental Surgery

基　　金：广西青年基金资助项目(0339001)

摘　　要：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 parathyroid hormone （PTH） gene and protein in vitro and evaluate the effect of gene therapy for hypoparathyroidsm （HPT）. Methods （1） Recombinant plasmid pcDPG was transformed into 293 cells by Lipofectamine2000TM once and multi-times, and fluorescent inverted microscope was used to observe the GFP expression. （2）PTH cDNA and protein expression at 24, 48, 72 and 96 h was assayed by real-time quantitative RT-PCR technique and Western Blot. （3） HPT rats＇ models were developed and plasmid pcDPG was injected into muscles once and multitimes. Serum calcium and PTH were assayed. Results Twenty-four h after transfection GFP expression could be detected and reached peaks at 72 h, then declined at 96 h, while GFP expression rate got to more than 90%. In real-time quantitative RT-PCR, cDNA copies were 5 × 10^3, highest （8 × 10^4 at 72 h） and decreased at 96 h, while copies were much higher after multi transfections （P 〈 0.01）. PTH protein expression could be detected by Western Blot at 48 h, highest at 72 h after multi-transfections, which made the signs of HPT disappear. Serum calcium and PTH levels were lower （P 〈 0.05）, returned to normal levels 48 h after treatment at the plasmid pcDPG doses of 300 μg/kg and 500μg/kg. Conclusion Recombinant PTH gene could treat HPT model with a satisfactory therapeutic effect.

关 键 词：甲状旁腺激素 基因治疗 甲状旁腺功能低下症 绿色荧光蛋白 用药剂量 

分 类 号：R582.2[医药卫生—内分泌]