CN105878283A - 人源干细胞生物活性物质及其制备方法 - Google Patents
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Abstract
本发明公开了一种人源干细胞生物活性物质及其制备方法,由胚胎干细胞生物活性物质、全反式维甲酸(all‑trans retinoic acid,ATRA)、兔软骨细胞、胰岛素样生长因子、基质金属蛋白酶、2‑8μmol/L的甲状腺素和杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合而成。本发明还公开了人源干细胞生物活性物质的制备方法。本发明人源干细胞生物活性物质具有很高的活性能力,可以制成不同的保健品或者其他产品,该人源干细胞生物活性物质能活化人体自身干细胞来进行生理性修复或替代机体损伤、病变和衰老的组织细胞的作用,同时增强机体免疫力。
Description
技术领域
本发明涉及人源干细胞生物活性物质及其制备方法,属于生物技术领域。
背景技术
干细胞(Stem Cell)是一类具有自我更新、高度增殖和多向分化潜能的细胞群体,是形成人体各种组织、器官的“祖宗细胞”、“万能细胞”。根据其发育阶段可分为胚胎干细胞和成体干细胞,根据分化潜能大小又可以分为全能干细胞、多能干细胞和单能干细胞。
间充质干细胞 (Mesenchymal stem cells,MSCs) 是一类具有多向分化潜能的组织干细胞,可以从骨髓、外周血、脂肪及皮肤等多种组织中获得,作为一类多能干细胞,它可以向骨、软骨、肌肉、神经及胰岛样细胞等方向增殖分化。目前 MSCs 主要来源于成人骨髓,但成人骨髓 MSCs 细胞数量及增殖分化潜能随供体年龄的增加而下降,病毒感染率较高,且采集具有创伤,使其来源受限。最近,已开发出从脐带提取 MSCs,它也是一类具有多向分化潜能的干细胞,越来越多的研究表明其极具开发潜质,与骨髓 MSCs 相比,具有以下明显的优势 : (1) 来源充足,取材方便,成本低廉 ;(2) 人脐带间充质干细胞含量丰富,较为原始,分化能力强,可在体外进行分离、培养,扩增迅速,且生物学性状稳定,多次传代扩增仍能保持旺盛功能,可以提供充足的细胞来源 ;(3) 免疫原性极低,免疫排斥的概率极低,且脐带间充质干细胞还具有一定的免疫抑制功能 ;(4) 脐带间充质干细胞可以支持造血和促进移植。造血干细胞 (Hematopoietic stem cells,HSCs) 是一类存在于造血组织中的多能干细胞,可以定向分化、增殖为不同的血细胞系,并进一步生成血细胞。造血干细胞在治疗白血病、恶性肿瘤、重症非恶性血液系统疾病以及某些遗传性疾病中有广泛的应用。造血干细胞根据来源又可分为骨髓造血干细胞、外周血造血干细胞和脐带血造血干细胞。与骨髓和外周血造血干细胞相比,脐血造血干细胞更为原始,自我增殖能力最强,且脐带血具有采集方便、细胞免疫不成熟、移植过程中不需要严格配型等优点,因而脐带血造血干细胞具有极大的临床应用价值。间充质干细胞与造血干细胞共同移植可促进造血干细胞的植入,对于提高造血干细胞的移植成功率有重要意义。
干细胞可以产生和分泌大量的生物活性因子,这些干细胞生物活性因子可有效调控机体细胞信号传导、活化人体干细胞,进而生理性修复或替代机体损伤、病变及衰老的细胞。研究表明人体骨髓、血管、脂肪组织、骨骼肌、心脏和大脑,也包括在肺、肝、胰腺、 消化道、皮肤、视网膜、乳房、卵巢、前列腺和睾丸的上皮内均可分离到相应的干细胞。理论上,干细胞可以修复身体任何部位的组织缺损,可以利用干细胞去治疗遗传疾病、恶性疾病 和退变性疾病等。2007 年 10 月 10 日,国际干细胞权威杂志《Cell》系列刊物《Cell-StemCell》的研究论文揭示干细胞功能下降可导致人类衰老,并证明干细胞衰老过程是受到外在和内在因素共同调控的。
鉴于上述材料,我们研究开发从人脐带组织和脐带血中提取干细胞,经体外培养、扩增, 然后提取其生物活性物质,进行制备,利用人源干细胞活性物质组合物发挥综合效应,可以制成药物或者保健品,用来活化人体自身干细胞,进行生理性修复或替代机体损伤、病变和衰老的组织细胞。
发明内容
本发明目的是提供一种人源干细胞生物活性物质及其制备方法,该人源干细胞活性物质起可以制成药物或者保健品,用来活化人体自身干细胞,进行生理性修复或替代机体损伤、病变和衰老的组织细胞。
本发明的方案如下:人源干细胞生物活性物质,由胚胎干细胞生物活性物质、全反式维甲酸(all-trans retinoic acid,ATRA)、兔软骨细胞、胰岛素样生长因子、基质金属蛋白酶、2-8μmol/L的甲状腺素和杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合而成。
进一步的,所述杜氏培养液(Dulbecco’s modified eagle medium,DMEM)是含90%低糖DMEM、5%胎牛血清、3%抗生素和8ng/mL碱性成纤维细胞生长因子(basic fibroblastgrowth factor ,bFGF)。
进一步的,所述胚胎干细胞生物活性物质、兔软骨细胞的质量比为(2-3):1。
上述人源干细胞生物活性物质的制备方法,包括以下步骤:
a 、胚胎干细胞生物活性物质的制备:
对足月儿产后脐带进行脐带被膜和动静脉血管剥离,切成1mm×1mm×1mm的碎块;用PBS漂洗至无色后,经37℃的水浴消化1.5-2h,分层离心、振荡,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),并放置于培养箱中;
b 、取新西兰兔的四肢关节软骨,切成1mm×1mm×1mm的碎块置于无菌试管,经PBS冲洗三次后离心,吸去PBS,加入3g/L胰酶5mL,置于CO2 孵箱0.5h,再进行分层离心,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),然后置于CO2 孵箱中培养;
c 、对步骤a和b中进行隔天半量换液,当细胞达到80%汇合时,使用0.33%磷酸酯酶对细胞进行消化,以1.5-2×105cells/ml 接种于培养皿中传代培养;
d 、重复4-5次后,分离、纯化获得高纯度间充质干细胞,并将浓度调整为2×106个/ ml密封,经超低温液氮反复冻融6-7 次至细胞破碎,再通过低温分离、纯化,便可获得高纯度的胚胎干细胞物活性物质;
e、将步骤d处理后的高纯度的胚胎干细胞物活性物质加入全反式维甲酸(all-transretinoic acid,ATRA)、胰岛素样生长因子、基质金属蛋白酶、2-8μmol/L的甲状腺素放入杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合、培养即得人源干细胞生物活性物质。
进一步的,所述的培养箱、CO2 孵箱和培养皿的培养环境为: pH值:7.2,相对饱和湿度92%,温度38°C;所述CO2 孵箱是体积分数为5%CO2 孵箱。
本发明人源干细胞生物活性物质具有很高的活性能力,可以制成不同的保健品或者其他产品,该人源干细胞生物活性物质能活化人体自身干细胞来进行生理性修复或替代机体损伤、病变和衰老的组织细胞的作用,同时增强机体免疫力。
具体实施方式
实施例1
人源干细胞生物活性物质,由胚胎干细胞生物活性物质、全反式维甲酸(all-transretinoic acid,ATRA)、兔软骨细胞、胰岛素样生长因子、基质金属蛋白酶、2μmol/L的甲状腺素和杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合而成。
所述杜氏培养液(Dulbecco’s modified eagle medium,DMEM)是含90%低糖DMEM、5%胎牛血清、3%抗生素和8ng/mL碱性成纤维细胞生长因子(basic fibroblast growthfactor ,bFGF)。
所述胚胎干细胞生物活性物质、兔软骨细胞的质量比为2:1。
上述人源干细胞生物活性物质的制备方法,包括以下步骤:
a 、胚胎干细胞生物活性物质的制备:
对足月儿产后脐带进行脐带被膜和动静脉血管剥离,切成1mm×1mm×1mm的碎块;用PBS漂洗至无色后,经37℃的水浴消化1.5h,分层离心、振荡,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),并放置于培养箱中;
b 、取新西兰兔的四肢关节软骨,切成1mm×1mm×1mm的碎块置于无菌试管,经PBS冲洗三次后离心,吸去PBS,加入3g/L胰酶5mL,置于CO2 孵箱0.5h,再进行分层离心,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),然后置于CO2 孵箱中培养;
c 、对步骤a和b中进行隔天半量换液,当细胞达到80%汇合时,使用0.33%磷酸酯酶对细胞进行消化,以1.5×105cells/ml 接种于培养皿中传代培养;
d 、重复4次后,分离、纯化获得高纯度间充质干细胞,并将浓度调整为2×106个/ ml密封,经超低温液氮反复冻融6 次至细胞破碎,再通过低温分离、纯化,便可获得高纯度的胚胎干细胞物活性物质;
e、将步骤d处理后的高纯度的胚胎干细胞物活性物质加入全反式维甲酸(all-transretinoic acid,ATRA)、胰岛素样生长因子、基质金属蛋白酶、2μmol/L的甲状腺素放入杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合、培养即得人源干细胞生物活性物质。
所述的培养箱、CO2 孵箱和培养皿的培养环境为: pH值:7.2,相对饱和湿度92%,温度38°C;所述CO2 孵箱是体积分数为5%CO2 孵箱。
实施例2
人源干细胞生物活性物质,由胚胎干细胞生物活性物质、全反式维甲酸(all-transretinoic acid,ATRA)、兔软骨细胞、胰岛素样生长因子、基质金属蛋白酶、2-8μmol/L的甲状腺素和杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合而成。
所述杜氏培养液(Dulbecco’s modified eagle medium,DMEM)是含90%低糖DMEM、5%胎牛血清、3%抗生素和8ng/mL碱性成纤维细胞生长因子(basic fibroblast growthfactor ,bFGF)。
所述胚胎干细胞生物活性物质、兔软骨细胞的质量比为3:1。
上述人源干细胞生物活性物质的制备方法,包括以下步骤:
a 、胚胎干细胞生物活性物质的制备:
对足月儿产后脐带进行脐带被膜和动静脉血管剥离,切成1mm×1mm×1mm的碎块;用PBS漂洗至无色后,经37℃的水浴消化2h,分层离心、振荡,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),并放置于培养箱中;
b 、取新西兰兔的四肢关节软骨,切成1mm×1mm×1mm的碎块置于无菌试管,经PBS冲洗三次后离心,吸去PBS,加入3g/L胰酶5mL,置于CO2 孵箱0.5h,再进行分层离心,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),然后置于CO2 孵箱中培养;
c 、对步骤a和b中进行隔天半量换液,当细胞达到80%汇合时,使用0.33%磷酸酯酶对细胞进行消化,以2×105cells/ml 接种于培养皿中传代培养;
d 、重复5次后,分离、纯化获得高纯度间充质干细胞,并将浓度调整为2×106个/ ml密封,经超低温液氮反复冻融7 次至细胞破碎,再通过低温分离、纯化,便可获得高纯度的胚胎干细胞物活性物质;
e、将步骤d处理后的高纯度的胚胎干细胞物活性物质加入全反式维甲酸(all-transretinoic acid,ATRA)、胰岛素样生长因子、基质金属蛋白酶、8μmol/L的甲状腺素放入杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合、培养即得人源干细胞生物活性物质。
所述的培养箱、CO2 孵箱和培养皿的培养环境为: pH值:7.2,相对饱和湿度92%,温度38°C;所述CO2 孵箱是体积分数为5%CO2 孵箱。
实施例3
人源干细胞生物活性物质,由胚胎干细胞生物活性物质、全反式维甲酸(all-transretinoic acid,ATRA)、兔软骨细胞、胰岛素样生长因子、基质金属蛋白酶、2-8μmol/L的甲状腺素和杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合而成。
所述杜氏培养液(Dulbecco’s modified eagle medium,DMEM)是含90%低糖DMEM、5%胎牛血清、3%抗生素和8ng/mL碱性成纤维细胞生长因子(basic fibroblast growthfactor ,bFGF)。
所述胚胎干细胞生物活性物质、兔软骨细胞的质量比为8:3。
上述人源干细胞生物活性物质的制备方法,包括以下步骤:
a 、胚胎干细胞生物活性物质的制备:
对足月儿产后脐带进行脐带被膜和动静脉血管剥离,切成1mm×1mm×1mm的碎块;用PBS漂洗至无色后,经37℃的水浴消化2h,分层离心、振荡,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),并放置于培养箱中;
b 、取新西兰兔的四肢关节软骨,切成1mm×1mm×1mm的碎块置于无菌试管,经PBS冲洗三次后离心,吸去PBS,加入3g/L胰酶5mL,置于CO2 孵箱0.5h,再进行分层离心,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),然后置于CO2 孵箱中培养;
c 、对步骤a和b中进行隔天半量换液,当细胞达到80%汇合时,使用0.33%磷酸酯酶对细胞进行消化,以1.8×105cells/ml 接种于培养皿中传代培养;
d 、重复5次后,分离、纯化获得高纯度间充质干细胞,并将浓度调整为2×106个/ ml密封,经超低温液氮反复冻融6次至细胞破碎,再通过低温分离、纯化,便可获得高纯度的胚胎干细胞物活性物质;
e、将步骤d处理后的高纯度的胚胎干细胞物活性物质加入全反式维甲酸(all-transretinoic acid,ATRA)、胰岛素样生长因子、基质金属蛋白酶、6μmol/L的甲状腺素放入杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合、培养即得人源干细胞生物活性物质。
所述的培养箱、CO2 孵箱和培养皿的培养环境为: pH值:7.2,相对饱和湿度92%,温度38°C;所述CO2 孵箱是体积分数为5%CO2 孵箱。
胚胎干细胞生物活性物质、兔软骨细胞的质量比的设置可以提高人源干细胞生物活性物质的活性,而且可以促进细胞的融合,一般4-5天即可达到92%以上的融合度。全反式维甲酸(all-trans retinoic acid,ATRA)、胰岛素样生长因子、基质金属蛋白酶的加入可以促进细胞的融合诱导,促进细胞分化;而甲状腺素的加入可以促进聚集蛋白多糖、II型胶原mRNA和蛋白质的表达水平,比单纯共培养具有明显的促进作用,而且本发明人源干细胞生物活性物质的活性高,分化能力强。
尽管上文对本发明的具体实施方式给予了详细描述和说明,但是应该指明的是,我们可以依据本发明的构想对上述实施方式进行各种等效改变和修改,其所产生的功能作用仍未超出说明书所涵盖的精神时,均应在本发明的保护范围之内。
Claims (5)
1.人源干细胞生物活性物质,其特征在于,由胚胎干细胞生物活性物质、全反式维甲酸(all-trans retinoic acid,ATRA)、兔软骨细胞、胰岛素样生长因子、基质金属蛋白酶、2-8μmol/L的甲状腺素和杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合而成。
2.根据权利要求1所述的人源干细胞生物活性物质,其特征在于,所述杜氏培养液(Dulbecco’s modified eagle medium,DMEM)是含90%低糖DMEM、5%胎牛血清、3%抗生素和8ng/mL碱性成纤维细胞生长因子(basic fibroblast growth factor ,bFGF)。
3.根据权利要求1所述的人源干细胞生物活性物质,其特征在于,所述胚胎干细胞生物活性物质、兔软骨细胞的质量比为(2-3):1。
4.如权利要求1至3任一所述的人源干细胞生物活性物质的制备方法,其特征在于,包括以下步骤:
a 、胚胎干细胞生物活性物质的制备:
对足月儿产后脐带进行脐带被膜和动静脉血管剥离,切成1mm×1mm×1mm的碎块;用PBS漂洗至无色后,经37℃的水浴消化1.5-2h,分层离心、振荡,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),并放置于培养箱中;
b 、取新西兰兔的四肢关节软骨,切成1mm×1mm×1mm的碎块置于无菌试管,经PBS冲洗三次后离心,吸去PBS,加入3g/L胰酶5mL,置于CO2 孵箱0.5h,再进行分层离心,去上清后加入杜氏培养液(Dulbecco’s modified eagle medium,DMEM),然后置于CO2 孵箱中培养;
c 、对步骤a和b中进行隔天半量换液,当细胞达到80%汇合时,使用0.33%磷酸酯酶对细胞进行消化,以1.5-2×105cells/ml 接种于培养皿中传代培养;
d 、重复4-5次后,分离、纯化获得高纯度间充质干细胞,并将浓度调整为2×106个/ ml密封,经超低温液氮反复冻融6-7 次至细胞破碎,再通过低温分离、纯化,便可获得高纯度的胚胎干细胞物活性物质;
e、将步骤d处理后的高纯度的胚胎干细胞物活性物质加入全反式维甲酸(all-transretinoic acid,ATRA)、胰岛素样生长因子、基质金属蛋白酶、2-8μmol/L的甲状腺素放入杜氏培养液(Dulbecco’s modified eagle medium,DMEM)混合、培养即得人源干细胞生物活性物质。
5.根据权利要求4所述的人源干细胞生物活性物质的制备方法,其特征在于,所述的培养箱、CO2 孵箱和培养皿的培养环境为: pH值:7.2,相对饱和湿度92%,温度38°C;所述CO2孵箱是体积分数为5%CO2 孵箱。
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