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目的探索用于RNA干扰的重组腺病毒Ad-shRNA-NgR转染大鼠缺血脑组织的适宜滴度。方法将低、中、高3种不同滴度(7.90×109、1.58×1010、3.16×1010pfu/ml)的重组腺病毒通过立体定位手术注射到大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)/再灌注模型大鼠(n=10)脑缺血区,于术后48 h和1周在荧光显微镜下观察各组大鼠脑组织中绿色荧光蛋白(green fluorescent protein,GFP)的表达强度并估算转染效率,通过HE染色检测炎性反应。结果中滴度和高滴度注射组大鼠脑组织腺病毒转染效率相似,为60%～70%,明显高于低滴度注射组(P<0.01)。在各组脑组织切片中,仅高滴度注射组有明显炎症反应,血管周围可见淋巴和巨噬细胞浸润。结论中等滴度(1.58×1010pfu/ml)的腺病毒能高效、稳定、低毒地转染大鼠缺血脑组织,推荐用于脑缺血基因干预治疗的实验研究。
Objective To explore the suitable titer of recombinant adenovirus Ad-shRNA-NgR transfected to ischemic brain in rats. Methods Three kinds of adenovirus with different titers (7.90 × 109, 1.58 × 1010 and 3.16 × 1010 pfu / ml) were injected into MCAO / The expression intensity of green fluorescent protein (GFP) in the brain tissue of each group was observed under a fluorescence microscope at 48 h and 1 week after reperfusion (model n = 10) Transfection efficiency, inflammatory response detected by HE staining. Results The transfection efficiencies of adenovirus in the middle titer and high titer injection groups were similar between 60% and 70%, significantly higher than those in the low titer injection group (P <0.01). In each group of brain tissue sections, only high titer injection group had obvious inflammatory reaction, visible perivascular infiltration of lymphocytes and macrophages. Conclusion The adenovirus with moderate titer (1.58 × 1010pfu / ml) can be transfered into ischemic brain tissue efficiently, stably and with low toxicity. It is recommended to be used in the experimental study of gene therapy for cerebral ischemia.