人神经生长因子基因血管内给药的安全性评价

Evaluation on safety of human nerve growth factor gene administered intravascularly

目的 :评价用高渗法向大鼠脑内导入二型重组腺相关病毒 人神经生长因子 (NGF) β基因(rAAV2 hNGFβ)的安全性。 方法 :大鼠经颈外动脉输入甘露醇诱导血脑屏障开放 ,分别经颈外动脉输入生理盐水 (对照组 )、rAAV2 hNGFβ或rAAV2 GFP。对导入rAAV2 hNGFβ的大鼠 ,观察饲养期间的一般情况 ,d 60称重 ,测定痛敏感度、出血时间和凝血时间。麻醉后腹腔取血 ,行血液学、血液生化检测 ;脏器称重 ,计算脏器系数 ,并做病理组织学检查。对导入rAAV2 GFP的大鼠处死后用 4 %多聚甲醛 PBS原位灌注固定 ,脏器连续冰冻切片 ,用激光共聚焦显微镜观察rAAV2 GFP在各脏器中的分布及表达。结果 :和对照组相比 ,导入rAAV2 hNGFβ大鼠的一般情况、痛敏感度、血液学和血液生化指标、脏器系数及各脏器病理组织学检查无明显差异。rAAV2 GFP分布器官包括肝、脾、肾、肾上腺、肺、甲状腺、垂体、胸腺、胃、小肠和胰腺组织。结论 :经颈外动脉将rAAV2 hNGFβ导入大鼠脑内d 60 ,rAAV2 hNGFβ对大鼠全身无明显毒性 ;rAAV2 hNGFβ除在大鼠脑内有表达外 ,其在体内的生物学分布较为广泛。

AIM: To evaluate the safety of human nerve growth factor gene β mediated by recombinant adeno-associated viruses type-2 (rAAV 2/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 rAAV 2/hNGFβ or rAAV 2/GFP with the titer of 1.5×10 15 vg·L -1 was infused into rat internal carotid artery respectively. In the long-term toxicity experiment (60 d), the rats infused rAAV 2/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 rAAV 2/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 rAAV 2/hNGFβ were not significantly different; the biological distribution of rAAV 2/GFP in the rats infused rAAV 2/GFP could be observed in brain, liver, spleen, kidneys, adrenal, lung, thyroid, hypophysis thymus, stomach, small intestine, and pancreas. CONCLUSION: The intravascular injection of rAAV 2/hNGFβ for does not display obvious toxicity on d 60 in rats. Meanwhile, the rAAV 2/hNGFβ can express not only in the brain, but also in many other tissues of rats.