腺相关病毒不同血清型对小鼠大脑动脉环感染效率的比较研究

Comparative Sstudy on the Infection Efficiency of Different Adeno-associated Virus on Circulus Arteriosus Cerebri in Mice

目的探讨通过立体定位注射腺相关病毒不同血清型对小鼠大脑动脉环(Willis环)血管的感染效率及特异性。方法 10周龄C57BL/6雄性小鼠依据脑定位图谱确定右侧基底池的定位坐标(前囟点喙侧1.2mm,右侧0.7mm),用微量注射泵以0.5μl/min泵速将AAV2-h Syn-GFP和AAV9-h Syn-GFP病毒注射入基底池。14天后将脑组织完整取出,采用荧光体视镜观察各处理组小鼠颅底Willis环附近绿色荧光强度及组织分布。小鼠脑组织固定后切片,行免疫荧光染色进一步观察AAV不同血清型对脑血管平滑肌细胞感染的效率及特异性。结果溴酚蓝染料示踪发现立体定位方法将AAV顺利注入到小鼠Willis环附近。采用荧光体视显微镜发现AAV2和AAV9注射后的小鼠颅底均激发出较强的绿色荧光。免疫荧光染色提示AAV9处理小鼠的脑组织GFP表达与SM22α表达存在明显共定位,与神经细胞几乎无共定位。AAV2处理小鼠脑组织GFP表达与SM22α表达仅存在部分共定位。结论 AAV9对大脑动脉环血管感染效率高且特异性较强,该研究为颅内动脉瘤的机制探索在动物模型水平提供了新的更特异的基因编辑途径。

Objective To explore the infection efficiency and specificity of different adeno-associated virus on circulus arteriosus cerebr i i n m ice.Methods C57 BL/6 m ice were f i xed i n the stereot axic f rame with mouse ad aptor to place sk ull hor izont ally.We stereotaxically placed the tip of the needle at the right basal cister n using the coordinate obtained from Mouse Brain Atlas（1.2 mm anterior to the bregma,0.7 mm lateral to the midline）,AAV2-or AAV9-h Syn-GFP vector were injected by ultra micro pump.After 14 days,the intensity and distribution of green f luorescence around circulus arteriosus cerebri were observed by f luoroscope.The infection eff iciency and specif icity of AAV on cerebrovascular smooth muscle cells were assessed by immunof luorescence staining.Results In mice that received bromophenol blue solution into the right basal cistern using our stereotactic coordinates,blue dye were seen within circulus arteriosus cerebri in all brains.Strong green f luorescence were observed around circulus arteriosus cerebri both in AAV2 and AAV9 treated groups.Using immunof luorescence staining,we found that GFP was predominantly detected in cerebrovascular smooth muscle cells in A AV9 treated groups.In contrast,the majority of GFP were found both in cerebrovascular smooth muscle cells and neurocytes in AAV2 treated groups.Conclusion Higher infection efficiency and specificity of AAV9 on circulus arteriosus cerebri were observed,which may provide a new and specific method of gene editing on animal models of intracranial aneurysms.