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CN106520674A - 一种用于表皮黑素细胞体外培养的无血清培养基 - Google Patents

一种用于表皮黑素细胞体外培养的无血清培养基 Download PDF

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CN106520674A
CN106520674A CN201611210540.8A CN201611210540A CN106520674A CN 106520674 A CN106520674 A CN 106520674A CN 201611210540 A CN201611210540 A CN 201611210540A CN 106520674 A CN106520674 A CN 106520674A
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Abstract

本发明属于细胞培养基领域，特别涉及一种用于表皮黑素细胞体外培养的无血清培养基。本发明培养基组成包括：基础培养基人转铁蛋白、胰岛素、谷氨酰胺、β‑成纤维细胞生长因子、表皮生长因子、肾上腺素，链霉素、氯化钙、硝酸铁、亚硒酸钠、硫酸铜、硫酸锌、维生素E、乙醇胺、磷酸乙醇和双丁酰环磷酸腺苷。本发明培养基培养黑素细胞增殖能力与现有黑素细胞培养基功能相当，解决了含血清培养基中，血清中可能带有朊病毒，血清的批次缺少稳定性等问题，与此同时，本发明培养基对黑素细胞分泌黑色素具有促进作用。

Description

一种用于表皮黑素细胞体外培养的无血清培养基
技术领域
本发明属于细胞培养基领域，特别涉及一种用于表皮黑素细胞体外培养的无血清培养基。
背景技术
皮肤颜色主要由皮肤制造的色素和皮肤表层所含天然色素的组合所决定。皮肤上皮层表面的表皮中含有能制造皮肤色素(即黑色素)的表皮黑素细胞。色素减退或无色素性皮肤病患者的皮肤黑素细胞缺失、被破坏或无功能，从而在身体各部位出现白斑。
白癜风是一种特殊的皮肤色素性疾病，以出现白斑和表皮黑素细胞缺失为主要特征。当前治疗包括光敏剂(如补骨脂素)联合UVA照射、窄波UVB照射或者局部使用皮质类固醇(肾上腺)，但疗效低，并伴随一定副作用。
外科移植技术已经应用于白癜风治疗，如负压吸疱移植、刃厚皮片移植或微小皮片移植。这些治疗在局限性和小面积病损的患者中有效。但对白斑面积大或者白斑数量多的患者，获得足够的移植皮片具有一定困难。
要成功地进行大面积的自体表皮移植，需有大的移植皮片，内含大量细胞，特别是黑素细胞。或者，可通过细胞培养大量扩增自体黑素细胞用于移植。这种方法需要从病人健康皮肤取得黑素细胞，在体外将其培养在合适环境中(可促进细胞增殖、迁移和黑素制造)使之大量扩增，再在合适条件下将细胞移植至病人病变处皮肤，使之恢复正常颜色。
关于黑素细胞培养基已有报道，Olsson等报告的表皮黑素细胞培养基配方，包含PC-1，碱性成纤维细胞生长因子(bFGF)，丁酰环腺苷酸(dbcAMP)，青霉素-链霉素。该培养基用于在自体黑素细胞移植中扩增分离得到表皮黑素细胞。此培养基不包含促进黑素细胞生长和分化必需的血清。此外，bFGF的浓度(5ng/ml)太低，不能刺激表皮黑素细胞作足够的生长。而dbcAMP不是一种天然物质，作用时间也很短。另外，对所培养细胞的数量和黑素含量均无量化指标报道，因此，很难评价这一培养基的效率。
另外的培养基，如HU16(FIC培养基)已被我们用于培养表皮黑素细胞。HU16培养基包括F12培养基(一种商品化的基础培养基)，补充有效浓度的bFGF、IBMX，霍乱毒素和胎牛血清。该培养基已经在中国台湾成功应用于120例白癜风患者黑素细胞移植治疗中的黑素细胞培养。
另一种表皮黑素细胞培养基(HU74)，含有bFGF、肝细胞生长因子(HGF)、肾上腺素和α-MSH，已经建立并在美国成功申报专利(美国专利6943024和7132279)。
虽然培养表皮黑素细胞的培养基很多，但都不是最佳的。因此，一些患者因为细胞生长不佳，必须长时间等待；另一小部分患者细胞生长差，达不到移植需要。另外，一些移植成功的病人在移植区域的边缘缺乏色素形成，这可能与培养基不能有效刺激细胞的迁移有关。
中国专利CN101735976B公布了一种用于表皮黑素细胞体外培养的培养基，可用于培养增殖能力、迁移能力和黑素合成能力增强的表皮黑素细胞。但依旧含有5-30％的血清，血清的整个流程极其复杂，造价高昂，而且血清中可能带有朊病毒，血清的批次质量也缺少稳定性。
发明内容
本发明目的是提供一种用于表皮黑素细胞体外培养的无血清培养基新配方，解决了血清中可能带有朊病毒，血清的批次缺少稳定性等问题。
本发明采用的技术方案是：
一种用于表皮黑素细胞体外培养的无血清培养基，1包括基础培养基及以下成分和浓度：人转铁蛋白15-100mg/L、胰岛素10-30mg/L、谷氨酰胺1-10mg/L、β-成纤维细胞生长因子0.1-5ug/L、表皮生长因子5-10ug/L、肾上腺素50-300ug/L，链霉素1-30mg/L、氯化钙10-50mg/L、硝酸铁0.1-1mg/L、亚硒酸钠1-10ug/L、硫酸铜1-10ug/L、硫酸锌0.1-1mg/L、维生素E 0.1-30mg/L、乙醇胺0.1-50mg/L磷酸乙醇0.1-10mg/L和双丁酰环磷酸腺苷5.3-6.2ug/L。
优选地，所述用于表皮黑素细胞体外培养的无血清培养基，由基础培养基及以下成分和浓度组成：人转铁蛋白25mg/L、胰岛素18mg/L、β-成纤维细胞生长因子1ug/L、表皮生长因子6.5ug/L、肾上腺素75ug/L，链霉素8mg/L、氯化钙 15mg/L、硝酸铁0.8mg/L、亚硒酸钠4ug/L、硫酸铜1.5ug/L、硫酸锌0.6mg/L、维生素E 9mg/L、乙醇胺3mg/L、磷酸乙醇10mg/L和双丁酰环磷酸腺苷为5.4ug/L。
优选地，所述的基础培养基为Ham’s F12
与现有技术相比，本发明培养基具有以下优点：
1.本发明培养基，是一种用于表皮黑素细胞体外培养的无血清培养基，解决了血清中可能带有朊病毒，血清的批次缺少稳定性等问题。
2.本发明培养基培养黑素细胞能力与现有黑素细胞培养基功能相当，且本发明培养基对黑素细胞分泌黑素有意外的促进作用。
具体实施例
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
实施例1一种用于表皮黑素细胞体外培养的无血清培养基
一种用于表皮黑素细胞体外培养的无血清培养基由以下成分及其浓度组成：人转铁蛋白15mg/L、胰岛素10mg/L、谷氨酰胺1mg/L、β-成纤维细胞生长因子0.1ug/L、表皮生长因子5ug/L、肾上腺素50ug/L，链霉素1mg/L、氯化钙10mg/L、硝酸铁0.1mg/L、亚硒酸钠1ug/L、硫酸铜1ug/L、硫酸锌0.1mg/L、维生素E 0.1mg/L、乙醇胺0.1mg/L、磷酸乙醇0.1mg/L和双丁酰环磷酸腺苷6.2ug/L，溶剂为Ham’s F12基础培养基。
实施例2一种用于表皮黑素细胞体外培养的无血清培养基
一种用于表皮黑素细胞体外培养的无血清培养基由以下成分及其浓度组成：人转铁蛋白100mg/L、胰岛素30mg/L、谷氨酰胺10mg/L、β-成纤维细胞生长因子5ug/L、表皮生长因子10ug/L、肾上腺素300ug/L，链霉素30mg/L、氯化钙50mg/L、硝酸铁1mg/L、亚硒酸钠10ug/L、硫酸铜10ug/L、硫酸锌1mg/L、维生素E 30mg/L、乙醇胺50mg/L、磷酸乙醇0.1mg/L和双丁酰环磷酸腺苷5.3ug/L，溶剂为Ham’s F12基础培养基。
实施例3一种用于表皮黑素细胞体外培养的无血清培养基
一种用于表皮黑素细胞体外培养的无血清培养基由以下成分及其浓度组成：人转铁蛋白25mg/L、胰岛素18mg/L、β-成纤维细胞生长因子1ug/L、表皮生长因子6.5ug/L、肾上腺素75ug/L，链霉素8mg/L、氯化钙15mg/L、硝酸铁0.8mg/L、亚硒酸钠4ug/L、硫酸铜1.5ug/L、硫酸锌0.6mg/L、维生素E 9mg/L、乙醇胺3mg/L、磷酸乙醇0.1mg/L和双丁酰环磷酸腺苷5.4ug/L，溶剂为Ham’s F12基础培养基。
对比例1一种用于表皮黑素细胞体外培养的无血清培养基
一种用于表皮黑素细胞体外培养的无血清培养基由以下成分及其浓度组成：人转铁蛋白25mg/L、胰岛素18mg/L、β-成纤维细胞生长因子1ug/L、表皮生长因子6.5ug/L、肾上腺素75ug/L，链霉素8mg/L、氯化钙15mg/L、硝酸铁0.8mg/L、亚硒酸钠4ug/L、硫酸铜1.5ug/L、硫酸锌0.6mg/L、维生素E 9mg/L、乙醇胺3mg/L、磷酸乙醇0.1mg/L和双丁酰环磷酸腺苷6.5ug/L，溶剂为Ham’s F12基础培养基。
与实施例3相比，双丁酰环磷酸腺苷浓度增加。
试验一、培养的表皮黑素细胞的细胞生长检测
1.试验对象：实施例1-3得到的表皮黑素细胞体外培养的无血清培养基
2.试验方法：
(1)表皮黑素细胞的分离和培养
细胞样本在0.25％(w/v)胰酶(GIBCO，Carlsbad，Calif.)中在37℃温度下孵育15分钟，0.2％(w/v)EDTA(Sigma，St.Louis，Mo.)中继续孵育10分钟。用镊子小心的将皮片上的表皮细胞分离下来，形成表皮细胞悬液。表皮细胞离心后，种植在25cm2的培养瓶中。培养瓶放置在37℃温度下的CO2培养箱中(95％空气，5％CO2)。3天后，加入100μg/ml基因素以去除角质形成细胞和成纤维细胞。每3天换液一次。黑素细胞一般在2周后基本融合。细胞融合后，用胰酶-EDTA溶液脱下，离心，稀释，传代培养。在移植时，黑素细胞用胰酶-EDTA溶液脱下，离心后用F12培养基重悬。
(2)表皮黑素细胞的细胞生长检测
表皮黑素细胞以2×104/孔的密度接种于24孔板，24h后，分别采用实施例1、2、3及对比例1中的培养基进行培养。每3天换液一次。6天后，用胰酶-EDTA脱下细胞，以含10％血清的培养基终止消化。细胞悬液离心后，用200μl F12培养基重悬沉淀，用Pasteur移液管转移20μL的细胞悬液至血球计数器，在光学显微镜下观察。计数4个1mm3区域中的细胞。细胞数量用下面的公式1计算，四次计数，取平均值。所有实验样本都重复3次。公式1：c＝0.2×n×104c＝细胞量(cells/mL)；n＝细胞计数。
(3)实验结果，不同培养基中表皮黑素细胞量检测结果，如表1所示：
表1不同培养基中表皮黑素细胞量
所用培养基 细胞量(104cells/mL)
实施例1 540
实施例2 530
实施例3 550
对比例1 408
由表1可知，在培养6天后，本发明配制得到的细胞基中细胞量均高于对比例培养基中黑素细胞量，并且实施例3配制得到的培养基培养黑素细胞量最多。
试验二、培养的表皮黑素细胞的黑色素含量检测
1.试验对象：实施例1-3及对比例1得到的表皮黑素细胞体外培养的无血清培养基
2.试验方法：
表皮黑素细胞以2×104/孔的密度接种于24孔板。24h后，分为四组，组1、组2、组3、组4，分别采用实施例1、2、3及对比例1配制得到的培养基。每3天换液一次。6天后将黑素细胞用胰酶-EDTA脱下并用血球计数器进行计数。再将细胞悬液离心，沉淀用1N的NaOH溶液溶解。通过分光光度计在475nm处检测溶液的光密度值，对照用合成的黑素(Sigma，St.Louis，Mo.)所做的标准曲线算出黑素含量。实验结果，如表2所示。
表2不同培养基中表皮黑素细胞的黑色素含量
由表2可知，采用本发明配制得到的培养基中黑素细胞分泌黑色素的量均高于对比例1，实施例3中培养基所得黑色素含量最高，与对比例1中培养基所得黑色素含量相比提高了1.69倍。由此可知，本发明配制得到的培养基对黑素细胞分泌黑色素有意外的促进作用。
以上列举的仅是本发明的具体实施例，显然本发明不限于以上实施例，还可有许多改动。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有改动，均应认为是本发明的保护范围。

Claims (3)

1.一种用于表皮黑素细胞体外培养的无血清培养基，其特征在于，包括基础培养基及以下成分和浓度：人转铁蛋白15-100mg/L、胰岛素10-30mg/L、谷氨酰胺1-10mg/L、β-成纤维细胞生长因子0.1-5ug/L、表皮生长因子5-10ug/L、肾上腺素50-300ug/L，链霉素1-30mg/L、氯化钙10-50mg/L、硝酸铁0.1-1mg/L、亚硒酸钠1-10ug/L、硫酸铜1-10ug/L、硫酸锌0.1-1mg/L、维生素E 0.1-30mg/L、乙醇胺0.1-50mg/L磷酸乙醇0.1-10mg/L和双丁酰环磷酸腺苷5.3-6.2ug/L。
2.根据权利要求1中所述的用于表皮黑素细胞体外培养的无血清培养基，其特征在于，由基础培养基及以下成分和浓度组成：人转铁蛋白25mg/L、胰岛素18mg/L、β-成纤维细胞生长因子1ug/L、表皮生长因子6.5ug/L、肾上腺素75ug/L，链霉素8mg/L、氯化钙15mg/L、硝酸铁0.8mg/L、亚硒酸钠4ug/L、硫酸铜1.5ug/L、硫酸锌0.6mg/L、维生素E 9mg/L、乙醇胺3mg/L、磷酸乙醇10mg/L和双丁酰环磷酸腺苷为5.4ug/L。
3.根据权利要求1或2所述的用于表皮黑素细胞体外培养的无血清培养基，其特征在于，所述的基础培养基为Ham’s F12。
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