CN116515747B - 一种牙髓间充质干细胞培养方法 - Google Patents
一种牙髓间充质干细胞培养方法 Download PDFInfo
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- inonotus obliquus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
本发明提供一种间充质干细胞的培养方法,包括向培养基中加入桦褐孔菌提取物和银杏叶提取物的混合物;所述的间充质干细胞为牙髓间充质干细胞。本发明的桦褐孔菌来源极其广泛,安全性高,易获得;桦褐孔菌提取物及银杏提取物混合物培养牙髓干细胞,可以提高细胞增殖能力,缩短培养时间,减少时间成本,同时可以提高细胞活性;且不会诱导牙髓干细胞分化,保证牙髓干细胞的纯度。
Description
技术领域
本发明属于干细胞培养领域,具体涉及一种牙髓间充质干细胞培养方法。
背景技术
牙髓干细胞(Dental pulp stem cells,DPSC)是具有多向分化、高增殖能力、自我更新的外胚间充质干细胞,可为组织器官修复和多种疾病治疗提供细胞来源,易于获得,易保存,较人体其他间充质干细胞更具优势。
桦褐孔菌是一类药用真菌,对多种疾病有防治效果,适合应用于医疗行业,其在抗高血糖、炎症、癌症等方面具有较好的药理作用,尤其是在代谢调节方面特别显著。同时,桦褐孔菌还具有抗肿瘤、抗炎、抗氧化、免疫调节和抗突变的特性等多种功能。桦褐孔菌中含有20多种成分,包括多糖、桦褐孔菌素、桦褐孔菌醇、多种氧化三萜类、类固醇类及低分子多酚类等有效成分。其中,多糖类化合物研究较多,活性较为显著。三萜类成分也是桦褐孔菌中的重要活性成分,它可以调节血压、降低胆固醇,对病毒的增殖有明显的抑制作用。而酚类化合物也是桦褐孔菌重要的有效成分,研究表明其在预防癌症、抗炎和降胆固醇等方面的作用更加安全有效。
桦褐孔菌多糖是桦褐孔菌中主要的生物活性成分,在降血糖、降血脂、抗氧化、抗肿瘤、提高免疫等方面具有重要作用和广泛的食药价值,而其大部分生物活性与其含有的多糖成分与结构密切相关。桦褐孔菌的抗氧化效果在很多实验中得到了验证,其可预防细胞的核酸、内源性DNA等氧化,可对抗H2O2破坏的作用,对氧自由基、羟基自由基、脂质过氧化发挥清除作用。同时还可升高过氧化氢酶活性,加强自由基清除,延长传代细胞的分裂代数,增进细胞寿命,促进代谢。
银杏叶中富含大量的次生代谢产物,如类黄酮、酚类、萜内酯类、异戊二烯类等,其中最具药用活性的成分是银杏类黄酮与萜内酯。银杏类黄酮物质约有38种,多以糖苷和甲基化形式存在,有黄酮、二氢黄酮、黄酮醇及其苷类、儿茶素类、双黄酮等,黄酮类化合物是植物中重要的次生代谢产物,具有增强免疫力、抗癌活性和抗衰老活性等作用,可用于预防和治疗心血管疾病,并且被认为是重要化学防御机制的组成部分。
利用牙髓干细胞再生为修复性牙本质,可以恢复牙髓疾病患者的生理状态和功能。牙髓干细胞由于缺少特异性标记,目前无法获得精确的定性、定位和纯化,因此在体外培养存在易变性、很难进行大量的扩增等问题,这限制了牙髓干细胞的发展。但是牙髓干细胞的临床应用一个必然的前提是容易培养,因此优化牙髓间充质干细胞的培养十分必要。
申请号为CN202111394843.0的中国专利中公开了一种牙髓间充质干细胞培养基,按体积分数计算,包括5%-20%血小板裂解液、0.05%-0.3%表皮生长因子溶液、0.05%-0.3%碱性成纤维生长因子溶液、0.1%-1%L-丙氨酰-L-谷氨酰胺添加剂和细胞基础培养基。该牙髓间充质干细胞培养基通过添加并限定上述各组分的含量,从供给细胞营养、防止细菌污染和促进细胞生长方面,能够促进牙髓间充质干细胞快速增殖。但其细胞增殖能力有限,有进一步优化的空间。
现有技术中还未有将桦褐孔菌提取物和银杏叶提取物应用于牙髓间充质干细胞培养的先例。
发明内容
为了解决上述问题,本发明提供了一种牙髓间充质干细胞的培养方法。
本发明中,“α-MEM”指α-MEM培养基,是一种改良的MEM培养基,与MEM培养基相比α-MEM培养基营养更为丰富,在MEM的基础上又添加了NEAA(非必需氨基酸)、丙酮酸钠、硫酸锌、VB12、生物素、抗坏血酸等成分,广泛应用于各种哺乳动物悬浮和贴壁细胞的培养。不含核苷和脱氧核苷的α-MEM培养基常常用作DG44和其他DHFR-缺陷型细胞的筛选培养基。
本发明中,“培养基基础体系”是指培养基最基本的组成,在培养基基础体系的基础上,可以根据需要添加其他需要的组分。
本发明中,“提取物”是指通过提取方法获得的产品,且该产品不为单一组分,如“桦褐孔菌提取物”为通过提取方法获得的混合物,通过桦褐孔菌提取的单一组分如某一种多糖或者某一种类固醇,不在本申请“提取物”含义包括的范围内。
一方面,本发明提供了一种间充质干细胞的培养方法。
所述的培养方法中包括向培养基中加入桦褐孔菌提取物和银杏叶提取物的混合物;所述的间充质干细胞为牙髓间充质干细胞。
优选地,所述的混合物的添加量为1%-10%;进一步优选为5%。
优选地,所述的桦褐孔菌提取物和银杏叶提取物的混合比例为1:2-2:1;进一步为1:1。
优选地,所述的培养基基础体系为α-MEM。
具体地,所述的培养基中还包括细胞生长因子、L-谷氨酰胺。
所述的细胞生长因子为10%胎牛血清。
优选地,所述的L-谷氨酰胺的添加量为1%。
所述的培养方法可以用于牙髓间充质干细胞原代细胞培养或传代细胞培养。
另一方面,本发明提供了一种间充质干细胞培养物。
所述的间充质干细胞培养物通过前述的培养方法进行制备。
所述的间充质干细胞培养物为牙髓间充质干细胞培养物。
再一方面,本发明提供了前述间充质干细胞培养物在制备牙周修复、牙周再生、骨损伤修复或神经修复药物中的应用。
所述的间充质干细胞培养物为牙髓间充质干细胞培养物。
所述的牙周再生为牙髓再生、牙槽骨再生、牙齿再生中的一种或多种。
牙髓间充质干细胞具有牙周修复、牙周再生、骨损伤修复或神经修复的功效,因此包含牙髓间充质干细胞也具有牙周修复、牙周再生、骨损伤修复或神经修复的功效。
又一方面,本发明提供了桦褐孔菌提取物和银杏叶提取物的混合物在培养间充质干细胞中的应用。
所述的间充质干细胞为牙髓间充质干细胞。
所述的应用为将桦褐孔菌提取物和银杏叶提取物加入培养基对牙髓间充质干细胞进行培养。优选地,所述的混合物的添加量为1%-10%;进一步优选为5%。
优选地,所述的桦褐孔菌提取物和银杏叶提取物的混合比例为1:2-2:1;进一步为1:1。
优选地,所述的培养基基础体系为α-MEM。
具体地,所述的培养基中还包括细胞生长因子、L-谷氨酰胺。
所述的细胞生长因子为10%胎牛血清。
优选地,所述的L-谷氨酰胺的添加量为1%。
所述的培养方法可以用于牙髓间充质干细胞原代细胞培养或传代细胞培养。
本发明的有益效果:
1、本发明所用到的桦褐孔菌是一类具有多种生物活性的药用真菌,其适合应用于医疗方面,来源极其广泛,安全性高,易获得;
2、本发明通过反复验证,添加桦褐孔菌提取物及银杏提取物混合物培养牙髓干细胞,可以提高细胞增殖能力,缩短培养时间,减少时间成本,同时可以提高细胞活性;
3、本发明通过添加桦褐孔菌提取物及银杏提取物混合物来培养牙髓干细胞,不会诱导牙髓干细胞分化,保证牙髓干细胞的纯度。
附图说明
图1为实施例1中样本1对照组的流式细胞法检测结果。
图2为实施例1中样本2实验组的流式细胞法检测结果。
图3为实施例1中样本3实验组的流式细胞法检测结果。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中:
桦褐孔菌提取物参照Q/GYY01-2018黑龙江科学院高技术研究院企业标准-桦褐孔菌提取物进行提取。以下实施例中采用的提取方法:桦褐孔菌子实体烘干粉碎,1:20m/V料液比加入蒸馏水,90℃保温2.5h,提取液12000r/min离心10min,上清液水浴蒸发浓缩至原体积1/3,加入终浓度为60%的无水乙醇,4℃静置24h,12000r/min离心10min,沉淀烘干,参照上述标准检测合格后进行以下实施例的实验。
银杏叶提取物购自阿拉丁,货号G195711,CAS号90045-36-6。
α-MEM:购自Gibco,货号12571063。
胎牛血清:购自Hyclone,货号SV30208.02。
L-谷氨酰胺:购自Gibco,25030081。
胰蛋白酶:购自Gibco,25200072。
实施例1一种牙髓间充质干细胞培养方法
按照以下步骤进行:
1、牙髓干细胞分离制备
在生物安全柜中放置2个10cm无菌培养皿,加入30mL含双抗(青霉素钠100μg/mL,硫酸链霉素100mg/mL)的0.9%氯化钠注射液。用无菌镊子取出采集瓶中的牙齿,放入到培养皿中清洗和消毒2-3min,充分去除牙周软组织和血污后,将牙齿放入第二个培养皿中再消毒一次。
将清洗后的牙齿放在无菌纱布上,然后用纱布包裹牙齿,用台虎钳将牙齿的牙釉质和牙本质钳开,打开无菌纱布,用镊子从牙髓腔内找出牙髓组织,将其装入装含无血清培养基(α-MEM)的EP管中。
在无血清培养基充分浸润条件下,用无菌眼科剪将牙髓组织剪成约1.0mm-3.0mm大小的组织块。在T25培养皿内滴加2mL培养基,培养体系为α-MEM,10%胎牛血清,1% L-谷氨酰胺,桦褐孔菌提取物,银杏叶提取物分别为1mL,0.1mL,10μL,25μg,25μg。
同时培养对照组(不添加桦褐孔菌提取物及银杏提取物混合物)。用牙科探针将组织碎块移入培养皿中的培养液中,使每滴培养液内约含8-10小块组织。放置培养箱中,于37℃、5%CO2饱和湿度条件下培养。
在培养的第6天,更换培养基,在培养12天时培养基进行半量换液,培养至18天时,显微镜下观察对比两组细胞的汇合度。
2、细胞传代培养
将两组原代细胞收集传代,接种到T25内,密度为1×105个,培养条件为37℃、5%CO2,所用的培养基保持不变,培养3-4天后,使用0.25%胰蛋白酶对细胞进行消化,收集所有细胞,再进行传代,将细胞传至第3代,对每组细胞量对比发现,在相同的培养时间内,通过在培养基内添加桦褐孔菌提取物及银杏提取物,可以提高细胞的增殖。
共选取了3个样本进行实验,各样本培养完成后检测细胞性能,结果如下。
样本1:
(1)细胞量(细胞增殖/个)对比:
(2)流式细胞法检测牙髓干细胞表面标志物,具体方法为:
1)收集准备好牙髓干细胞细胞,制成5×106个/mL的细胞态液。
2)取7支流式专用管进行标记,各自加入100μL的细胞悬液样本,1-4号分别加FITC、PE、Percp-cy、APC单色抗体5μL,5号为阴性管无添加,6号加入阳性和阴性同型对照抗体各20μL,7号加入阳性和阴性检测抗体各20μL。
3)充分混匀,置4℃冰箱避光孵育30分钟。
4)孵育结束以后加入500μL PBS,充分混匀,314g离心5分钟。
5)弃上清液,加入300μL PBS。充分混匀,待上机检测。
流式细胞法检测结果显示牙髓干细胞表面标志CD73、CD90、CD105阳性表达率97%以上,阴性指标表达率低于2%,添加桦褐孔菌提取物及银杏提取物可以培养牙髓干细胞不会诱导牙髓干细胞分化。具体见图1。
样本2:
(1)细胞量对比:
(2)流式细胞法检测结果显示牙髓干细胞表面标志CD73、CD90、CD105阳性表达率95%以上,阴性指标表达率低于2%,添加桦褐孔菌提取物及银杏提取物可以培养牙髓干细胞不会诱导牙髓干细胞分化。具体见图2。
样本3:
(1)细胞量对比
(2)流式细胞法检测结果显示牙髓干细胞表面标志CD73、CD90、CD105阳性表达率98%以上,阴性指标表达率低于2%,添加桦褐孔菌提取物及银杏提取物可以培养牙髓干细胞不会诱导牙髓干细胞分化。具体见图3。
对比例
根据实施例1的方法设置对比例,具体差别如下:
对比例 | 与实施例1的差别 |
对比例1 | 培养基中添加50μg银杏提取物,不添加桦褐孔菌提取物 |
对比例2 | 培养基中添加50μg桦褐孔菌提取物,不添加银杏提取物 |
参照实施例1的方法对同一样本来源的牙髓干细胞(样本1)进行培养。
细胞量对比(1×105):
流式细胞法检测结果:
Claims (3)
1.一种间充质干细胞的培养方法,其特征在于,包括向培养基中加入桦褐孔菌提取物和银杏叶提取物的混合物;所述的间充质干细胞为牙髓间充质干细胞;
所述的混合物的添加量为5%;
所述的桦褐孔菌提取物和银杏叶提取物的混合比例为1:1;
所述的培养基基础体系为α-MEM;
所述的培养基中还包括细胞生长因子、L-谷氨酰胺,所述的细胞生长因子为10%胎牛血清,所述的L-谷氨酰胺的添加量为1%;
所述的桦褐孔菌提取物的制备方法:桦褐孔菌子实体烘干粉碎,1:20m/V料液比加入蒸馏水,90℃保温2.5h,提取液12000r/min离心10min,上清液水浴蒸发浓缩至原体积1/3,加入终浓度为60%的无水乙醇,4℃静置24h,12000r/min离心10min,沉淀烘干;
所述的银杏叶提取物CAS号90045-36-6。
2.根据权利要求1所述的培养方法,其特征在于,用于原代细胞培养或传代细胞培养。
3.桦褐孔菌提取物和银杏叶提取物的混合物在培养间充质干细胞中的应用,其特征在于,所述的间充质干细胞为牙髓间充质干细胞;向牙髓间充质干细胞培养基中加入桦褐孔菌提取物和银杏叶提取物的混合物;
所述的混合物的添加量为5%;
所述的桦褐孔菌提取物和银杏叶提取物的混合比例为1:1;
所述的培养基基础体系为α-MEM;
所述的培养基中还包括细胞生长因子、L-谷氨酰胺,所述的细胞生长因子为10%胎牛血清,所述的L-谷氨酰胺的添加量为1%;
所述的桦褐孔菌提取物的制备方法:桦褐孔菌子实体烘干粉碎,1:20m/V料液比加入蒸馏水,90℃保温2.5h,提取液12000r/min离心10min,上清液水浴蒸发浓缩至原体积1/3,加入终浓度为60%的无水乙醇,4℃静置24h,12000r/min离心10min,沉淀烘干;
所述的银杏叶提取物CAS号90045-36-6。
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