CN111304167A - 人源脂肪干细胞来源的神经元前体细胞及其制备方法和应用 - Google Patents
人源脂肪干细胞来源的神经元前体细胞及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种诱导培养基、采用该培养基诱导培养人源脂肪干细胞分化为神经元前体细胞,包括NEAA、β‑巯基乙醇、谷氨酰胺、生长因子和神经营养因子,所述NEAA与β‑巯基乙醇的体积比为4:1‑13:1,所述NEAA与谷氨酰胺的体积比为1:2‑2:1,所述生长因子由10‑20ng/mL的bFGF和10‑20ng/mL的EGF组成,所述神经营养因子由10‑20ng/mL的BDNF、10‑20ng/mL的NGF和10‑20ng/mL的NT3组成。本发明人源脂肪干细胞来源的神经元前体细胞群通过特定的诱导培养基诱导人源脂肪干细胞分化而得,神经元前体细胞健康状况好,活性及纯度高。
Description
技术领域
本发明涉及干细胞及生物医药技术领域,尤其涉及人源脂肪干细胞来源的神经元前体细胞、制备方法及其在治疗神经系统疾病方面的应用。
背景技术
人源脂肪干细胞(Human-Adipose Derived Adult Stemcell,hADSC),因其易于取材,体外培养老化率低,低免疫原性,生物安全性高,不涉及伦理问题等优势,已成为最有前景的细胞来源之一。脂肪干细胞向神经分化是其向临床转化治疗神经系统疾病的关键一步。目前,诱导脂肪干细胞向神经分化的方案有化合物法、基因重编程法。化合物法的弊端是化合物对细胞的毒副作用,影响分化获得神经细胞的健康状况,同时化合物法具有无序性和低效性。基因重编程法,虽然具有针对性和高效性,但基因及基因载体的选择是个难度较大的问题。
目前,通过诱导脂肪干细胞分化成的神经细胞健康状况欠佳,诱导获得的神经细胞得率不高,而且会伴有细胞的坏死和凋亡现象,影响后续临床上的应用。
另外,由脂肪干细胞诱导获得的神经元的分类定性存在欠缺,诱导分化后获得的细胞仍然是个复杂的细胞群体,无法根据临床要求制备特定的神经元或前体细胞。
如何通过诱导脂肪干细胞获得健康状况和活性较好、纯度较高的神经元前体细胞成为目前亟待解决的问题。
发明内容
为了解决现有的脂肪干细胞诱导的神经元样细胞健康状况欠佳,得率、纯度及活性较低的问题,本发明的目的是提供人源脂肪干细胞来源的神经元前体细胞、制备方法及其在治疗神经系统疾病方面的应用。
为了实现上述目的,本发明采用如下技术方案:
本发明的第一方面,提供一种诱导培养基,包括NEAA、β-巯基乙醇、谷氨酰胺、生长因子和神经营养因子,所述NEAA与β-巯基乙醇的体积比为4:1-13:1,所述NEAA与谷氨酰胺的体积比为1:2-2:1,所述生长因子由10-20ng/mL的 bFGF和10-20ng/mL的EGF组成,所述神经营养因子由10-20ng/mL的BDNF、 10-20ng/mL的NGF和10-20ng/mL的NT3组成。
本发明的第二方面,提供采用上述诱导培养基将人源脂肪干细胞分化成神经元前体细胞的方法,包括以下步骤:
S1,向超低吸附培养皿中加入诱导培养基后,注入脂肪干细胞,对脂肪干细胞进行诱导培养;
S2,收集成球生长的悬浮脂肪干细胞,洗掉血清后进行消化处理,处理后的细胞悬浮于诱导培养基中继续培养,获得神经元前体细胞。
本发明的第三方面,提供采用上述方法制备的神经元前体细胞群。
本发明的第四方面,提供人源脂肪干细胞来源的神经元前体细胞群的用途,在用于制备治疗神经系统疾病的药物上的应用。
其中,所述神经系统疾病包括阿尔茨海默,帕金森,中风,急性脑损伤和自闭症。
本发明的第五方面,提供一种药物组合物,包含上述的人源脂肪干细胞来源的神经元前体细胞群和药学上可接受的载体,所述人源脂肪干细胞来源的神经元前体细胞群的存在量足以在给予患者所述人源脂肪干细胞来源的神经元前体细胞群后促进神经系统的功能恢复。
其中,所述人源脂肪干细胞来源的神经元前体细胞群中神经元前体细胞的数量为5×107-108个。
选取康宁公司生产的超低吸附培养皿,这种培养皿可最大程度地降低细胞的吸附性,使细胞容易脱离表面成球培养,加入诱导培养基促使这些成球培养的脂肪干细胞向神经细胞转变,成功地将脂肪干细胞转化为球状的悬浮神经干细胞,此细胞为中间细胞,球状的悬浮神经干细胞继续培养后获得的神经元前体细胞健康状况得到极大地改善,移植入中枢神经系统后成活率和分化能力将得到提高,且容易具有电生理活性。
与现有技术相比,本发明实现的有益效果:本发明人源脂肪干细胞来源的神经元前体细胞群通过特定的诱导培养基诱导人源脂肪干细胞分化而得,将人源脂肪干细胞分化而得的神经元前体细胞原位移植入中风鼠,检测到神经元前体细胞在中风鼠活脑组织中的动作电位和突触电位,说明神经元前体细胞健康状况好;同时观察到神经元前体细胞能够显著改善中风鼠的行为情况。
附图说明
以下结合附图和具体实施方式来进一步详细说明本发明:
图1是通过细胞形态和CD表面标记物表征hADSC;
图2显示了hADSC分化成中间体球状细胞;
图3显示了hADSC分化成神经元前体细胞;
图4显示了植入神经元前体细胞后,脑中风小鼠的行为变化情况。
图5显示了神经元前体细胞植入中风治疗组后,神经元前体细胞在活脑组织中的电生理活性。
具体实施方式
发明人经过广泛而深入地研究,意外地发现通过选取超低吸附培养皿,在超低吸附培养皿中加入由NEAA、β巯基乙醇,谷氨酰胺(Glutamax)、生长因子、神经营养因子组成的诱导培养基,诱导培养人源脂肪干细胞分化成中间体球状细胞,消化后成悬浮细胞继续培养,获得纯度较高、健康状况及活性好的神经元前体细胞。
人源脂肪干细胞(hADSC)是从脂肪组织中分离得到的一种具有多向分化潜能的干细胞,它是来源于脂肪组织的一种间充质干细胞,可以分化为成骨细胞、软骨细胞或脂肪细胞等。hADSC能够在体外稳定增殖且衰亡率低,同时具有取材容易、少量组织即可获取大量干细胞,适宜大规模培养,对机体损伤小等优点,而且来源广泛,体外储备量大,适宜自体移植。
本发明的“脂肪干细胞来源的神经元前体细胞”是指通过诱导脂肪干细胞分化得到的神经元前体细胞。
本发明所用的“脂肪干细胞来源的神经元前体细胞群”是指本发明方法所得的脂肪干细胞来源的神经元前体细胞的群体,而非指单个神经元前体细胞。
现有技术由脂肪干细胞诱导分化的神经元前体细胞健康状况及活性不佳,或者所得到的为复杂的细胞群体,不仅包括神经元前体细胞,还包括处于不同阶段的神经元细胞,无法根据临床要求获得纯度较高的神经元前体细胞。
发明人经过不断摸索,开发了一种诱导培养基,诱导人源脂肪干细胞分化。诱导培养基是以一种商业化的培养基作为基础培养基(BrainPhys Neuron Basel Medium,购自BrainPhysTMNeuronal Medium,Stem cell technology,型号为XX),在基础培养基内加入NEAA、β-巯基乙醇、谷氨酰胺、生长因子和神经营养因子,NEAA与β-巯基乙醇的体积比为4:1-13:1,NEAA与谷氨酰胺的体积比为1:2-2:1,具体实验时,NEAA的体积百分数为1%-2%,β-巯基乙醇的体积百分数为 0.15-0.25%,谷氨酰胺的体积百分数为1%-2%,生长因子由10-20ng/mL的bFGF 和10-20ng/mL的EGF组成,神经营养因子由10-20ng/mL的BDNF、10-20ng/mL 的NGF和10-20ng/mL的NT3组成。
本发明还提供了一种采用上述诱导剂诱导人源脂肪干细胞分化成神经元前体细胞的方法,包括以下步骤:
S1,向超低吸附培养皿中加入含有诱导剂的神经元培养基后,注入脂肪干细胞,诱导培养3天;
S2,收集悬浮着呈球样生长的脂肪干细胞,洗掉血清后,,进行消化处理,处理后的细胞悬浮于神经元培养基中继续培养6天。
本发明的神经元前体细胞群可以用于治疗神经系统疾病,此处的神经系统疾病包括阿尔茨海默,帕金森,中风,急性脑损伤和自闭症。
本发明还提供了一种药物组合物,包含上述的人源脂肪干细胞来源的神经元前体细胞群和药学上可接受的载体,所述人源脂肪干细胞来源的神经元前体细胞群的存在量足以在给予患者所述人源脂肪干细胞来源的神经元前体细胞群后促进神经系统的功能恢复。
其中,所述人源脂肪干细胞来源的神经元前体细胞群中神经元前体细胞的数量为5×107-108个。
实施例1
诱导培养基的制备过程如下:
向基础培养基内加入NEAA、β-巯基乙醇、谷氨酰胺、生长因子和神经营养因子,其中NEAA的体积百分数为1%,β-巯基乙醇的体积百分数为0.15%,谷氨酰胺的体积百分数为1%,生长因子由10ng/mL的bFGF和20ng/mL的EGF 组成,神经营养因子由20ng/mL的BDNF、10ng/mL的NGF和10ng/mL的NT3 组成。
实施例2
诱导培养基的制备过程如下:
向基础培养基内加入NEAA、β-巯基乙醇、谷氨酰胺、生长因子和神经营养因子,其中NEAA的体积百分数为2%,β-巯基乙醇的体积百分数为0.25%,谷氨酰胺的体积百分数为2%,生长因子由20ng/mL的bFGF和10ng/mL的EGF 组成,神经营养因子由10ng/mL的BDNF、20ng/mL的NGF和20ng/mL的NT3 组成。
实施例3
诱导培养基的制备过程如下:
向基础培养基内加入NEAA、β-巯基乙醇、谷氨酰胺、生长因子和神经营养因子,其中NEAA的体积百分数为1.5%,β-巯基乙醇的体积百分数为0.2%,谷氨酰胺的体积百分数为1.5%,生长因子由15ng/mL的bFGF和15ng/mL的 EGF组成,神经营养因子由15ng/mL的BDNF、15ng/mL的NGF和15ng/mL 的NT3组成。
实施例4 hADSC的分离和表征
采用差速贴壁分离方法进行人源脂肪干细胞的分离和后续扩增培养,并应用流式细胞仪及细胞免疫组化方法检测细胞的特性,检测结果如图1。从图1中可知,制备的脂肪干细胞表达间充质干细胞表面标记蛋白CD44、CD73、CD105,不表达CD34、CD45、CD133。
实施例5 hADSC诱导为中间体球状细胞
将hADSC诱导为中间体球状细胞的过程如下:
S1,向超低吸附培养皿中加入实施例3制备的诱导培养基后,注入脂肪干细胞,诱导培养3天;
S2,收集悬浮着呈球样生长的脂肪干细胞,将细胞铺展在凝胶包被的玻璃片上,继续培养3天后,去掉培养基,用4%的多聚甲醛固定后进行免疫细胞化学染色来检测中间体细胞的特性。
如图2为中间体细胞的细胞形态及标记情况。从图2中可知,获得的中间体细胞为球状细胞,表达干细胞相关标记Nanog和OCT4,但不表达神经元相关标记Tubulin和Synapsin。
实施例6将中间体球状细胞继续诱导为神经元前体细胞
将消化后的中间体球状细胞继续悬浮培养3天,获得神经元前体细胞。用免疫细胞化学染色来检测神经元前体细胞的特性,如图3所示。
由图3可知,神经元前体细胞可表达神经元标记蛋白MAP2,星形胶质细胞标记物或神经干细胞标记物GFAP和神经元标记物Synapsin,表明诱导培养基能够将成球培养的脂肪干细胞诱导成为神经元前体细胞。
实施例7将神经元前体细胞用于治疗脑中风
1、采用经典方法进行大脑中动脉栓塞建立小鼠脑中风模型;
2、将实施例3制备的神经元前体细胞消化为单细胞,单细胞的数量为5×107-108,单细胞标记绿色荧光蛋白后,原位移植到中风鼠的脑内。移植后每隔7天进行小鼠神经功能评分,一个月后用水迷宫方法检测小鼠的学习记忆能力,测试结果如图4。
如图4,正常小鼠(MCAO-Sham),中风未治疗组(MCAO-Ctrl-PBS),中风治疗组(MCAO-hADSC)间的比较可得出,中风治疗组(MCAO-hADSC)在水迷宫测试过程中的平台潜伏期较中风未治疗组(MCAO-Ctrl-PBS)明显缩短,而接近正常小鼠 (MCAO-Sham),其路径效率较中风未治疗组(MCAO-Ctrl-PBS)明显提高而接近正常小鼠(MCAO-Sham)。表明由脂肪干细胞分化而来的神经元前体细胞置入中风小鼠的损伤区后,与未治疗组相比,能够显著提高中风小鼠的学习记忆能力。
采用全细胞膜片钳检测神经元前体细胞移植入中风治疗组(MCAO-hADSC)后,神经元前体细胞在活脑组织中的电生理活性,结果如图5。
从图5中可以看出,全细胞膜片钳检测到神经元前体细胞在活脑组织中的动作电位和突触电位,免疫组化能检测到移植细胞在脑内长期存活,且表达成熟神经元标记NeuN,MAP2。
上述的具体实施方式只是示例性的,是为了更好地使本领域技术人员能够理解本专利,不能理解为是对本专利包括范围的限制;只要是根据本专利所揭示精神的所作的任何等同变更或修饰,均落入本专利包括的范围。
Claims (7)
1.一种诱导培养基,其特征在于,包括NEAA、β-巯基乙醇、谷氨酰胺、生长因子和神经营养因子,所述NEAA与β-巯基乙醇的体积比为4:1-13:1,所述NEAA与谷氨酰胺的体积比为1:2-2:1,所述生长因子由10-20ng/mL的bFGF和10-20ng/mL的EGF组成,所述神经营养因子由10-20ng/mL的BDNF、10-20ng/mL的NGF和10-20ng/mL的NT3组成。
2.一种采用权利要求1所述的诱导培养基将人源脂肪干细胞分化成神经元前体细胞的方法,其特征在于,包括以下步骤:
S1,向超低吸附培养皿中加入诱导培养基后,注入脂肪干细胞,对脂肪干细胞进行诱导培养;
S2,收集成球生长的悬浮脂肪干细胞,洗掉血清后进行消化处理,处理后的细胞悬浮于诱导培养基中继续培养,获得神经元前体细胞。
3.一种人源脂肪干细胞来源的神经元前体细胞群,其特征在于,所述细胞群采用权利要求2所述的方法制备。
4.如权利要求3所述的人源脂肪干细胞来源的神经元前体细胞群的用途,其特征在于,在用于制备治疗神经系统疾病的药物上的应用。
5.如权利要求4所述的用途,其特征在于,所述神经系统疾病包括阿尔茨海默,帕金森,中风,急性脑损伤和自闭症。
6.一种药物组合物,其特征在于,包含权利要求5所述的人源脂肪干细胞来源的神经元前体细胞群和药学上可接受的载体,所述人源脂肪干细胞来源的神经元前体细胞群的存在量足以在给予患者所述人源脂肪干细胞来源的神经元前体细胞群后促进神经系统的功能恢复。
7.如权利要求6所述的药物组合物,其特征在于,所述人源脂肪干细胞来源的神经元前体细胞群中神经元前体细胞的数量为5×107-108个。
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