CN106834214B - 一种诱导多能干细胞形成角质形成细胞的诱导培养基及诱导方法 - Google Patents
一种诱导多能干细胞形成角质形成细胞的诱导培养基及诱导方法 Download PDFInfo
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Abstract
本发明提供了一种诱导多能干细胞形成角质形成细胞的诱导培养基和诱导方法,本发明所述诱导培养基包括以下浓度的各组分:EGF 18‑25ng/ml、b FGF 10‑20ng/ml、IGF‑1 5ng/ml、地塞米松0.05‑0.2μmol/L、全转式维A酸0.5‑2μmol/l、BMP‑4 20‑30ng/ml、CaCl2 0.2‑0.5mM、SCGF 20‑35ng/ml、TGF‑β 0.6‑1.0ng/ml、VEGF 8‑15ng/ml、谷氨酰胺1.0‑2.0mM、转铁蛋白3‑8μg/ml、三碘甲状腺原氨(1‑4)×10‑7mol/L、腺嘌呤(1‑2)×10‑4mol/L。本发明所述诱导培养基是在K‑SFM培养基的基础上通过添加一些特定的细胞因子和补充物从而提高诱导多能干细胞(ips)向角质形成细胞分化的概率,为皮肤组织工程提供种子细胞来源。
Description
技术领域
本发明涉及一种诱导培养基和诱导方法,具体涉及一种诱导多能干细胞形成角质形成细胞的诱导培养基和诱导方法。
背景技术
皮肤组织工程是近几年的研究热点,但是皮肤的相关细胞都属于终末分化细胞,很难再体外进行大规模的培养扩增,种子细胞来源问题阻碍了组织工程的发展,自体的种子细胞来源有限,异体的种子细胞存在排斥反应,干细胞又存在伦理问题,因此从尿液中收集尿液细胞,将尿液细胞诱导成为IPS细胞,ips细胞再诱导成为角质形成细胞、成纤维细胞、汗腺细胞、毛乳头细胞等即可解决皮肤组织工程的种子来源问题,从尿液中获得细胞,方便快捷,不用通过创伤获取,来源广泛。
发明内容
本发明的目的在于克服现有技术存在的不足之处而提供了一种诱导多能干细胞形成角质形成细胞的诱导培养基和诱导方法。
为实现上述目的,本发明所采取的技术方案为:一种诱导多能干细胞形成角质形成细胞的诱导培养基,所述诱导培养基包括以下浓度的各组分:
其中,EGF为表皮细胞生长因子,b FGF为碱性成纤维细胞生长因子,IGF-1为类胰岛素一号增长因子,BMP-4为骨形态发生蛋白4,CaCl2为氯化钙,SCGF为干细胞生长因子,TGF-β为转化生长因子-β,VEGF为血管内皮生长因子。
优选地,上述所述诱导多能干细胞形成角质形成细胞的诱导培养基,包括以下浓度的各组分:
采用上述所述的诱导多能干细胞形成角质形成细胞的诱导培养基可以最大程度、最高效地诱导多能性干细胞分化为角质形成细胞。
优选地,所述诱导培养基还包括基础培养液,所述基础培养液为K-SFM。
本发明诱导培养基是在K-SFM培养基的基础上通过添加一些特定的细胞因子和补充物从而提高诱导多能干细胞(ips)向角质形成细胞分化的概率,为皮肤组织工程提供种子细胞来源。
另外,本发明提供了一种诱导多能干细胞形成角质形成细胞的诱导方法,所述方法是将诱导多能干细胞的拟胚体接种于上述所述的诱导培养基中进行诱导培养的。
优选地,所述诱导培养时间为6~8天。
优选地,所述诱导培养时间为7天。
优选地,所述拟胚体的获得方法为将将单个复苏的诱导多能干细胞接种于含有拟胚体形成培养基中,离心之后放入培养箱中培养48h后,即形成拟胚体。
优选地,所述单个复苏的诱导多能干细胞的接种量为1×106个细胞/孔。
优选地,所述培养板离心的参数条件为:700r/min,5min。
优选地,所述培养箱的温度为37℃、二氧化碳含量为5%CO2。
具体地,拟胚体的获得方法为:(1)、复苏细胞:选择P30的IPS细胞进行复苏,用无基质的人胚胎干细胞培养基(mTeSR1)进行细胞培养,细胞达到80%汇合度时,用Accutase细胞消化液消化为单细胞,计数;(2)、然后按每个AggreWellTM800培养板孔中加入1×106个细胞,加入EB形成培养基,按产品使用操作手册,将AggreWellTM800培养板以700r/min低速
离心5min,然后放入含5%CO2、37℃培养箱中培养,培养48h后即形成拟胚体。
另一方面,本发明提供一种上述所述的诱导多能干细胞形成角质形成细胞的诱导培养基在诱导多能干细胞形成角质形成细胞时的应用。
本发明的有益效果在于:本发明提供了一种诱导多能干细胞形成角质形成细胞的诱导培养基和诱导方法,采用该诱导培养基和诱导方法能显著提高诱导多能干细胞(ips)向成角质形成细胞分化的概率,为皮肤组织工程提供种子细胞来源。
附图说明
图1为本发明实施例1中在本发明所述诱导培养基和对照培养基中诱导分化简单拟胚体发生形变所需天数的柱状统计图;
图2为在本发明所述诱导培养基和对照培养基中诱导后抗角蛋白CK19的免疫荧光图,图中白色地方即为CK19表达位点;
图3为在本发明所述诱导培养基和对照培养基中角质形成细胞的增殖速度曲线对比图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图及具体实施例对本发明作进一步说明。
实施例1
一种诱导多能干细胞形成角质形成细胞的诱导培养基,所述诱导培养基包括以下浓度的各组分:
实施例2
一种诱导多能干细胞形成角质形成细胞的诱导培养基,所述诱导培养基包括以下浓度的各组分:
实施例3
一种诱导多能干细胞形成角质形成细胞的诱导培养基,所述诱导培养基包括以下浓度的各组分:
实施例4体外诱导ips向角质形成细胞分化
一、实验组培养基的配制
按照500ml K-SFM培养基中加入,EGF2μg,bFGF1.5μg,IGF-1 0.5μg,地塞米松0.05μmol,转铁蛋白2.5mg,全转式维A酸0.5μmol,BMP-4 12.5μg,CaCl20.2mol,SCGF 15μg,TGF-β0.25μg,VEGF 5μg,谷氨酰胺0.75mmol,腺嘌呤90μmol,三碘甲状腺原氨0.1μmol的比例配制本发明所述的诱导多能干细胞形成角质形成细胞的诱导培养基,并作为以下实验中的实验组培养基。
二、对照组培养基的配制
按照500ml K-SFM培养基中加入10ml FBS的比例配制对照组培养基。
三、复苏细胞
选择P30的IPS细胞进行复苏,用无基质的人胚胎干细胞培养基(mTeSR1)进行细胞培养,细胞达到80%汇合度时,用Accutase细胞消化液消化为单细胞,计数。
四、拟胚体的形成:按每个AggreWellTM800培养板孔中加入1x106个细胞,加入EB形成培养基,按产品使用操作手册,将AggreWellTM800培养板以700r/min低速离心5min,然后放入含5%CO2、37℃培养箱中培养,培养48h后即形成拟胚体。
五、诱导分化
将拟胚体转移至低吸附24孔板中,24孔板分为两组,分别为实验组和对照组,每组12孔,实验组用实验组培养基培养,对照组用对照组培养基培养,隔天换液,记录细胞第几天发生形变,挑选形变细胞进行免疫荧光鉴定角质形成细胞相关基因的表达,确定是角质形成细胞后传代连续培养,每次传代都进行细胞计数,比较两组的细胞增殖速度。
其中,免疫荧光鉴定诱导后角质形成细胞的CK19的抗原表达的相关步骤为:
1、诱导15天后,分别将实验组、对照组的培养基弃去,用PBS洗三遍,每次5min;
2、每孔加入4%多聚甲醛1ml固定1h,用PBS洗三遍,每次5min;
3、0.1%Triton破膜10min,用PBS洗三遍,每次5min;
4、弃去PBS,实验组、对照组分别加入加入CK19(1:50)500ul,4℃湿盒孵育过夜;
5、用PBS洗三遍,每次5min,实验组、对照组分别加入二抗兔抗鼠IgG(1:200)500ul,室温避光孵育1h;
6、用PBS洗三遍,每次5min;
7、对照组加入hoechst33258,室温避光孵育15min;
8、用PBS洗三遍,每次5mi;
9、50%甘油封片,荧光显微镜下观察,并对发出荧光的细胞进行计数。
六、结果
如图1所示,采用对照组培养基诱导ips形成角质形成细胞时,诱导后出现细胞形变所需的天数为17天,然而采用本发明所述诱导培养基诱导多能干细胞诱导形成角质形成细胞后出现细胞形变所需的天数仅为7天;
并且,如图2所示,实验组被染色的细胞比对照组被染色的细胞的数量多,即采用本发明所述诱导培养基诱导多能干细胞诱导形成的角质形成细胞比采用对照组培养基诱导形成的角质形成细胞的数量多;
此外,如图3可见,实验组培养基细胞增殖的速度快于对照组培养基中细胞的增殖速度;
因此,以上实验结果说明,在对照组的基础培养基上添加的细胞因子和补充物达到复合增效作用,可以提高ips细胞诱导为角质形成细胞的概率,并且能稳定传代,提高细胞的增殖速度,为皮肤组织工程提供种子细胞来源,同时整个过程没有用到血清,有效的避免了动物源性病原体带来的风险,提高了临床应用的安全性。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
3.如权利要求1或2所述的诱导多能干细胞形成角质形成细胞的诱导培养基,其特征在于,所述诱导培养基还包括基础培养液,所述基础培养液为K-SFM。
4.一种诱导多能干细胞形成角质形成细胞的诱导方法,其特征在于,所述方法是将诱导多能干细胞的拟胚体接种于如权利要求1所述的诱导培养基中进行诱导培养的。
5.根据权利要求4所述的诱导方法,其特征在于,所述诱导培养的时间为6~8天。
6.根据权利要求4所述的诱导方法,其特征在于,所述诱导培养的时间为7天。
7.根据权利要求4所述的诱导方法,其特征在于,所述拟胚体的获得方法:将单个复苏的诱导多能干细胞接种于含有拟胚体形成培养基中,离心之后放入培养箱中培养48h后,即形成拟胚体。
8.根据权利要求7所述的诱导方法,其特征在于,所述单个复苏的诱导多能干细胞的接种量为1×106个细胞/孔。
9.根据权利要求7所述的诱导方法,其特征在于,所述离心的参数条件为:700r/min,5min。
10.如权利要求1或2所述的诱导多能干细胞形成角质形成细胞的诱导培养基在诱导多能干细胞形成角质形成细胞时的应用。
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