关键词: 毒理学, 安全性评价, 重组腺相关病毒, 神经生长因子基因, 血脑屏障, 生物学分布, 大鼠

Abstract: AIM: To evaluate the safety of human nerve growth factor gene β mediated by recombinant adeno- associated viruses type-2 (rAAV2 /hNGFβ) delivered into rat brain via external carotid artery after the mannitol inducing the blood-brain barrier (BBB) opening. METHODS: 25 % mannitol was infused into left internal carotid artery of rats via left external carotid artery. After 2 min when 25 % mannitol was infused in 30 s at a speed of 0.12 ml·s ^-1, 1 ml normal saline (NS) or rAAV2 /hNGFβ or rAAV2 GFP with the titer of 1.5 ×1015 vg ·L ^-1 was infused into rat internal carotid artery respectively.In the long-term toxicity experiment (60 d), the rats infused rAAV₂ /hNGFβ were observed the general state during breeding.The weight, the pain sensitivity, bleeding time and clotting time, blood biochemistry and hematology, the viscera index, and viscera-pathologic change were examined on 60d.In the biological distribution experiment, the rats infused rAAV₂ /GFP (green fluorescence protein, GFP) were perfused transcardially with 4 % solution of paraformaldehyde in 0.1 mol·L ^-1 phosphate buffer at 4 ℃.Tissue sections were cut on a freezing microtome set at 10 μm.The expression of GFP in every organ was observed under laser confocal microscope. RESULTS: Compared to control, the general state, pain sensitivity, hematology, blood-biochemical indices, viscera index and histopathologic examination of the rats infused rAAV2 hNGFβ were not significantly different;the biological distribution of rAAV₂ /GFP in the rats infused rAAV₂ GFP could be observed in brain, liver, spleen, kidneys, adrenal, lung, thyroid, hypophysis thymus, stomach, small intestine, and pancreas. CONCLUSION: The intravascular injection of rAAV₂ /hNGFβ for does not display obvious toxicity on d 60 in rats.Meanwhile, the rAAV₂ /hNGFβ can express not only in the brain, but also in many other tissues of rats.

Key words: toxicology, safety evaluation, recombinant adeno-associated viruses, nerve growth factor gene, blood-brain barrier, biological distribution, rats