Basic research in kidney-specific gene transfer using renal transplantation procedure

本研究的目的是建立使用肾移植程序实现最有效的肾脏特异性基因转移的条件。实现我们目标的方法分为三个步骤。(I)我们研究了温度对阳离子脂质体基因转移效率的影响，这些基因转移到来自HeLa、LLC-PK I、正常系膜和正常内皮细胞系的各种细胞中。结果表明：（1）阳离子脂质体的基因转移效率几乎不受温度的影响;（2）随着温度的升高，不同细胞之间的效率差异变小。(II)本研究尝试将报告基因导入急性肾小管坏死（ATN）恢复期大鼠肾脏，并进行原位移植。在此之前，我们用氯化汞（HgCl_2）处理大鼠，造成ATN，移植后10天取出肾脏，用RT-PCR和免疫组织化学方法检测报告基因的表达。结果，3只HgCl_2染毒大鼠和6只未染毒大鼠中的5只均检测到。此外，在肾小球和间质细胞中也很明显，偶见肾小管细胞。(III)我们以肾囊肿为研究对象，分析了肾囊肿形成的分子基础，获得了新的认识。

The purpose of this study was to establish the conditions where we can realize the most efficient kidney-specific gene transfer using renal transplantation procedure. The approach to our goal was divided into three steps.(I) We investigated the effects of temperatures on the efficiency of gene transfer with cationic liposomes into a variety of cells derived from HeLa, LLC-PK I, normal mesangial, and normal endothelial cell lines. At a result, the following became clear : (1) The efficiency of gene transfer with cationic liposomes was almost independent of temperature ; (2) Differences of the efficiency among a variety of cells became smaller with a rise of temperature.(II) We tried to transfer reporter genes into rat kidneys which was in the convalescent stage from acute tubular necrosis (ATN) and transplanted them orthotopicafly. In advance we had treated rats with HgCl_2 to develop ATN.The kidneys were removed 10 days after transplantation and reporter gene expression was determined using RT-PCR and immunohistochemistry. At a result it was detected in all of three HgCl_2-treated rats and 5 of 6 untreated rats. In addition it was evident in glomerular and interstitial cells, occasionally tubular cells.(III) We assumed renal cystic disease as a subject of this therapy, then analyzed the molecular basis of cystogenesis and obtained new informations on it.