CN112080470B - 一种神经干细胞向神经元分化的体外培养方法 - Google Patents
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Abstract
本发明涉及细胞工程技术领域，具体涉及一种神经干细胞向神经元分化的体外培养方法。本发明提供一种神经干细胞向神经元分化的体外培养方法，其包括将神经干细胞置于含神经生长因子NT‑3的分化培养基中并在微重力旋转细胞培养系统中培养的步骤。本发明发现微重力旋转细胞培养系统(RCCS)联合含神经生长因子NT‑3的分化培养基能够非常显著地提高三维胶原海绵支架上培养的神经干细胞向神经元分化的能力以及神经干细胞的迁移能力，其作用效果较单独使用微重力旋转细胞培养系统和含神经生长因子NT‑3分化培养基均具有显著的提升。该方法对临床细胞移植治疗具有重要意义。
Description
技术领域
本发明涉及细胞工程技术领域，具体涉及一种神经干细胞的体外培养方法。
背景技术
神经干细胞作为一种具备自我更新能力及多向分化潜能的细胞,来源于神经组织并可生成神经组织，在适当条件下可分化成神经元、少突胶质细胞和星形细胞。神经干细胞为中枢神经系统疾病的功能重建和神经再生提供了一条新的途径，具有广泛的临床应用前景。近年来随着组织工程学的发展，寻找能够培养获得具有更高分化和迁移能力的种子细胞的方法对于细胞移植治疗具有非常重要的意义。
微重力旋转细胞培养系统(RCCS)是一种微重力培养系统，利用旋转使培养皿内的细胞或材料在旋转力及重力双重影响下而保持悬浮状态。RCCS系统在药物、医学研究、再生医学、细胞疗法中都有着广泛的应用前景。神经生长因子-3(NT-3)是一种神经营养生长因子。。胶原海绵支架材料具有良好的孔隙度，有利于细胞培养过程中营养物质输送和代谢产物排出；在未来的细胞移植治疗中，接种在细胞支架上的细胞还有利于将移植的细胞固定在损伤部位，避免移植的细胞被血液和组织液冲散。
发明内容
本发明的目的是提供一种神经干细胞向神经元分化的体外培养方法，该体外培养方法可显著提高神经干细胞向神经元方向分化能力和迁移能力。
为实现上述目的，本发明对神经干细胞的体外培养方法进行了大量的摸索。本发明发现，虽然微重力旋转细胞培养系统可模拟微重力环境，有利于细胞增殖、提高细胞培养密度，便于大规模细胞培养，但是在微重力旋转细胞培养系统中培养神经干细胞对于其向神经元的分化能力和迁移能力的促进作用并不理想。本发明意外发现，在采用微重力旋转细胞培养系统培养神经干细胞时，在培养基中同时添加神经生长因子NT-3，NT-3和微重力培养环境可产生协同增效作用，极大地提升神经干细胞向神经元的分化能力和神经干细胞的迁移能力。
具体地，本发明提供以下技术方案：
本发明提供一种神经干细胞向神经元分化的体外培养方法，其包括将神经干细胞置于含神经生长因子NT-3的分化培养基中并在微重力旋转细胞培养系统中培养的步骤。
本发明在研发过程中尝试过其他神经生长因子(NGF)，但发现其他多种细胞培养常用的NGF均不能与微重力旋转培养环境产生协同作用，其对于神经干细胞向神经元的分化能力和迁移能力的促进作用十分有限。
具体地，所述含神经生长因子NT-3的分化培养基中，NT-3的浓度为30-80ng/ml。将NT-3的浓度控制在上述范围内，可使得NT-3与微重力旋转培养环境产生更优的协同作用。
优选地，所述含神经生长因子NT-3的分化培养基中，NT-3的浓度为40-60ng/ml。
作为本发明的优选方案，所述含神经生长因子NT-3的分化培养基中，NT-3的浓度为50ng/ml。NT-3的浓度为50ng/ml时，NT-3与微重力旋转培养环境的协同增效作用最强，神经干细胞向神经元的分化能力和迁移能力的提升效果最佳。
以上所述的含神经生长因子NT-3的分化培养基包括基础培养基，还包括如下组分：非必需氨基酸0.1-0.2mM，丙酮酸钠1-2mM，B272-3％(体积百分比)，谷氨酸2-3mM，葡萄糖30-40mM，NT-3 40-60ng/ml。采用上述分化培养基培养神经干细胞能够在微重力旋转培养环境下更好地促进神经干细胞向神经元的分化能力和迁移能力的提高。
优选地，所述非必需氨基酸为甘氨酸、L-丙氨酸、L-精氨酸、L-天冬氨酸、L-脯氨酸和L-丝氨酸。
优选地，所述基础培养基为DMEM/F12。更优选为DMEM/F12(1:1)。
本发明的培养方法中，在微重力旋转细胞培养系统中培养的过程中，细胞贴附的材料保持悬浮状态。
为更好地适应微重力旋转细胞培养系统，本发明的培养方法中，神经干细胞优选为以胶原海绵为支架进行三维培养的神经干细胞。
本发明所述的胶原海绵可采用本领域常规方法制备得到。优选为采用由包括如下步骤的方法制备得到的胶原海绵：取猪皮肤组织的内层组织置于0.4-0.6M醋酸中浸泡4.5-5.5h后取出进行匀浆处理；然后加入1-2％的胃蛋白酶，15℃-18℃处理45-55小时；离心收集上清，上清中加入1.3-1.8M NaCl处理1-2小时；离心收集沉淀，将沉淀置于水中采用分子量为3000-3500的透析袋于15℃-18℃透析，将透析后的物质冻干；冻干物质于0.4-0.6M的乙酸中溶解；将pH调整至6.8-7.2后再冻干；加入1-1.5％戊二醛交联2.5-3.5h，清洗后再次冻干。
采用上述方法制备得到的胶原海绵具有更高的孔隙度和有序性，能够与NT-3和微重力旋转细胞培养环境更好地配合，促进神经干细胞向神经元的分化能力和迁移能力的提高。
在以上所述的胶原海绵支架培养过程中，所述神经干细胞的接种量为1×105细胞/0.19cm3胶原海绵。本发明所述的培养方法中，在微重力旋转细胞培养系统培养前，先对神经干细胞进行贴壁培养，将细胞贴附在胶原海绵支架材料上。
具体地，所述贴壁培养为将接种神经干细胞的胶原海绵支架先在37℃、5％CO2培养箱中贴壁培养3-5h后，再补充含有体积百分比为10％胎牛血清的高糖DMEM培养基，继续培养至24小时。
本发明的有益效果在于：本发明发现微重力旋转细胞培养系统(RCCS)联合含神经生长因子NT-3的分化培养基能够非常显著地提高三维胶原海绵支架上培养的神经干细胞向神经元分化的能力以及神经干细胞的迁移能力，其作用效果较单独使用微重力旋转细胞培养系统和含神经生长因子NT-3的分化培养基均具有非常大的提升。本发明提供的神经干细胞的体外培养方法为用于细胞移植的高质量神经细胞的获得提供了有效方法，对临床细胞移植治疗具有重要意义。
附图说明
图1为本发明实验例1中免疫荧光检测各组神经干细胞Tuj1、Map2和GFAP表达情况，其中，NT3+RCCS代表实施例1，RCCS代表对比例2，static代表对比例3，NT3+static代表对比例1；柱形图为Tuj1、Map2和GFAP阳性细胞统计结果。
图2为本发明实验例2中Transwell检测各组神经干细胞的迁移情况，其中，NT3+RCCS代表实施例1，RCCS代表对比例2，static代表对比例3，NT3+static代表对比例1；柱形图为迁移细胞统计结果。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中，神经干细胞的分离和体外扩增方法如下：
取8只SD乳鼠(出生12小时以内的乳鼠)(购自北京维通利华实验动物技术有限公司)，在75％酒精中浸泡处死，在超净台内断头，取出端脑。将端脑转移至预冷的含糖PBS(葡萄糖的浓度为0.1M)中，仔细地剔除血管和脑膜，在预冷的含糖PBS中洗3次，将PBS吸干。弯剪剪碎脑组织，将组织转移至15ml离心管，加0.25％胰酶，37℃消化40分钟，消化20分钟时取出吹打一次，消化40分钟后用胰酶抑制剂终止消化，加基础培养基，吹散沉淀，注意尽量不要产生气泡，200目细胞筛过滤后，500×g离心5分钟，去掉上清。加入添加有B27、20ng/mlbFGF和20ng/ml EGF的DMEM/F12培养基重悬细胞，接种于培养瓶中,置于37℃、5％CO2、饱和湿度的培养箱中悬浮培养，培养3天后换液，继续培养至7天，形成直径100-200μm的神经球。将含神经球的悬浮培养基倒入离心管中，250×g离心5分钟，去掉上清，收集沉淀。加入0.25％胰酶消化，室温孵育15-20分钟，在第8分钟时，轻轻将沉底的细胞团块吹散，继续孵育，孵育至20分钟后，用含体积百分比为10％胎牛血清的高糖DMEM培养基终止胰酶作用，显微镜下计数细胞。
实施例1神经干细胞的体外分化培养方法
本实施例提供一种神经干细胞的体外分化培养方法，具体步骤如下：
1、胶原海绵的制备
取猪皮肤组织，刮去毛发，去除外层组织，保留内层皮肤；加入0.5M的醋酸浸泡5小时，将组织冷冻匀浆，20000rpm，5分钟。加入1％的胃蛋白酶，16℃处理48小时；将组织15000×g离心2小时，去除沉淀，保留上清；上清加入1.5M NaCl作用1小时，然后离心15000×g离心30分钟；收集沉淀，去离子水16℃透析3天，透析袋分子量为3500；将透析后物质冻干；按2％(质量/体积)加入0.5M的乙酸溶解。利用NaOH调pH值到7，冻干；加入1％的戊二醛交联3小时，清洗10次，每次5分钟，再次冻干，即得到胶原海绵。
2、神经干细胞的贴壁培养及分化
(1)在每块胶原海绵(0.19cm3)上接种20μl的5×106/mL的神经干细胞，将胶原海绵材料置于10cm的细胞皿内，在37℃、5％CO2培养箱中贴壁培养4小时后，在内含胶原海绵材料的10cm的细胞皿内补充10ml含有体积百分比为10％胎牛血清的高糖DMEM培养基，继续培养。
(2)将步骤(1)获得的贴壁培养的胶原海绵支架材料置于含NT3的神经干细胞分化培养基中，在RCCS生物反应器内继续培养；
含NT3的神经干细胞分化培养基(100ml)的配方如下：非必需氨基酸0.1mM，丙酮酸钠1mM，双抗(青链霉素混合液)100U/ml，2％的B27(体积百分比)，谷氨酸2mM，葡萄糖33mM，NT-3 50ng/ml，DMEM/F12(1:1)补足至100ml。
对比例1
本对比例提供一种神经干细胞的体外分化培养方法，其与实施例1的区别仅在于神经干细胞分化培养基中不含有NT-3。
对比例2
本对比例提供一种神经干细胞的体外分化培养方法，其与实施例1的区别仅在于将贴壁培养的海绵支架材料置于静止培养的培养皿内培养(将RCCS生物反应器替换为静止培养的培养皿)。
对比例3
本对比例提供一种神经干细胞的体外分化培养方法，其与实施例1的区别仅在于：(1)神经干细胞分化培养基中不含有NT-3；(2)将贴壁培养的海绵支架材料置于静止培养的培养皿内培养(将RCCS生物反应器替换为静止培养的培养皿)。
实验例1神经干细胞向神经元分化能力检测
取利用各实施例和对比例的培养方法培养14天的细胞，通过免疫荧光方法检测神经干细胞的神经元分化相关基因(Tuj1，Map2，GFAP)的表达情况，进而分析神经干细胞向神经元分化的能力，具体方法如下：
(1)将各实施例和对比例培养细胞的胶原海绵支架取出，采用4％多聚甲醛固定30分钟；
(2)PBS洗涤2次，每次5分钟；
(3)在含体积百分比为0.5％的TritonX-100的PBS缓冲液中室温孵育5min；
(4)PBS洗涤2次，每次5分钟；
(5)在含体积百分比为5％正常羊血清的PBS缓冲液中孵育30min；
(6)PBS洗涤2次，每次5分钟；
(7)滴加免疫荧光一抗(Tuj1，ab18207；Map2，ab32454,GFAP,7260)，37℃孵育2小时；
(8)PBS洗涤3次，每次5分钟；
(9)加入免疫荧光二抗(Thermo fisher A32731)和Hochest，37℃孵育30min；
(10)PBS洗涤3次，每次5分钟。
(11)显微镜下观察并随机选择4个视野统计阳性细胞数，拍照，以PBS代替免疫荧光一抗作为空白对照。
结果如图1所示，根据神经元分化分子标志物Tuj1和Map2以及胶质纤维酸性蛋白(GFAP)的检测结果，采用RCCS联合NT-3神经生长因子分化培养基的实施例1的神经干细胞向神经元分化比例最高，显著高于各对比例；单独使用RCCS的对比例1的神经元分化比例高于静止培养的对比例3。与之相反，实施例1的神经干细胞向星形胶质细胞的分化比例最低，单独使用RCCS的对比例1的神经干细胞向星形胶质细胞分化比例低于静止培养组。以上结果表明，在RCCS生物反应器内采用含有NT-3神经生长因子的分化培养基培养可以明显提高在三维支架材料上培养的神经干细胞定向分化为神经元的能力，减少其向星形胶质细胞分化的能力。
实验例2神经干细胞的迁移能力检测
1、Transwell实验检测神经干细胞的迁移情况
取利用各实施例和对比例的培养方法培养14天的细胞，通过Transwell实验检测各实施例和对比例的培养方法得到的神经干细胞的迁移能力，具体方法如下：
(1)制备细胞悬液：将细胞从三维胶原海绵支架消化下来后离心弃去细胞液(用PBS洗1～2遍)，用含1％FBS的培养基重悬，调整细胞浓度至1×106/ml；
(2)接种细胞：取细胞悬液100μl加入Transwell小室，24孔板的下室内加入600μl含0.5μg/mL的SDF-1(stromal derived factor-1)的增殖培养基(注意下层培养液和小室中不要产生气泡)；
(3)培养18小时，取出Transwell小室，弃去孔中培养液，用棉签将残留在上室内表面上的细胞拭去；上室经PBS洗涤后,用4％多聚甲醛固定迁移至上室外表面上的细胞；
(4)甲醇固定：甲醇固定30min；
(5)苏木素、伊红显色：苏木精染色10s，酒精脱色，伊红染色10s；
(6)结果统计：在显微镜下计算迁移细胞数,结果取5个视野(10×20)细胞计数的平均值。
结果如图2所示，RCCS联合NT-3神经营养因子分化培养基的实施例1的方法中的神经干细胞迁移能力最高，显著高于各对比例；单独使用RCCS培养的对比例1的迁移能力高于静止培养的对比例3。以上结果表明，在RCCS生物反应器内采用含有NT-3神经生长因子的分化培养基培养的方法可以显著提高在三维支架材料上培养的神经干细胞的迁移能力。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (4)
1.一种提高神经干细胞向Tuj1+神经元分化、减少其向星形胶质细胞分化并提高神经干细胞迁移能力的体外培养方法，其特征在于，其包括将神经干细胞置于含神经生长因子NT-3的分化培养基中并在微重力旋转细胞培养系统中培养的步骤；
每100ml所述含神经生长因子NT-3的分化培养基由以下组分组成：非必需氨基酸0.1mM，丙酮酸钠1mM，青链霉素混合液100U/ml，B27 2%，谷氨酸2 mM，葡萄糖33mM，NT-350ng/ml，以DMEM/F12补足至100ml；其中，所述非必需氨基酸为甘氨酸、L-丙氨酸、L-精氨酸、L-天冬氨酸、L-脯氨酸和L-丝氨酸；
所述神经干细胞以胶原海绵为支架材料进行三维培养；
所述胶原海绵的制备方法包括如下步骤：取猪皮肤组织的内层组织置于0.5M醋酸中浸泡5h后取出进行匀浆处理；然后加入1%的胃蛋白酶，16℃处理48h；离心收集上清，上清中加入1.5M NaCl处理1h；离心收集沉淀，将沉淀置于水中采用分子量为3500的透析袋于16℃透析，将透析后的物质冻干；冻干物质于0.5M的乙酸中溶解；将pH调整至7后再冻干；加入1%戊二醛交联3h，清洗后再次冻干。
2.根据权利要求1所述的方法，其特征在于，在微重力旋转细胞培养系统中培养的过程中，细胞贴附的材料保持悬浮状态。
3.根据权利要求1~2任一项所述的方法，其特征在于，所述神经干细胞的接种量为1×105细胞/0.19cm3胶原海绵。
4.根据权利要求1~2任一项所述的方法，其特征在于，在微重力旋转细胞培养系统培养前，先对神经干细胞进行贴壁处理，使其粘附于胶原海绵支架材料上。
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| CN106367460A (zh) * | 2016-08-26 | 2017-02-01 | 杭州易文赛科拓干细胞技术研究有限公司 | 一种酸性条件下胶原蛋白海绵的制备方法 |
| CN110699321B (zh) * | 2019-11-14 | 2021-04-09 | 信阳师范学院 | 一种体外诱导神经干细胞定向分化为神经元的方法 |
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