显微注射增强绿色荧光蛋白-逆转录病毒检测缺血缺氧新生大鼠皮质神经干细胞的增殖

Detection of neural stem cell proliferation through microinjection of enhanced green fluorescent protein- retrovirus retrovirus in neonatal hypoxic-ischemic rat cortex

目的用显微注射增强绿色荧光蛋白（EGFP）-逆转录病毒的方法检测缺血缺氧对新生大鼠内源性神经干细胞增殖的影响。方法新生7dSD大鼠结扎左侧颈总动脉，并给予氧浓度为8％的氧氮混合气体缺氧处理2h，制作新生大鼠缺血缺氧模型；将绿色荧光蛋白（GFP）基因片段定向插入逆转录病毒载体pLNCX2，转染DH5a感受态细胞，用lipofectamine将pLNCX2-EGFP逆转录病毒载体导入PA317包装细胞，用G418筛选获得抗性细胞克隆，扩增抗性细胞并收集病毒上清；用显微注射的方法往正常组及缺血缺氧模型动物皮质注射EGFP-逆转录病毒；取脑组织制作冰冻切片，用4’，6-二脒基-2-苯基吲哚染细胞核，荧光显微镜下观察缺血缺氧前后动物大脑皮质EGFP阳性细胞（内源性神经干细胞）的增殖情况。结果成功构建了EGFP-逆转录病毒，并将病毒注射到对照组及模型组动物皮质，荧光显微镜观察显示，新生鼠缺血缺氧后EGFP阳性细胞（内源性神经干细胞）的数目比正常动物多，正常组皮质GFP标记的细胞数为（104±9）；模型组皮质GFP标记的细胞数为（169±12），差异有统计学意义（P〈0．01）。结论显微注射EGFP-逆转录病毒是观察内源性神经干细胞增殖的理想方法；缺血缺氧能促进新生大鼠皮质神经干细胞的增殖。

Objective To observe the endogenous neural stem cell proliferation in neonatal hypoxic-ischemic rats through enhanced green fluorescent protein（EGFP）-retrovirus microinjection. Methods Postneonatal 7 d SD rats received left common carotid artery ligation and 2 h hypoxia with oxygen （ concentration of 8% ） and nitro- gen mixed gas to establish neonatal rat model of ischemia and hypoxia. The green fluorescent protein gene fragment was inserted into pLNCX2 retroviral vector, and ligation product was transformed in to DH5cx competent cells; re- sistant cells were screened with CA18; then resistant cells were amplified and viral supernatants were collected. pLNCX2-EGFP retrovirus vector was transfceted into PA317 packaging cells by lipofectamin. EGFP retroviruses were injected into cortex of normal and hypoxia^ischemia animals with method of microinjection. Frozen brain tissue sections were made and DAPI was used to stain the nucleus. The difference of neural stem cells（ endogenous neural stem cells） proliferation between the norulal and hypoxic-ischemic animals was observed with a Fluorescence microscopy. Results EGFP-retroviral vector was constructed and viruses were injected into animal cortex; fluores- cence microscopy showed that the number of EGFP-positive cells（ endogenous neural stem cells）in hypoxia-ischemia neonatal rat cortex was more than that in the normal animals. The number of GFP-labeled cells in the cortex of the normal group was （ 104 ±9） ; while the number of GFP-labeled cells in the model group was （ 169±12） ; the difference was statistically significant （P 〈0. 01 ）. Conclusion EGFP-retroviral microinjection is an ideal method for the observation of endogenous neural stem cell proliferation ; hypoxic-ischemia can promote the proliferation of neonatal rat cortical neural stem cells.