Study in gene therapy for lower urinary tract dysfunction

研究背景：毒蕈碱M3受体（M3 receptor）是膀胱平滑肌收缩的主要受体。一氧化氮（NO）是膀胱重要的舒张因子之一。假设nNOS和M3受体在膀胱中的过表达分别抑制和增强膀胱收缩。因此，我们采用电穿孔法将nNOS和M3受体基因导入大鼠膀胱，并对其功能表达进行了研究。方法：将表达荧光素酶、绿色荧光蛋白（GFP）、nNOS和M3受体的质粒分别注入大鼠膀胱，并立即施加方波电脉冲。基因转移后两天，我们分析了基因表达。采用nNOS和M3受体免疫组化染色，观察氨甲酰胆碱（carbachol）和电场刺激（EFS）对离体膀胱肌条收缩反应的影响。还评估了从离体膀胱条释放的NO ...更多信息 采用微透析和HPLC。结果：电穿孔的最佳条件为：脉冲数8，电压45，脉冲数50毫秒，频率1 Hz。在这些条件下，与仅注射DNA相比，荧光素酶基因表达增强约300倍。关于用抗nNOS和抗M3受体的免疫组织化学，与对照大鼠的膀胱相比，在nNOS和M3受体基因转移的大鼠膀胱中观察到免疫活性增加。在大鼠与nNOS基因注射电穿孔有显着的nNOS免疫反应，和NOx释放的膀胱条显着大于对照组。在转移M3受体基因的大鼠中，卡巴胆碱和EFS诱导的膀胱平滑肌条的最大反应显着增加。与对照组相比，M3受体转移去神经大鼠的逼尿肌对卡巴胆碱的收缩力和EFS显著增加。膀胱测压结果显示，排尿间隔缩短，膀胱压力增加。结论：这些结果表明，在体内EP程序是一种有用的方法，基因转移到膀胱。通过该方法转移的nNOS基因功能性地表达并有助于NO的产生，并且大鼠膀胱中M3受体的过表达增强膀胱收缩性。该技术有望成为治疗逼尿肌功能不全的一种新方法。少

Background : Muscarinic M3(M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Nitric oxide(NO) is one of the important relaxing factor in bladder. Under the hypothesis that overexpression of nNOS and M3 receptor in the bladder would inhibition and enhancement of bladder contractions, respectively. So, we have transferred the nNOS and M3 receptor gene into rat bladders using electroporation(EP) and evaluated the functional expression of the gene.Methods : Plasmids expressing luciferase, GFP, nNOS and M3 receptor were injected into the rat bladder and square-wave electric pulses were immediately applied. Two days after gene transfer, we analyzed gene expression. Immunohistochemical staining for nNOS and M3 receptors was performed and the contractile responses from isolated bladder strips, which were carbachol and electrical field stimulation(EFS), were evaluated. NO released from isolated bladder strips was also assessed … More using microdialysis and HPLC. M3 gene transfer also preformed into bladder of deneravation(detrusor underactivity) model rat.Results : The optimal conditions of electroporation were 8 pulses, 45 voltages, 50 millisec/pulses and 1 Hz. Under these conditions, luciferase gene expression was enhanced approximately 300-fold, compared to an injection of DNA only. Regarding immunohistochemistry with an anti-nNOS and anti-M3 receptor, increases in immunoactivity wer observed in the nNOS and M3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the nNOS gene injected by electroporation there was marked nNOS immunoreactivity, and NOx released from bladder strips was significantly greater than in the control groups. In rats with the transferred M3 receptor gene, carbachol- and EFS-induced maximum responses of bladder smooth muscle strips significantly increased. In the M3 receptor transferred denervation rats, detrusor contractility for carbachol and EFS significantly increased, as compared with the control rats. The cystometric findings showed, decreased micturituion interval and increased in bladder pressure.Conclusions : These findings suggest that an in vivo EP procedure is a useful method for gene transfer into the bladder. nNOS gene transferred by this procedure functionally expresses and contributes to NO production, and that an overexpression of M3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity. Less