尾静脉注射重组腺相关病毒载体小鼠肾组织病毒表达的观察

Observation on Gene Delivery in Murine Renal Tissue Using Recombinant Adeno-associated Viral Vector Via Tail Vein Injection

目的:通过小鼠尾静脉注射r AAV8-GFP病毒,观察腺相关病毒作为基因治疗载体在肾组织的表达情况,探讨此造模方法使腺相关病毒作为基因载体在肾脏表达的可行性,为进一步基因治疗肾脏疾病提供依据。方法:取16只C57-BL6小鼠,随机分为两组(n=8),对照组小鼠尾静脉注射PBS 0.2 ml,模型组小鼠尾静脉注射含有1×1011Iu/ml r AAV8-GFP 0.2 ml,4周后处死小鼠,用HE染色和免疫组化方法分别观察携有绿色荧光蛋白(GFP)r AAV-8在肾脏、肝脏、脾脏、肺脏和心脏等各组织,形态结构变化和荧光表达情况,并用PCR和Real-Time PCR方法检测r AAV8-GFP在肾脏、肝脏、脾脏、肺脏和心脏DNA表达情况。结果:与对照组比较,HE染色提示,模型组各脏器组织结构无明显变化。免疫组化提示肝脏有明显绿色荧光蛋白表达,肾脏,脾脏,肺脏和心脏绿色荧光蛋白表达不明显。Real-Time PCR方法发现,与对照组比较,肝脏表达r AAV-8表达最高,心脏,肾脏,肺脏和脾脏都有不同层度表达,但表达水平较低。结论:尾静脉注射r AAV8-GFP造模方法可使r AAV8-GFP在肾组织中有一定程度表达,但表达量偏少,尚不能作为一个较好腺相关病毒在肾脏表达的造模方法。

Objective：The purpose of thepresent study was to evaluate gene expression in the rat kidneyafter tail veininjec-tion of a rAAV（serotype 8）vectorencoding green fluorescence protein（GFP）. Methods：C57-BL6 mice（n=8）were treated with either PBS or transduced with rAAV8 -GFP（0. 2 ml,5× 10^11 infectious units/ml）through tail vein injection. At 4 weeks after transduction,the mice were sacrificed,and the morphology and GFP expression of different organs including was measured by IHC and HE methods. The DNA expressions of rAAV8 -GFP were also detected by PCR and Real-Time PCR methods. Results：The morphology of all tissues was normal,in contrast to the control group. The expressions of GFP were only found to exist in liver,and no transduction was observed in the kidney,lung,spleen and heart tissues. However,the DNA expression in kidney was detectable by Real-Time PCR method,and the highest copy of genome vector was also detected from the transduced liver. Conclusion：This study demonstrated a certain level of transgene expressions in the kidney after tail vein injection,but the GFP expression in kidney was un-detectable. It seems that it is not a good modeling method to transduce rAAV into kidney via tail vein injection.