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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 investigate the efficiency and specificity of intracerebral injection of adeno-associated virus (RSV) in different serogroups on blood vessels of mouse cerebral artery rings (Willis rings). Methods The 10-week-old C57BL / 6 male mice were used to determine the location of the right basal cistern (1.2mm on the beak and 0.7mm on the right) based on the brain localization map. AAV2 h Syn-GFP and AAV9-h Syn-GFP viruses were injected into the basal cistern. After 14 days, the brain tissue was removed completely. The fluorescence intensity and tissue distribution of Willis ring in the skull base were observed by fluorescence microscope. The brain tissue of mice was fixed and sliced, and the efficiency and specificity of different serotypes of AAV on cerebrovascular smooth muscle cells infection were further observed by immunofluorescence staining. Results Bromphenol blue dye tracing found that AAV was successfully injected into the vicinity of the Willis ring of mice by stereotaxic method. Fluorescent microscopy revealed that both AAV2 and AAV9 injected mouse skull base were stimulated with strong green fluorescence. Immunofluorescence staining indicated that the expression of GFP in brain tissue of AAV9-treated mice was significantly colocalized with that of SM22α, and there was almost no colocalization with nerve cells. AAV2-treated mouse brain tissue GFP expression and SM22α expression only partial co-localization. CONCLUSION: AAV9 is highly efficient and specific for vasculogenic infection of the cerebral arteries. This study explored the mechanism of intracranial aneurysm to provide new and more specific gene editing approaches at the animal model level.