一种获取精确胚胎天数鼠脑皮质神经细胞原代培养方法

A Method for Primary Culture of Rat Cerebral Cortical Nerve Cells at Precise Embryonic Days

目的:探讨获取精确胚胎天数鼠脑皮质神经细胞原代培养方法。方法:首先将性成熟雌雄SD大鼠1:1合笼于代谢饲养盒内,次日观察有无阴栓(同时做阴道涂片),若有记为E 0。取E 17孕鼠断颈处死,无菌状态下取出子宫,剥离羊膜与胚外膜,取得胚胎。胚胎断头,在解剖显微镜下用尖镊沿双眼、小脑和脑干腹侧切取大脑皮质,用0.125%胰酶进行温和消化,采用MEM加10%胎牛血清、10%马血清和1%L-Glutamine混合培养基,用0.73 M Poly-L-Ornithine Hydrobromide处理的培养板入37℃,10%CO2孵箱内培养。结果:雌鼠一次可排出阴栓3-5个不等,阴栓为奶黄色、锥体形、质韧。同时做阴道涂片可见活动精子;获取得胚胎脑皮质细胞呈贴壁生长,其胞体饱满,折光性好,突起明显,与周围细胞形成网络连接。结论:用阴栓并精子涂片判断胚胎胎龄简便而准确;E 17胚胎适宜体外培养获得脑皮质细胞;组合培养基加处理过的培养板和10%CO2孵箱浓度的新方法获得了状态良好的脑皮质神经细胞。

Objective： To get the primary culture method on accurate embryonic days of rat cerebral cortical nerve cells. Methods： To fed sexually mature male and female SD rats in the metabolism box with 1：1, and observe the vaginal suppository＇s existence （while doing the vaginal smear） in the next day, if can see all these, recorded it embryonic 0 day. Broken neck of 17 days of pregnant rat to death and removed the uterus under sterile.We had stripped amniotic membrane and extraembryonic membrane before getting the embryos. Cut the head of embryo and used angled tweezers along the two eyes, cerebella and the ventral of brainstem to cut the cerebral cortex under dissecting microscope. Later mildly digested these tissue with pancreatic enzymes, using mixed media （10% FBS, 10 % HI, 1% L-Glutamine and 79 % MEM）. We applied 0.73 M Poly-L-Ornithine Hydrobromide to pack plates. Put all these cells into 37℃, 10 % CO2 incubator. Results： Female rat discharged 3-5 vaginal suppositories at one time, which was milk-yellow, vertebral shape and toughness. At the same time, we saw living sperms through vaginal smear. Cerebral cortical nerve cells were adherent to the surface of culture dish, which showed satiation body, prominent processes and Clear network with surrounding cells. Conclusion： Judge fetal day by the vaginal suppository and vaginal smear, which is simple and accurate. The 17th day＇s embryo was fit for in vitro culture of cerebral cortical never cells. It was a new method of mixed medium, treated plate and 10 % CO2 concentration that get the cerebral cortical nerve cells in good condition.