脑损伤模型中基于形态学多重免疫荧光技术的神经细胞研究方法

Multi-overlap immunofluorescence techniques used in nerve cells based on morphology in brain injury models

目的探讨多重免疫荧光技术在神经细胞形态学研究中的应用，为神经细胞靶向研究提供新方法。方法Balb／c小鼠10只按随机数字表法分为重度创伤性脑损伤（STBI）组、假模对照组（各5只），其中STBI组采用控制性经脑皮质撞击法进行造模，神经功能评级为8分。各组均于第7天终止实验取脑切片，应用多重免疫荧光技术方法，在激光共聚焦显微镜下同时观测神经元核抗原抗体（NeuN）、5-溴脱氧尿嘧啶核苷（BrdU）、胶质纤维酸性蛋白（GFAP）及4’，6-二脒基-2-苯基吲哚（DAPI）的形态学表达并定量比较。结果利用多重免疫荧光技术获得的图像色彩鲜明、清晰明辨、形态感强。STBI组大脑损伤区图像显示BrdLI＋NeuN＋GFAP＋DAPI表达较多；假模对照组大脑对应区域图像上几乎没有BrdU＋NeuN＋DAPI表达，GFAP表达也非常少。STBI组与假模对照组BrdU＋NeuN＋DAPI和GFAP相对表达量比较差异均有统计学意义（P〈0．05）。结论小鼠遭受重度脑创伤后可诱导神经细胞或星形胶质细胞不同程度自行增殖。多重免疫荧光技术可作为靶向研究神经细胞发育、增殖、凋亡或死亡机制的一种新的科学研究手段。

Objective To explore the application of multi-overlap immunofluorescence techniques in the morphological research of nerve cells, and provide a new method of targeted research on nerve cells. Methods Ten Balb/c mice were selected and randomly divided into experimental group of severe traumatic brain injury （STBI） and sham control group （n=5）; mice in the experimental group were induced STBI models by controlled cortical impact （CCI） method, and the neurological severity scale （NSS） scores were 8 in them. All animals were terminated on day 7. The gene and protein expressions of neuronal nuclei antigen （NeuN）, glial fibrillary acidic protein （GFAP） and bromodeoxyuridine （BrdU）, and the morphology expression of 4＇,6-diamidino-2-phenylindole （DAPI） were observed under confocal microscopy by applying multi-overlap immunofluorescence techniques. Results The images got by multi-overlap immunofluorescence techniques possessed the characteristics of bright and clear color, strong morphological sense on the expressions. The more obvious expressions of BrdU＋NeuN＋GFAP＋DAPI in the STBI group could be noted as compared with those in the sham control group; the relative expression quantity of BrdU＋NeuN＋DAPI was significantly different between the two groups （P〈0.05）. Conclusion STBI in mice could induce self-regenerated phenomenon on cerebral neural cells or astrocyte; these new techniques provide new scientific means in the targeted study of development, proliferation, apoptosis or death of nerve cells and its mechanism.