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目的:建立在外周伤害性感受器特异性表达GCa MP6f蛋白的方法,并在疼痛刺激条件下,应用钙成像技术实时监测小鼠伤害性感受神经元的细胞内钙活动。方法:包装并纯化r AAV2/9-CAG-DIO-GCamp6f病毒,并通过立体定位注射该病毒于SNS-Cre小鼠L4/L5背根神经节(dorsal root ganglion,DRG)内。动物存活3~4周,然后制备L4/L5整节DRG标本,检测GCa MP6f蛋白表达情况,并观察疼痛刺激条件下诱致的伤害性DRG神经元的钙反应情况。结果:SNS-Cre小鼠L4/L5 DRG内注射r AAV2/9-CAG-DIO-GCamp6f病毒后,荧光显微镜观察显示GCamp6f病毒特异性地表达在中、小型的DRG神经元。给予DRG神经元高钾刺激(KCl 30 mmol/L)可以诱致神经元胞内的钙离子水平显著上升。进一步给予伤害性DRG神经元特异性标志物TRPV1受体的激动剂Capsaicin(1μmol/L),结果显示其可以诱致GCamp6f蛋白标记的DRG神经元呈现显著的钙反应。结论:本研究建立的SNS-Cre小鼠DRG在体注射r AAV2/9-CAG-DIO-GCamp6f病毒特异性标记伤害性DRG神经元的方法是成功的,该方法的成功建立对于活体特异性监测伤害性DRG神经元的功能活动提供了重要工具。
OBJECTIVE: To establish a method to specifically express GCa MP6f protein in peripheral nociceptors and to detect intracellular calcium activity of nociceptive neurons in real-time using calcium imaging technique under pain-stimulating conditions. Methods: The rAAV2 / 9-CAG-DIO-GCamp6f virus was packaged and purified and injected into the SNS-Cre mouse L4 / L5 dorsal root ganglion (DRG) by stereotactic injection. Animals were allowed to survive for 3 to 4 weeks. Whole L4 / L5 DRG specimens were prepared to detect the expression of GCa MP6f protein. The calcium response of nociceptive DRG neurons induced by pain was observed. Results: Fluorescence microscopy showed that GCamp6f virus was specifically expressed in moderate and small DRG neurons after injecting rAAV2 / 9-CAG-DIO-GCamp6f into L4 / L5 DRG of SNS-Cre mice. Administration of KCl 30 mmol / L to DRG neurons induced a significant increase in intracellular calcium levels. Further, Capsaicin (1μmol / L), an agonist of TRPV1 receptor, a nociceptive DRG neuron specific marker, showed that it could induce significant Ca2 + response to GCamp6f protein-labeled DRG neurons. CONCLUSIONS: The SNS-Cre mouse DRG established in this study was successful in the in vivo labeling of nociceptive DRG neurons with rAAV2 / 9-CAG-DIO-GCAb6f virus. The successful establishment of this method is of great importance for the detection of in vivo-specific Functional activity of nociceptive DRG neurons provides an important tool.