CN109022355B - 一种提高间充质干细胞向软骨细胞分化的培养方法 - Google Patents
一种提高间充质干细胞向软骨细胞分化的培养方法 Download PDFInfo
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Abstract
本发明属于干细胞技术领域,具体涉及一种提高间充质干细胞向软骨细胞分化的培养方法(1)提取软骨细胞,将所述软骨细胞接种在细胞培养液中进行培养;(2)提取间充质干细胞,将所述间充质干细胞接种于插入式细胞培养皿中;(3)再将步骤(2)所述的插入式细胞培养皿与步骤(1)培养的软骨细胞共同放在细胞诱导分化培养基中一起进行培养;所述细胞诱导分化培养基中含有人骨成型蛋白2和生长激素HGH。本发明在诱导培养基中共同时进行软骨细胞和间充质干细胞的培养,两种细胞在培养基诱导下模拟体内环境,从而达到发生协同作用,提高了软骨细胞增殖率,并且同时提高了诱导分化后细胞的胶原的表达率与表达量。
Description
技术领域
本发明属于干细胞技术领域,具体涉及一种提高间充质干细胞向软骨细胞分化的培养方法。
背景技术
关节软骨主要是由软骨细胞和细胞外基质组成的无血管组织,主要依靠关节的运动和挤压吸收营养物质,所以软骨损伤后自我修复能力比较弱,其损伤后的修复移植一直以来是医学界的难题之一。修复软骨组织的传统方法是采用自体软骨细胞,此法必须在关节镜下切取非负重区的关节软骨,对其中的软骨细胞进行扩增,这种手术唯一的优点为无抗原性,但造成机体损伤很大。另外软骨组织中绝大部分为基质,软骨细胞数量少且难分离,体外传代次数有限,一般最多培养3~4代,所以难以获得大量细胞。
为了对体外软骨细胞进行培养和扩增,出现了多种培养软骨细胞的软骨细胞培养基。比如加入自体血清等,但是软骨细胞去分化趋势严重,诱导后效率较低。
发明内容
本发明的目的在于克服现有技术的上述不足,提供一种提高间充质干细胞向软骨细胞分化的培养方法。
为了实现上述发明目的,本发明实施例的技术方案如下:
一种提高间充质干细胞向软骨细胞分化的培养方法,包括以下步骤:
(1)提取软骨细胞,将所述软骨细胞接种在细胞培养液中进行培养;
(2)提取间充质干细胞,将所述间充质干细胞接种于插入式细胞培养皿中;
(3)再将步骤(2)所述的插入式细胞培养皿与步骤(1)培养的软骨细胞共同放在细胞诱导分化培养基中一起进行培养;所述细胞诱导分化培养基中含有2×10-7~5×10- 7mol/L人骨成型蛋白2和4×10-7~6×10-7mol/L生长激素HGH。
其中,所述细胞诱导分化培养基以高糖型DMEM培养液为基础培养基,还包含以下成分:
浓度为30-180μg/ml的维生素C;
浓度为25-80ng/ml的碱性成纤维生长因子bFGF;
浓度为20-30ng/ml的转化生长因子TGF-α;
浓度为10-35ng/ml的转化生长因子TGF-β;
浓度为5-25μg/ml的胰岛素;
浓度为2-25ng/ml的胰岛素生长因子IGF-1;
浓度为1-10ng/ml的骨形态形成蛋白质类BMP;
浓度为5~25v/v%胎牛血清或者自体血清;
浓度为50~100nmol/L的地塞米松;
浓度为1~2w%的丙酮酸钠;
浓度为5-15ng/ml的白细胞介素IL-1。
进一步的,步骤(1)中,提取的所述的软骨细胞,为原代培养或传代培养的软骨细胞中的一种或两种混合。所述软骨细胞的接种量为0.3×104~0.8×104个/cm2。软骨细胞在在细胞培养液中1~2天,所述的细胞培养液为软骨细胞培养液。
步骤(2)中,提取的所述的间充质干细胞,为原代培养或传代培养的间充质干细胞中的一种或两种混合。所述间充质干细胞的接种量为3×104~5×104个/cm2。
步骤(3)中,所述的培养在培养箱中进行,培养条件为温度37℃、CO25%,PH值7.2~7.4,无菌恒温培养。
以上为本发明的方法,但是经过发明人多次试验和总结,为了细胞诱导分化培养的效果更好,还可以包括浓度为1-3mg/L丹参酮、浓度为0.5-1.0mg/L红花粉、浓度为1.0-2.5mg/L牛膝总皂苷、浓度为2-3v/v%L-谷氨酰胺。
其中,在此基础上,丹参酮是从中药丹参(唇形科植物丹参Salvia miltiorrhizaBunge根)中提取的具有抑菌作用的脂溶性菲醌化合物,单体具有抗菌作用,尚有抗炎、降温作用。总丹参酮有抗菌、消炎、活血化瘀、促进伤口愈合等多方面作用,对于细胞培养过程中的抗菌作用非常明显。
本发明提供的一种提高间充质干细胞向软骨细胞分化的培养方法,在诱导培养基中共同时进行软骨细胞和间充质干细胞的培养,两种细胞在培养基诱导下模拟体内环境,从而达到发生协同作用,提高了软骨细胞增殖率,并且同时提高了诱导分化后细胞的胶原的表达率与表达量。
具体实施方式
结合实施例说明本发明的具体技术方案。
实施例1
一种提高间充质干细胞向软骨细胞分化的培养方法,包括以下步骤:
(1)提取软骨细胞,将所述软骨细胞接种在细胞培养液中进行培养;提取的所述的软骨细胞,为原代培养的软骨细胞。所述软骨细胞的接种量为0.6×104~0.8×104个/cm2。软骨细胞在在细胞培养液中1天,所述的细胞培养液为软骨细胞培养液。
(2)提取间充质干细胞,将所述间充质干细胞接种于插入式细胞培养皿中;提取的所述的间充质干细胞,为原代培养的间充质干细胞。所述间充质干细胞的接种量为3.5×104~4×104个/cm2。
(3)再将步骤(2)所述的插入式细胞培养皿与步骤(1)培养的软骨细胞共同放在细胞诱导分化培养基中一起进行培养;所述的培养在培养箱中进行,培养条件为温度37℃、CO25%,PH值7.2~7.4,无菌恒温培养。
其中,所述细胞诱导分化培养基以高糖型DMEM培养液为基础培养基,还包含以下成分:
浓度为3.5×10-7mol/L人骨成型蛋白2;
浓度为4.8×10-7mol/L生长激素HGH;
浓度为90μg/ml的维生素C;
浓度为60ng/ml的碱性成纤维生长因子bFGF;
浓度为25ng/ml的转化生长因子TGF-α;
浓度为24ng/ml的转化生长因子TGF-β;
浓度为15μg/ml的胰岛素;
浓度为15ng/ml的胰岛素生长因子IGF-1;
浓度为9ng/ml的骨形态形成蛋白质类BMP;
浓度为25v/v%胎牛血清或者自体血清;
浓度为50nmol/L的地塞米松;
浓度为2w%的丙酮酸钠;
浓度为5.5ng/ml的白细胞介素IL-1。
诱导分化为软骨细胞中的阳性表达II型胶原细胞进行流式检测,诱导第5天时,II型胶原阳性表达率为54.29%,诱导第8天时,II型胶原阳性表达率为93.24%;免疫荧光强度测定:在诱导第5天时,阳性表达II型胶原细胞的荧光强度与DAPI的荧光强度的比值提高了15.57%,在诱导第8天时,提高了21.76%;增殖率的测定,所得细胞的数量分别为1.246×105;分泌的GAG含量测定:在诱导第5天时,分泌的GAG含量分别为326.82μg;在诱导第8天时,分泌的GAG含量分别为418.94μg。
实施例2
一种提高间充质干细胞向软骨细胞分化的培养方法,包括以下步骤:
(1)提取软骨细胞,将所述软骨细胞接种在细胞培养液中进行培养;提取的所述的软骨细胞,为传代培养的软骨细胞。所述软骨细胞的接种量为0.3×104~0.8×104个/cm2。软骨细胞在在细胞培养液中2天,所述的细胞培养液为软骨细胞培养液。
(2)提取间充质干细胞,将所述间充质干细胞接种于插入式细胞培养皿中;提取的所述的间充质干细胞,为原代培养间充质干细胞中。所述间充质干细胞的接种量为3×104~4×104个/cm2。
(3)再将步骤(2)所述的插入式细胞培养皿与步骤(1)培养的软骨细胞共同放在细胞诱导分化培养基中一起进行培养;所述的培养在培养箱中进行,培养条件为温度37℃、CO25%,PH值7.2~7.4,无菌恒温培养。
其中,所述细胞诱导分化培养基以高糖型DMEM培养液为基础培养基,还包含以下成分:
浓度为5×10-7mol/L人骨成型蛋白2;
浓度为4.5×10-7mol/L生长激素HGH;
浓度为150μg/ml的维生素C;
浓度为40ng/ml的碱性成纤维生长因子bFGF;
浓度为20ng/ml的转化生长因子TGF-α;
浓度为15ng/ml的转化生长因子TGF-β;
浓度为15μg/ml的胰岛素;
浓度为21ng/ml的胰岛素生长因子IGF-1;
浓度为6ng/ml的骨形态形成蛋白质类BMP;
浓度为20v/v%胎牛血清或者自体血清;
浓度为60nmol/L的地塞米松;
浓度为1w%的丙酮酸钠;
浓度为7ng/ml的白细胞介素IL-1。
还包括浓度为3mg/L丹参酮、浓度为0.8mg/L红花粉、浓度为1.5mg/L牛膝总皂苷、浓度为3v/v%L-谷氨酰胺。
诱导分化为软骨细胞中的阳性表达II型胶原细胞进行流式检测,诱导第5天时,II型胶原阳性表达率为46.23%,诱导第8天时,II型胶原阳性表达率为89.24%;免疫荧光强度测定:在诱导第5天时,阳性表达II型胶原细胞的荧光强度与DAPI的荧光强度的比值提高了15.38%,在诱导第8天时,提高了18.65%;增殖率的测定,所得细胞的数量分别为1.286×105;分泌的GAG含量测定:在诱导第5天时,分泌的GAG含量分别为326.81μg;在诱导第8天时,分泌的GAG含量分别为435.27μg。
实施例3
一种提高间充质干细胞向软骨细胞分化的培养方法,包括以下步骤:
(1)提取软骨细胞,将所述软骨细胞接种在细胞培养液中进行培养;提取的所述的软骨细胞,为原代培养或传代培养的软骨细胞两种混合。所述软骨细胞的接种量为0.3×104~0.8×104个/cm2。软骨细胞在在细胞培养液中1天,所述的细胞培养液为软骨细胞培养液。
(2)提取间充质干细胞,将所述间充质干细胞接种于插入式细胞培养皿中;提取的所述的间充质干细胞,为原代培养的间充质干细胞中的一种或两种混合。所述间充质干细胞的接种量为3×104~5×104个/cm2。
(3)再将步骤(2)所述的插入式细胞培养皿与步骤(1)培养的软骨细胞共同放在细胞诱导分化培养基中一起进行培养;所述的培养在培养箱中进行,培养条件为温度37℃、CO25%,PH值7.2~7.4,无菌恒温培养。
其中,所述细胞诱导分化培养基以高糖型DMEM培养液为基础培养基,还包含以下成分:
浓度为2×10-7mol/L人骨成型蛋白2;
浓度为4.8×10-7mol/L生长激素HGH;
浓度为80μg/ml的维生素C;
浓度为70ng/ml的碱性成纤维生长因子bFGF;
浓度为20ng/ml的转化生长因子TGF-α;
浓度为15ng/ml的转化生长因子TGF-β;
浓度为5.5μg/ml的胰岛素;
浓度为25ng/ml的胰岛素生长因子IGF-1;
浓度为10ng/ml的骨形态形成蛋白质类BMP;
浓度为5v/v%胎牛血清或者自体血清;
浓度为70nmol/L的地塞米松;
浓度为1w%的丙酮酸钠;
浓度为15ng/ml的白细胞介素IL-1。
诱导分化为软骨细胞中的阳性表达II型胶原细胞进行流式检测,诱导第5天时,II型胶原阳性表达率为56.13%,诱导第8天时,II型胶原阳性表达率为87.91%;免疫荧光强度测定:在诱导第5天时,阳性表达II型胶原细胞的荧光强度与DAPI的荧光强度的比值提高了15.38%,在诱导第8天时,提高了18.94%;增殖率的测定,所得细胞的数量分别为1.652×105;分泌的GAG含量测定:在诱导第5天时,分泌的GAG含量分别为324.86μg;在诱导第8天时,分泌的GAG含量分别为486.25μg。
实施例4
一种提高间充质干细胞向软骨细胞分化的培养方法,包括以下步骤:
(1)提取软骨细胞,将所述软骨细胞接种在细胞培养液中进行培养;提取的所述的软骨细胞,为传代培养的软骨细胞中。所述软骨细胞的接种量为0.3×104~0.8×104个/cm2。软骨细胞在在细胞培养液中2天,所述的细胞培养液为软骨细胞培养液。
(2)提取间充质干细胞,将所述间充质干细胞接种于插入式细胞培养皿中;提取的所述的间充质干细胞,为原代培养和传代培养的间充质干细胞中两种混合。所述间充质干细胞的接种量为3×104~5×104个/cm2。
(3)再将步骤(2)所述的插入式细胞培养皿与步骤(1)培养的软骨细胞共同放在细胞诱导分化培养基中一起进行培养;所述的培养在培养箱中进行,培养条件为温度37℃、CO25%,PH值7.2~7.4,无菌恒温培养。
其中,所述细胞诱导分化培养基以高糖型DMEM培养液为基础培养基,还包含以下成分:
浓度为3.5×10-7mol/L人骨成型蛋白2;
浓度为5.6×10-7mol/L生长激素HGH;
浓度为120μg/ml的维生素C;
浓度为60ng/ml的碱性成纤维生长因子bFGF;
浓度为28ng/ml的转化生长因子TGF-α;
浓度为25ng/ml的转化生长因子TGF-β;
浓度为25μg/ml的胰岛素;
浓度为2ng/ml的胰岛素生长因子IGF-1;
浓度为3ng/ml的骨形态形成蛋白质类BMP;
浓度为17v/v%胎牛血清或者自体血清;
浓度为70nmol/L的地塞米松;
浓度为1w%的丙酮酸钠;
浓度为12ng/ml的白细胞介素IL-1。
还包括浓度为3mg/L丹参酮、浓度为0.50mg/L红花粉、浓度为2.5mg/L牛膝总皂苷、浓度为3v/v%L-谷氨酰胺。
诱导分化为软骨细胞中的阳性表达II型胶原细胞进行流式检测,诱导第5天时,II型胶原阳性表达率为61.43%,诱导第8天时,II型胶原阳性表达率为92.48%;免疫荧光强度测定:在诱导第5天时,阳性表达II型胶原细胞的荧光强度与DAPI的荧光强度的比值提高了27.24%,在诱导第8天时,提高了16.49%;增殖率的测定:所得细胞的数量分别为1.5832×105;分泌的GAG含量测定:在诱导第5天时,分泌的GAG含量分别为341.56μg;在诱导第8天时,分泌的GAG含量分别为453.28μg。
Claims (7)
1.一种提高间充质干细胞向软骨细胞分化的培养方法,其特征在于,包括以下步骤:
(1)提取软骨细胞,将所述软骨细胞接种在细胞培养液中进行培养;
(2)提取间充质干细胞,将所述间充质干细胞接种于插入式细胞培养皿中;
(3)再将步骤(2)所述的插入式细胞培养皿与步骤(1)培养的软骨细胞共同放在细胞诱导分化培养基中一起进行培养;
其中,所述细胞诱导分化培养基以高糖型DMEM培养液为基础培养基,包含以下成分:
浓度为2×10-7mol/L人骨成型蛋白2;
浓度为4.8×10-7mol/L生长激素HGH;
浓度为80μg/ml的维生素C;
浓度为70ng/ml的碱性成纤维生长因子bFGF;
浓度为20ng/ml的转化生长因子TGF-α;
浓度为15ng/ml的转化生长因子TGF-β;
浓度为5.5μg/ml的胰岛素;
浓度为25ng/ml的胰岛素生长因子IGF-1;
浓度为10ng/ml的骨形态形成蛋白质类BMP;
浓度为5v/v%胎牛血清或者自体血清;
浓度为70nmol/L的地塞米松;
浓度为1w%的丙酮酸钠;
浓度为15ng/ml的白细胞介素IL-1。
2.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的培养方法,其特征在于,步骤(1)中,所述软骨细胞的接种量为0.3×104~0.8×104个/cm2。
3.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的培养方法,其特征在于,步骤(1)软骨细胞在细胞培养液中1~2天,所述的细胞培养液为软骨细胞培养液。
4.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的培养方法,其特征在于,步骤(1)提取的所述的软骨细胞,为原代培养或传代培养的软骨细胞中的一种或两种混合。
5.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的培养方法,其特征在于,步骤(2)中,所述间充质干细胞的接种量为3×104~5×104个/cm2。
6.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的培养方法,其特征在于:步骤(2)提取的所述的间充质干细胞,为原代培养或传代培养的间充质干细胞中的一种或两种混合。
7.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的培养方法,其特征在于,步骤(3)中,所述的培养在培养箱中进行,培养条件为温度37℃、CO25%,pH 值7.2-7.4,无菌恒温培养。
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