In vivo HVJ-liposome mediated gene transfer into intestine and carcinoma.

目前已经有几种外源基因的体内转染方法，但都存在一定的局限性。本研究旨在探讨日本血吸虫交配病毒（HVJ）脂质体介导的基因转移在肠道疾病基因治疗中的应用价值。荧光异硫氰酸酯标记寡脱氧核苷酸和大肠杆菌β-半乳糖苷酶采用HVJ-脂质体将β-gal基因导入大鼠肠道，通过两种途径检测转染效率;一种是经肠系膜动脉递送，另一种是在用Pronase制备以去除粘液屏障后的腔内递送，在这两种方法中，FITC标记的ODN和β-gal基因的表达不仅在肠固有层中，而且在大鼠肠的肌肉层中被观察到。结果表明HVJ-脂质体法是一种有效的外源基因导入肠道的方法。并建立了模拟人炎症性肠病（IBD）的半抗原（TNB）诱导的慢性结肠炎模型，作为基因治疗的靶点。在基因治疗之前，我们分析了各种细胞因子如IL-12、IL-18、TNF-α、INF-γ，并利用IL- 18敲除小鼠和IL- 18的中和抗体确定了细胞因子在该模型中的作用。我们的结论是，在这个模型中，TNF-α和INF-γ似乎发挥了诱导慢性结肠炎的主要作用。为了探讨HVJ脂质体介导的基因治疗炎症性肠病的可能性，我们在该模型中转移HGF，结果减少了结肠炎。为进一步的研究，我们建立了一种用于基因治疗的放射性结肠炎模型。

Several transfection methods have been developed to deliver exogenous genes into gastro-intestinal tract in vivo, but all have limitations. We evaluated the ability of Hemagglutmating virus of Japan (HVJ)-liposome mediated gene transfer to be used for gene therapy of incurrable intestinal diseases.We first investigated the possibilities of HVJ-liposome mediated gene trasfere in vivo. Fluorescent isothiocyanate (FTTC )-labeled oligodeoxynucleotides (ODN) and Escherichia Coli beta -galactosidase (beta-gal) gene was introduced into rat intestine by HVJ-liposome in vivo to examine transfer efficacy throughtwo approaches ; one was delivered via mesenteric artery and the other was intraluminal delivery after preparation with Pronase to remove mucus barrier, In the both method the localization of FITC-Labeled ODN and expression of beta-gal gene was observed not only in intestinal lamina propria but also muscle layer of the rat intestine. Our results demonstrate that HVJ-liposome method is useful for introduction of foreign gene into intestinal tract. Furthermore we established the model of hapten (TNB) induced chronic colitis as a target site of gene therapy, which mimic human inflammatory bowel disease (IBD). Prior to gene therapy, we analyzed various cytokines such as IL-12, IL-18, TNF-alpha, INF-gamma and determined role of cytokine in this model utilizing IL- 18 knock out mouse and neutralization antibody for IL- 18. We conclude that in this model TNF-alpha and INF-gamma seems to play a major role of inducing chronic colitis. To investigate possibilities of HVJ-liposome mediated gene therapy against inflammatory bowel disease we trasfer HGF in this model and it result in decreasing colitis. For the future study we are estabished a model of radiation colitis utilizing for gene thrapy.