胎鼠纹状体神经干细胞分离培养方法的改进

Improvement in the methods for separation and culture of neural stem cells in striatum of embryonic rats

目的:利用有血清和多聚赖氨酸培养液中加入SD14d胚胎大鼠纹状体区神经干细胞,探讨其分离、纯化神经干细胞操作技术的改进。方法:实验于2003-03/2004-11在广州第一军医大学珠江医院神经外科实验室完成。①选用SD大鼠20只,取孕13～14d的SD大鼠胚胎纹状体,吸管吹打(不超过10次)后,用组织剪剪成1mm3块状,以1.25mL/L的胰酶,在37℃下消化20min,以S-Dulbecco改良的Eagle培养基-F12中止消化,采用1000r/min离心10min,显微镜下细胞记数,以1×104/cm2接种到改良的有血清和多聚赖氨酸铺被的12孔培养板上,培养液为Dulbecco改良的Eagle培养基-F12加上B27,加入血清和20μg/L表皮生长因子。7d后用吸管吸取漂浮的细胞球,机械吹打成单细胞悬液,按每培养孔1×104/cm2细胞行传代再培养,此后5～7d再传代,方法同前。②利用神经干细胞与星形胶质细胞、神经细胞以及成纤维细胞贴壁之间的差异,使细胞分为贴壁和漂浮的两层细胞,分离去掉贴壁的神经细胞、成纤维细胞、星形胶质细胞以及部分的神经干细胞,得到漂浮的细胞球。③机械吹打后制成单细胞悬液,再传代培养,细胞重新增殖成细胞球,应用免疫细胞化学技术特异性抗原巢蛋白检测最初的细胞球、吹打后的单细胞和重新增殖的细胞球巢蛋白抗原的表达和分化后特异性成熟神经细胞抗原的表达。巢蛋白为神经干细胞特异性标记物,其阳性表达表明神经干细胞的存在。结果:①原代培养的纹状体细胞第2天可见贴壁生长的神经细胞、星形胶质细胞和增殖的细胞球(约10～16个细胞大小),漂浮的细胞开始出芽并增殖;第3天贴壁的细胞球继续增殖(约50个细胞大小),漂浮的细胞则增殖成4～8个大小的细胞球;第4～7天贴壁的细胞球开始分化,漂浮的细胞则继续增殖成约30个细胞大小的细胞球,巢蛋白染色阳性。②漂浮的细胞,吹打后行传代培养,加入表皮生长因子、血清和多聚赖氨酸,细胞仍漂浮生长,1周后增殖成约70～80个细胞大小的细胞球,吸管吹打继续再培养,细胞仍漂浮增殖成细胞球,巢蛋白抗原表现阳性。③免疫细胞化学染色显示原代培养中贴壁的细胞球、吹打后的单细胞、漂浮的细胞球巢蛋白染色阳性,吹打后的细胞若不加入表皮生长因子,则细胞染色显示胶质纤维酸性蛋白和神经丝蛋白阳性。分离纯化后的细胞具有自我更新能力和多分化潜能,有很强的增殖能力。结论:利用有血清和多聚赖氨酸加入的方法分离培养的原代细胞球巢蛋白表达阳性,具有纯化度高,存活时间长的特点。

AIM： To culture the neural stem cells （NSCs） from striatum of 14 days＇ embryonic rats in the culture medium containing blood serum and polylysine so as to probe into the improvement of technique for separation and purification of NSCs. METHODS： The experiment was conducted in the Laboratory for Neurosurgery, Zhujiang Hospital, First Military Medical University from March 2003 to November 2004. ①The striatum, which had been separated from 20 SD embryonic rats （13 to 14 days）, was beaten upon through a pipette for less than 10 times, and then was sheared into 1 mm^3 pieces by a pair of scissors. Then the pieces of striatum were digested with pancreatin 1.25 mL/L at 37 ℃ for 20 minutes followed by final digestion in Dulbecco＇s modified Eagle＇s medium plus Ham＇s F12 medium （DMEM/F12）. Then the striatum was centrifuged at 1 000 rotations per minute for 10 minutes to count the number of cells under microscope. The cells at 1 × 10^4/cm^2 were inoculated onto a modified 12-well culture plate containing blood serum and ploylysine, and then the culture plate was dipped into the DMED/F12 plus B27 culture medium, added with blood serum and 20 μg/L epidermal growth factors. Seven days later, the floating cell balls were imbibed with the pipette, and beaten upon into single cell suspension mechanically. The cells of 1×10^4/cm^2 per well were subcuhured for 5 to 7 days followed by another subculture with the same method. ② According to the difference of NSCs from astrocytes, nerve cells and fibroblasts in their adherence to the wall, the NSCs were divided into adhesive and floating layers, and then the nerve cells, fibroblasts, astrocytes and some NSCs which were all adhered to the wall were separated to get the floating cell balls. ③ The above cell balls were made into single cell suspension by beating upon mechanically followed by subculture, then the cells proliferated into cell balls again. The expression of nestin antigens and the expression of differentiated specific mature nerve cell antigens were detected in the mechanically beaten single cell suspension, suhcultured cells and re-proliferated cell balls. Nestin was the specific marker of NSCs, and the expression of it indicated the existence of NSCs. RESULTS： ①At 2 days after the striatum cells were cultured primarily, the adhesive nerve cells, astrocytes and proliferative cell balls （10 to 16 cells） were found, and the floating cells began to bud and proliferate. At 3 days, the adhesive cell balls continued to proliferate（about 50 cells）, and the floating cells proliferated into cell balls（4 to 8 ceils）. From 4 to 7 days, the adhesive cell balls began to differentiate, the floating cells continued to proliferate into cell balls（about 30 cells）, and the nestin was stained white. ②The floating cells were subeuhured after beaten upon, which were still floating after added with epidermal growth factors, blood serum and polylysine, and they proliferated into the cell balls of 70 to 80 cells; After another beating upon, the cells were still floating and then proliferated into cell balls, and the nestin was positive. ③Shown by immunohistochemistry, the nestin protein was stained positive in the adhesive subeuhured cell balls, beaten single cells and floating cell balls, and if not added with epidermal growth factors, the glial fibriliary acidic protein and neurofilament protein were positive in the beaten cells. The separated and purified cells were of self-renewing ability, multi-differentiating potential, and strong proliferative ability. CONCLUSION： The nestin is positive in the primarily cultured cell balls after separation and culture by means of adding blood serum and polylysine, and these ceils are of high purification and can survive for a long term.