Zhou Yi, Lü Bing-Jie, Xu Ping, Song Chun-Fang
Department of General Surgery, First Clinical Hospital, Harbin Medical University, Harbin 150001, China.
Chin Med J (Engl). 2005 Feb 5;118(3):204-9.
The treatment of hypoparathyroidism (HPT) is still a difficult clinical problem, which necessitates a new therapy. Gene therapy of HPT has been valuable, but how to improve the gene transfer efficiency and expression stability is a problem. This study was designed to optimize the gene therapy of HPT with hematopoietic stem cells (HSCs) recombined with the parathyroid hormone (PTH) gene.
The human PTH gene was amplified by polymerase chain reaction (PCR) from pcDNA3.1-PTH vectors and inserted into murine stem cell virus (MSCV) vectors with double enzyme digestion (EcoRI and XhoI). The recombinant vectors were transfected into PA317 packaging cell lines by the lipofectin method and screened by G418 selective medium. The condensed recombinant retroviruses were extracted and used to infect HSCs, which were injected into mice suffering from HPT. The change of symptoms and serum levels of PTH and calcium in each group of mice were investigated.
The human PTH gene was inserted into MSCV vectors successfully and the titres were up to 2 x 10(7) colony forming unit (CFU)/ml in condensed retroviral solution. The secretion of PTH reached 15 ng.10(-6).cell(-1) per 48 hours. The wild type viruses were not detected via PCR amplification, so they were safe for use. The mice suffering from HPT recovered quickly and the serum levels of calcium and PTH remained normal for about three months after the HSCs recombined with PTH were injected into them. The therapeutic effect of this method was better than simple recombinant retroviruses injection.
The recombinant retroviral vectors MSCV-PTH and the high-titre condensed retroviral solution recombined with the PTH gene are obtained. The recombinant retroviral solution could infect HSCs at a high rate of efficiency. The infected HSCs could cure HPT in mice. This method has provided theoretical evidence for the clinical gene therapy of HPT.
甲状旁腺功能减退症（HPT）的治疗仍是一个棘手的临床问题，需要新的治疗方法。HPT的基因治疗具有重要价值，但如何提高基因转移效率和表达稳定性是一个问题。本研究旨在优化利用重组甲状旁腺激素（PTH）基因的造血干细胞（HSCs）对HPT进行基因治疗。
通过聚合酶链反应（PCR）从pcDNA3.1-PTH载体中扩增人PTH基因，经双酶切（EcoRI和XhoI）插入小鼠干细胞病毒（MSCV）载体。采用脂质体法将重组载体转染至PA317包装细胞系，并用G418选择培养基筛选。提取浓缩的重组逆转录病毒，用于感染HSCs，将其注射到患有HPT的小鼠体内。观察每组小鼠症状变化以及血清PTH和钙水平。
人PTH基因成功插入MSCV载体，浓缩逆转录病毒溶液滴度高达2×）10（7）集落形成单位（CFU）/ml。每48小时PTH分泌量达15 ng·10（-6）·细胞（-1）。经PCR扩增未检测到野生型病毒，使用安全。将重组PTH的HSCs注射到患有HPT的小鼠体内后，小鼠恢复迅速，血清钙和PTH水平在约三个月内保持正常。该方法的治疗效果优于单纯注射重组逆转录病毒。
获得了重组逆转录病毒载体MSCV-PTH以及与PTH基因重组的高滴度浓缩逆转录病毒溶液。重组逆转录病毒溶液能高效感染HSCs。被感染的HSCs可治愈小鼠的HPT。该方法为HPT的临床基因治疗提供了理论依据。
