CN105866432B - 用于鉴定牙髓间充质干细胞的试剂盒 - Google Patents
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Abstract
本发明公开了一种用于鉴定牙髓间充质干细胞的试剂盒,包括流式表型检测试剂套装、细胞固定液、普通培养基、成脂分化诱导及检测试剂套装、成骨分化诱导及检测试剂套装、成软骨分化诱导及检测试剂套装。传统成骨、成脂诱导分化通常需要大约24天,时间较长且诱导效率较低,本发明将成骨、成脂诱导分化时间缩短至18天,本发明提高诱导分化效率,缩短诱导分化的时间;同时传统流式检测大多只针对间充质干细胞通用的表面标志,而牙髓间充质干细胞具有不同于其他来源间充质干细胞的阳性表达的表面标志CD146,本发明试剂盒中增加了CD146抗体,从而得出更为准确的鉴定结果。
Description
技术领域
本发明涉及一种试剂盒,尤其是一种用于鉴定牙髓间充质干细胞的试剂盒。
背景技术
目前越来越多的研究将牙髓分离间充质干细胞用于临床,牙髓间充质干细胞的发展前景也越来越好。国际细胞治疗协会(ISCT)规定间充质干细胞的鉴定包括形态学、流式表型和诱导分化能力三个方面。传统的间充质干细胞鉴定方法已经比较成熟,但是诱导分化过程中耗时较长,而且诱导效率较低,影响最终细胞鉴定结果。
发明内容
本发明所要解决的技术问题是,提供一种提高诱导分化效率,缩短诱导分化时间的用于鉴定牙髓间充质干细胞的试剂盒。
为了解决上述技术问题,本发明采用的技术方案是:一种用于鉴定牙髓间充质干细胞的试剂盒,包括流式表型检测试剂套装、细胞固定液、普通培养基、成脂分化诱导及检测试剂套装、成骨分化诱导及检测试剂套装、成软骨分化诱导及检测试剂套装;
所述流式表型检测试剂套装:包括CD73抗体、CD90抗体、CD105抗体、CD34抗体、CD45抗体、CD49d抗体、CD106抗体、IgG1抗体,每只均为200uL;
所述成脂分化诱导及检测试剂套装:包括100mL成脂诱导培养基和10mL成脂分化检测试剂,成脂诱导培养基为含有0.2-2mmoL/L谷氨酰胺、5-30μg/mL IGF-1、10-20mmol/L3-磷酸甘油、1-5mmoL/L氟轻松、0.1-0.5mmoL/L异丁基甲基黄嘌呤、100-200μmoL/L吲哚美辛、50-100μmoL/L乙酰CoA、体积分数2-10%胎牛血清的DMEM培养基;
所述成骨分化诱导及检测试剂套装:包括100mL成骨诱导培养基和10mL成骨分化检测试剂,成骨诱导培养基为含有0.5-2mmoL/L谷氨酰胺、0.25-1mmoL/L氟轻松、2-10μg/mLTGF-β、2-10mmol/Lβ-甘油磷酸钠、15-25mmol/L维生素D3、10-50mmol/L维生素C、体积分数2-10%胎牛血清的DMEM培养基;
所述成软骨分化诱导及检测试剂套装:包括100mL成软骨诱导培养基和10mL成软骨分化检测试剂,成软骨诱导培养基为含有15-30ng/ml TGF-β1、10-30mg/L维生素C、5-10nmol/L氟轻松、10-30mg/ml ITS、20-50mmol/L氨基葡萄糖、10-20mmol/L丙酮酸钠、10-20ug/mg亚油酸、5-10ng/ml人血清白蛋白、5-10mmol/Lβ-甘油磷酸钠、体积分数2-10%胎牛血清的DMEM培养基。
所述细胞固定液为20mL质量百分比浓度4%多聚甲醛的水溶液。
所述普通培养基为20mL含体积分数2-8%胎牛血清的DMEM培养基。
所述成脂分化检测试剂为含有1g/L茜素磺酸钠水溶液。
所述成骨分化检测试剂为含有质量体积分数为0.5%油红O的异丙醇溶液。
所述成软骨分化检测试剂为含有10g/L阿尔新兰、质量体积分数3%醋酸的水溶液。
本发明的有益效果是:提高诱导分化效率,缩短诱导分化的时间,传统成骨、成脂诱导分化通常需要大约24天,时间较长且诱导效率较低,本发明将成骨、成脂诱导分化时间缩短至18天;同时传统流式检测大多只针对间充质干细胞通用的表面标志,而牙髓间充质干细胞具有不同于其他来源间充质干细胞的阳性表达的表面标志CD146,本发明试剂盒中增加了CD146抗体,从而得出更为准确的鉴定结果。
附图说明
图1流式检测结果图。
图2成脂分化染色图。
图3成骨分化染色图。
图4成软骨分化染色图。
具体实施方式
下面结合具体实施方式对本发明作进一步详细说明:
本发明的用于鉴定牙髓间充质干细胞的试剂盒,包括流式表型检测试剂套装、细胞固定液、普通培养基、成脂分化诱导及检测试剂套装、成骨分化诱导及检测试剂套装、成软骨分化诱导及检测试剂套装;
所述流式表型检测试剂套装:包括CD73抗体、CD90抗体、CD105抗体、CD34抗体、CD45抗体、CD146抗体、IgG1抗体,每只均为200uL;
所述成脂分化诱导及检测试剂套装:包括100mL成脂诱导培养基和10mL成脂分化检测试剂,成脂诱导培养基为含有0.2-2mmoL/L谷氨酰胺、5-30μg/mL IGF-1、10-20mmol/L3-磷酸甘油、1-5mmoL/L氟轻松、0.1-0.5mmoL/L异丁基甲基黄嘌呤、100-200μmoL/L吲哚美辛、50-100μmoL/L乙酰CoA、体积分数2-10%胎牛血清的DMEM培养基;
所述成骨分化诱导及检测试剂套装:包括100mL成骨诱导培养基和10mL成骨分化检测试剂,成骨诱导培养基为含有0.5-2mmoL/L谷氨酰胺、0.25-1mmoL/L氟轻松、2-10μg/mLTGF-β、2-10mmol/Lβ-甘油磷酸钠、15-25mmol/L维生素D3、10-50mmol/L维生素C、体积分数2-10%胎牛血清的DMEM培养基;
所述成软骨分化诱导及检测试剂套装:包括100mL成软骨诱导培养基和10mL成软骨分化检测试剂,成软骨诱导培养基为含有为含有以下组分包括:15-30ng/ml TGF-β1、10-30mg/L维生素C、5-10nmol/L氟轻松、10-30mg/ml ITS、20-50mmol/L氨基葡萄糖、10-20mmol/L丙酮酸钠、10-20ug/mg亚油酸、5-10ng/ml人血清白蛋白、5-10mmol/Lβ-甘油磷酸钠、体积分数2-10%胎牛血清的DMEM培养基。
所述细胞固定液为20mL质量百分比浓度4%多聚甲醛的水溶液。
所述普通培养基为20mL含体积分数2-8%胎牛血清的DMEM培养基。
所述成脂分化检测试剂为含有1g/L茜素磺酸钠水溶液。
所述成骨分化检测试剂为含有质量体积分数为0.5%油红O的异丙醇溶液。
所述成软骨分化检测试剂为含有10g/L阿尔新兰、质量体积分数3%醋酸的水溶液。
本发明的试剂盒操作步骤如下:
(1)流式检测步骤:
步骤一:将细胞悬液按照每管1×105数量分装到EP管内,离心后加入1mL PBS重悬后离心;
步骤二:加入相应的流式抗体,室温避光孵育30min;
步骤三:每管加入1mL PBS重悬后离心;
步骤四:每管加入1mL PBS重悬后流式细胞仪检测。
(2)成脂分化诱导及检测步骤:
步骤一:准备6孔培养板,将P4细胞按照2×103个/平方厘米的规格接种于普通培养液中;
步骤二:待细胞融合度长至70-80%,更换成脂诱导培养基培养,每三天换液一次。
步骤三:18天后去除全部培养液,使用PBS清洗两次。
步骤四:4%多聚甲醛室温固定20分钟;PBS清洗3次;
步骤五:按照1ml/孔加入成脂分化检测试剂,室温孵育一小时;
步骤六:清除掉没有完全结合的染料,75%乙醇溶液清洗3次;
步骤七:倒置显微镜观察拍照记录。
(3)成骨分化诱导及检测步骤:
步骤一:准备6孔培养板,将P4细胞按照5×103个/平方厘米的规格接种于普通培养液中;
步骤二:待细胞融合度长至40-50%,更换成骨诱导培养基;每三天换液一次;
步骤三:18天后去除全部培养液,使用PBS清洗两次;
步骤四:使用4%多聚甲醛室温固定15min,完成后使用PBS冲洗两次;
步骤五:按照1ml/孔加入成骨分化检测试剂,室温孵育20min并轻微振荡;
步骤六:清除掉没有完全结合的染料,用PBS漂洗并振荡5min重复4次;
步骤七:倒置显微镜观察拍照记录。
(4)成软骨分化诱导及检测步骤:
步骤一:准备6孔培养板,将P4细胞按照5×103个/平方厘米的规格接种于普通培养液中;
步骤二:待细胞融合度长至50-60%,更换成软骨诱导培养基;每三天换液一次;
步骤三:13天后去除全部培养液,使用PBS清洗两次;
步骤四:用细胞固定液固定30min,再用3%醋酸水溶液洗2次后;(醋酸水溶液用户自己准备)
步骤五:按照1ml/孔加入成软骨分化检测试剂染色10~30min;
步骤六:清除掉没有完全结合的染料,3%醋酸水溶液洗3次;
步骤七:倒置显微镜观察拍照记录。
实施例1
所述成脂分化诱导及检测试剂套装:包括100mL成脂诱导培养基和10mL成脂分化检测试剂,成脂诱导培养基为含有2mmoL/L谷氨酰胺、30μg/mL IGF-1、20mmol/L 3-磷酸甘油、5mmoL/L氟轻松、0.5mmoL/L异丁基甲基黄嘌呤、200μmoL/L吲哚美辛、100μmoL/L乙酰CoA、体积分数10%胎牛血清的DMEM培养基;
所述成骨分化诱导及检测试剂套装:包括100mL成骨诱导培养基和10mL成骨分化检测试剂,成骨诱导培养基为含有2mmoL/L谷氨酰胺、1mmoL/L氟轻松、10μg/mL TGF-β、10mmol/Lβ-甘油磷酸钠、25mmol/L维生素D3、50mmol/L维生素C、体积分数10%胎牛血清的DMEM培养基;
所述成软骨分化诱导及检测试剂套装:包括100mL成软骨诱导培养基和10mL成软骨分化检测试剂,成软骨诱导培养基为含有为含有以下组分包括:30ng/ml TGF-β1、30mg/L维生素C、10nmol/L氟轻松、30mg/ml ITS、50mmol/L氨基葡萄糖、20mmol/L丙酮酸钠、20ug/mg亚油酸、10ng/ml人血清白蛋白、10mmol/Lβ-甘油磷酸钠、体积分数10%胎牛血清的DMEM培养基。
操作步骤如下:
(1)流式检测:
将细胞悬液按照每管1×105数量分装到EP管内,离心后加入1mL PBS重悬后离心;
加入相应的流式抗体,室温避光孵育30min;
每管加入1mL PBS重悬后离心;
每管加入1mL PBS重悬后流式细胞仪检测。IgG1为同型对照,检测结果CD73、CD90、CD105、CD146为阳性表达,CD34、CD45为阴性表达,见图1。
(2)成脂分化诱导及检测:
准备6孔培养板,将P4细胞按照2×103个/平方厘米的规格接种;
待细胞融合度长至80%,更换成脂诱导培养基培养,每三天换液一次。
18天后去除全部培养液,使用PBS清洗两次。
细胞固定液室温固定20分钟;PBS清洗3次;
按照1ml/孔加入成脂分化检测试剂,室温孵育一小时;
清除掉没有完全结合的染料,75%乙醇溶液清洗3次;
倒置显微镜观察拍照记录。检测结果出现红色液滴,见图2。
(3)成骨分化诱导及检测:
准备6孔培养板,将P4细胞按照5×103个/平方厘米的规格接种于普通培养液中;
待细胞融合度长至50%,更换成骨诱导培养基;每三天换液一次;
18天后去除全部培养液,使用PBS清洗两次;
细胞固定液室温固定15min,完成后使用PBS冲洗两次;
按照1ml/孔加入成骨分化检测试剂,室温孵育20min并轻微振荡;
清除掉没有完全结合的染料,用PBS漂洗并振荡5min重复4次;
倒置显微镜观察拍照记录。检测结果出现红色结节,见图3。
(4)成软骨分化诱导及检测:
准备6孔培养板,将P4细胞按照5×103个/平方厘米的规格接种于普通培养液中;
待细胞融合度长至60%,更换成软骨诱导培养基;每三天换液一次;
13天后去除全部培养液,使用PBS清洗两次;
用细胞固定液固定30min,再用3%醋酸水溶液洗2次后;
按照1ml/孔加入成软骨分化检测试剂染色30min;
清除掉没有完全结合的染料,3%醋酸水溶液洗3次;
倒置显微镜观察拍照记录。检测结果出现蓝色,见图4。
实施例2
所述成脂分化诱导及检测试剂套装:包括100mL成脂诱导培养基和10mL成脂分化检测试剂,成脂诱导培养基为含有0.2mmoL/L谷氨酰胺、5μg/mL IGF-1、10mmol/L 3-磷酸甘油、1mmoL/L氟轻松、0.1mmoL/L异丁基甲基黄嘌呤、100μmoL/L吲哚美辛、50μmoL/L乙酰CoA、体积分数2%胎牛血清的DMEM培养基;
所述成骨分化诱导及检测试剂套装:包括100mL成骨诱导培养基和10mL成骨分化检测试剂,成骨诱导培养基为含有0.5mmoL/L谷氨酰胺、0.25mmoL/L氟轻松、2μg/mL TGF-β、2mmol/Lβ-甘油磷酸钠、15mmol/L维生素D3、10mmol/L维生素C、体积分数2%胎牛血清的DMEM培养基;
所述成软骨分化诱导及检测试剂套装:包括100mL成软骨诱导培养基和10mL成软骨分化检测试剂,成软骨诱导培养基为含有为含有以下组分包括:15ng/ml TGF-β1、10mg/L维生素C、5nmol/L氟轻松、10mg/ml ITS、20mmol/L氨基葡萄糖、10mmol/L丙酮酸钠、10ug/mg亚油酸、5ng/ml人血清白蛋白、5mmol/Lβ-甘油磷酸钠、体积分数2%胎牛血清的DMEM培养基。
操作步骤如下:
(1)流式检测:
将细胞悬液按照每管1×105数量分装到EP管内,离心后加入1mL PBS重悬后离心;
加入相应的流式抗体,室温避光孵育30min;
每管加入1mL PBS重悬后离心;
每管加入1mL PBS重悬后流式细胞仪检测。IgG1为同型对照,检测结果CD73、CD90、CD105、CD146为阳性表达,CD34、CD45为阴性表达。
(2)成脂分化诱导及检测:
准备6孔培养板,将P4细胞按照2×103个/平方厘米的规格接种;
待细胞融合度长至70%,更换成脂诱导培养基培养,每三天换液一次。
18天后去除全部培养液,使用PBS清洗两次。
细胞固定液室温固定20分钟;PBS清洗3次;
按照1ml/孔加入成脂分化检测试剂,室温孵育一小时;
清除掉没有完全结合的染料,75%乙醇溶液清洗3次;
倒置显微镜观察拍照记录。检测结果出现红色液滴。
(3)成骨分化诱导及检测:
准备6孔培养板,将P4细胞按照5×103个/平方厘米的规格接种于普通培养液中;
待细胞融合度长至40%,更换成骨诱导培养基;每三天换液一次;
18天后去除全部培养液,使用PBS清洗两次;
细胞固定液室温固定15min,完成后使用PBS冲洗两次;
按照1ml/孔加入成骨分化检测试剂,室温孵育20min并轻微振荡;
清除掉没有完全结合的染料,用PBS漂洗并振荡5min重复4次;
倒置显微镜观察拍照记录。检测结果出现红色结节。
(4)成软骨分化诱导及检测:
准备6孔培养板,将P4细胞按照5×103个/平方厘米的规格接种于普通培养液中;
待细胞融合度长至50%,更换成软骨诱导培养基;每三天换液一次;
13天后去除全部培养液,使用PBS清洗两次;
用细胞固定液固定30min,再用3%醋酸水溶液洗2次后;
按照1ml/孔加入成软骨分化检测试剂染色10min;
清除掉没有完全结合的染料,3%醋酸水溶液洗3次;
倒置显微镜观察拍照记录。检测结果出现蓝色。
本发明提高诱导分化效率,缩短诱导分化的时间,传统成骨、成脂诱导分化通常需要大约24天,时间较长且诱导效率较低,本发明将成骨、成脂诱导分化时间缩短至18天,同时传统流式检测大多只针对间充质干细胞通用的表面标志,而牙髓间充质干细胞具有不同于其他来源间充质干细胞的阳性表达的表面标志CD146,本发明试剂盒中增加了CD146抗体,从而得出更为准确的鉴定结果。
综上所述,本发明的内容并不局限在上述的实施例中,相同领域内的有识之士可以在本发明的技术指导思想之内可以轻易提出其他的实施例,但这种实施例都包括在本发明的范围之内。
Claims (5)
1.一种用于鉴定牙髓间充质干细胞的试剂盒,其特征在于,包括流式表型检测试剂套装、细胞固定液、普通培养基、成脂分化诱导及检测试剂套装、成骨分化诱导及检测试剂套装、成软骨分化诱导及检测试剂套装;
所述流式表型检测试剂套装:包括CD73抗体、CD90抗体、CD105抗体、CD34抗体、CD45抗体、CD146抗体、IgG1抗体,每只均为200uL;
所述成脂分化诱导及检测试剂套装:包括100mL成脂诱导培养基和10mL成脂分化检测试剂,成脂诱导培养基为含有0.2-2mmoL/L谷氨酰胺、5-30μg/mL IGF-1、10-20mmol/L 3-磷酸甘油、1-5mmoL/L氟轻松、0.1-0.5mmoL/L异丁基甲基黄嘌呤、100-200μmoL/L吲哚美辛、50-100μmoL/L乙酰CoA、体积分数2-10%胎牛血清的DMEM培养基;
所述成骨分化诱导及检测试剂套装:包括100mL成骨诱导培养基和10mL成骨分化检测试剂,成骨诱导培养基为含有0.5-2mmoL/L谷氨酰胺、0.25-1mmoL/L氟轻松、2-10μg/mLTGF-β、2-10mmol/Lβ-甘油磷酸钠、15-25mmol/L维生素D3、10-50mmol/L维生素C、体积分数2-10%胎牛血清的DMEM培养基;
所述成软骨分化诱导及检测试剂套装:包括100mL成软骨诱导培养基和10mL成软骨分化检测试剂,成软骨诱导培养基为含有15-30ng/ml TGF-β1、10-30mg/L维生素C、5-10nmol/L氟轻松、10-30mg/ml ITS、20-50mmol/L氨基葡萄糖、10-20mmol/L丙酮酸钠、10-20ug/mg亚油酸、5-10ng/ml人血清白蛋白、5-10mmol/Lβ-甘油磷酸钠、体积分数2-10%胎牛血清的DMEM培养基;
所述普通培养基为20mL含体积分数2-8%胎牛血清的DMEM培养基。
2.根据权利要求1所述的用于鉴定牙髓间充质干细胞的试剂盒,其特征在于,所述细胞固定液为20mL质量百分比浓度4%多聚甲醛的水溶液。
3.根据权利要求1所述的用于鉴定牙髓间充质干细胞的试剂盒,其特征在于,所述成骨分化检测试剂为含有1g/L茜素磺酸钠水溶液。
4.根据权利要求1所述的用于鉴定牙髓间充质干细胞的试剂盒,其特征在于,所述成脂分化检测试剂为含有质量体积分数为0.5%油红O的异丙醇溶液。
5.根据权利要求1所述的用于鉴定牙髓间充质干细胞的试剂盒,其特征在于,所述成软骨分化检测试剂为含有10g/L阿尔新兰、质量体积分数3%的醋酸水溶液。
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