鼠胚神经干细胞体外培养方法的优化

Optimization of the method to culture rat embryonic neural stem cells in vitro

目的:神经干细胞能够分化为神经元和神经胶质细胞,但其纯化、培养技术尚不完善通过不同接种密度和传代方法,筛选优化神经干细胞的体外培养技术。方法:实验于2006-05/12在四川大学移植免疫实验室完成。①动物:选取清洁级孕12～16 d雌鼠,实验过程中对动物的处置符合动物伦理学标准。②实验方法:取孕鼠胚胎脑皮质,胰蛋白酶+乙二胺四乙酸消化组织,分离神经干细胞,加入含B27、碱性成纤维细胞生长因子、表皮生长因子的DMEM/F12培养基.传至第3代时.调整细胞密度至1×10~7L^(-1),1×10~8L^(-1),1×10~9L^(-1),1×10^(10)L^(-1)进行培养。另取原代培养7～10 d后的神经干细胞,收集形成的细胞球,机械吹打法传代是离心后用由粗到细的吸管轻柔吹打.或用带5号针头的无菌注射器吹打分离细胞.操作中避免产生气泡.使用注射器时吹打的次数保持5次。胰酶消化联合机械吹打法传代是离心后加入胰蛋白酶,37℃水浴10min,用由粗到细且经火焰抛光的吸管轻柔吹打.或用带5号针头的无菌注射器吹打.以胎牛血清终止消化。③实验评估:观察第3代神经干细胞生长状态.MTT法检测各密度下培养1,3,5,7 d时的增殖情况。计数两种传代方法亚代神经干细胞形成的克隆球数目,比较增殖效果。免疫荧光染包检测神经球巢蛋白、BrdU标记、神经元特异性烯醇化酶、胶质细胞原纤维酸性蛋白的表达。结果:①神经干细胞的培养和鉴定:从胚胎大鼠脑皮质分离培养出的细胞可聚集成球状.并在体外大量扩增和长期存活,巢蛋白、神经元特异性烯醇化酶、胶质细胞原纤维酸性蛋白均呈阳性表达,免疫荧光染色可见大量BrdU标记的阳性细胞。②不同接种密度对神经干细胞增殖的影响:按1×10~9L^(-1)密度培养的细胞分裂增殖最为迅速.于第7天达高峰,且克隆球生成数量明显多于1×10~7L^(-1),1×10~8 L^(-1),1×10^(10)L^(-1)密度(P均<0.05)。③不同传代方法对亚代神经干细胞克隆增殖效果的影响:采用机械吹打法传代的神经干细胞所增殖形成的细胞球结合紧密,抛光玻璃管+注射器吹打后细胞球能较好地分开.但不能完全分散成单细胞.传代后细胞能够继续生长,并形成亚代神经干细胞球。经胰酶消化的神经球在机械吹打后也不能完全形成单细胞,且其亚代克隆的形成能力明显低于机械吹打法(P<0.05)。结论:①从胚胎大鼠脑皮质分离培养出的细胞具有神经干细胞特性,可诱导分化为神经元和星形胶质细胞②1×10~9L^(-1)为最佳细胞接种密度,机械吹打法亚代克隆的活性和增殖能力明显优于胰酶消化联合机械吹打法。

AIM： Neural stem cells can be induced to differentiate into various types of neural cells such as neurons and neuroglia cells, but the technique of depuration and cultivation does not consummate. This article determines the optimal culture technique of neural stem cells by different culture concentrations and passage methods. METHODS： Experiments were conducted from May to December 2006 at Laboratory of Transplantation Immunity of Sichuan University. ①Clean pregnant female rats （embryonic age range from 12-16 days） and the disposition of animal met ethical standard. ② The cerebral cortex of rat embryos were collected, and digested with trypsin and ethylenediamine tetraacetic acid mixture to obtain signal cell suspension. They were cultured in serum-free medium （DMEM/F12 medium containing B27, basic fibroblast growth factor and epidermal growth factor）. The 3^rd passage cells were collected, and incubated at 1 × 10^7 L^-1, 1 × 10^8 L^-1, 1 × 10^9 L^-1, 1 × 10^10 L^-1, respectively. In addition, neural stem cells were collected 7-10 days after primary culture to harvest formative cell masses. Mechanical blow refers to soft blowing with haustorial tube from thick to thin after centrifugation, or sterile sydnge with No. 5 pinhead blowing cells when the blow was 5 times. Bubble production was avoided during the operation. Trypsin aspiration combined with mechanical blow refers to trypsin was added after centrifugation, at 37 ℃ for 10 minutes, the neural stem cells were lightly blown with haustorial tube polished with flame or blown with sterile syringe with No. 5 pinhead, and then fetal bovine serum was added to stop digestion. ③The growth characteristic of the 3^rd passage cells at different culture concentration was observed and proliferation was measured at days 1, 3, 5 and 7 by MTT assay. The neural clone spheres of subcultured was counted to determine the optimal passage way. Immunofluorescence was carried out to detect nestin （special marker to neural stem cells）, BrdU, neurone specific enolase, glial fibdllary acidic protein. RESULTS： ①Growth characteristics and identification of rat embryonic neural stem cells in vitro. The dissociated neural stem cells from the cerebral cortex of rat embryos were continuously harvested and purified by suspension cultures to get the daughter cell clone. Nestin positive cells could be found in the neurospheres and after attachment they could differentiate into neurone specific enolase and glial fibriUary acidic protein positive cells, and immunofluorescence showed a great of BrdU-positive cells. ②The effect of different incubated number of neural stem cells on proliferation： When the neural stem cells planted at the concentration of 1 × 10^9 L^-1, the growth rate of the cells was the highest of all concentrations. The number of clone spheres exceeded others at concentration 1× 10^7 L^-1,1× 10^8 L^-1 and 1× 10^10 L^-1（P 〈 0.05）, ③The neural stem cell proliferation ability affected by different passage ways： The mechanical blow method produced progeny neurospheres were active and tolerate passging. The progeny neurospheres combined tightly and could be triturated with fire polished glass pipette and injection syringe, then the neurospheres could not be fully separated into single cells, The trypsin digestion method produced progeny neurospheres were tolerate passging, but the ability of proliferation was lower than that in mechanical blow method and the neurospheres could not be separated into single cells absolutely in trypsin digestion method （P 〈 0.05）. CONCLUSION： ①The cells isolated from the cerebral cortex of rat embryos possess the characteristics of neural stem cells, and these neural stem cells can differentiate into neuron and astrocyte phenotypes.②The optimal culture concentration is 1× 10^9 L^-1, The mechanical blow method produced progeny neurospheres were active and tolerate passging, and this ability is better than that in trypsin digestion combined with mechanical blow method.