CN102399742B - 用于哺乳动物唾液腺上皮细胞和唾液腺干细胞培养的无血清培养液 - Google Patents
用于哺乳动物唾液腺上皮细胞和唾液腺干细胞培养的无血清培养液 Download PDFInfo
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- CN102399742B CN102399742B CN201110303107.XA CN201110303107A CN102399742B CN 102399742 B CN102399742 B CN 102399742B CN 201110303107 A CN201110303107 A CN 201110303107A CN 102399742 B CN102399742 B CN 102399742B
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Landscapes
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Abstract
本发明提供了一种用于哺乳动物唾液腺上皮细胞和唾液腺干细胞培养的无血清培养液，该培养液是以MCDB153HAA为基础培养基，并添加氨基酸、维生素、盐类、脂类、微量元素、缓冲液、激素类化合物、转金属蛋白、抗氧化剂、血清白蛋白、糖类、嘌呤、嘧啶碱类物质以及pH值指示剂制成。本发明的无血清细胞培养液不仅能使哺乳动物唾液腺上皮细胞生长旺盛，维持其良好的细胞活性和生理特性，而且也适合唾液腺上皮干细胞的培养，非常适用于生物组织工程相关研究领域，是一种可以商品化的细胞培养液。
Description
技术领域
本发明涉及细胞生物学研究领域，尤其涉及哺乳动物唾液腺上皮细胞和唾液腺干细胞培养技术领域。
背景技术
唾液腺是哺乳动物的重要外分泌腺之一，其分泌物可以帮助消化、保护黏膜及抗菌抑菌。唾液腺的主要功能是产生和分泌唾液，正常情况下，人每天唾液分泌量约1升至1.5升。唾液的25％由腮腺分泌，5％由舌下腺分泌，10％由各种小涎腺(唇腺、颊腺、腭腺等)分泌，60％由下颌下腺分泌。目前已从动物及人的颌下腺发现或分离出近30种生物活性多肽，如神经生长因子(NGF)、表皮生长因子(EGF)、内皮生长刺激因子(EGSF)、红细胞生成素(EPO)、骨髓克隆刺激因子(CSF)、肾素(renin)、激肽释放酶(kallikrein，kk)、生长抑素、胰岛素、淀粉酶、酸性磷酸酶、核糖核酸酶和酯肽酶等。这些多肽物质或直接分泌入血，或随唾液进入消化道再由胃肠吸收入血，对多种组织和细胞的生理活动起重要调节作用。
唾液腺的实质部分由腺泡和导管组成。腺泡是腺体的分泌部，由单层腺上皮细胞围绕而成。导管是排泄分泌物的管道，由导管上皮细胞构成。现有的研究成果显示，唾液腺干细胞很有可能存在于导管部位。因此唾液腺上皮细胞是形成腺体、分泌和排泄唾液的重要功能细胞，唾液腺上皮细胞的功能异常，将会导致多种唾液腺疾病：(1)随着年龄的增长，腺体的腺泡部分萎缩，导致简直纤维性变和脂肪细胞增多，唾液流量减少。这种脂肪细胞增多是腺泡萎缩后的一种替代现象；(2)米古利兹氏病、舍格林氏综合征(口干综合征)。这是唾液腺的自身免疫性疾病，淋巴细胞浸润并取代腺泡；(3)头颈部肿瘤患者在接受放射性治疗后，唾液腺上皮细胞受到损伤，唾液的分泌也会显著减少，影响患者的生存质量；(4)唾液腺上皮细胞发生恶变形成肿瘤，如多形性腺瘤、腺样囊性癌等。肿瘤细胞影响唾液腺的正常机能，但这些疾病的发病机理尚不清楚。因此，明确唾液腺上皮细胞的生物学特性，可以帮助在治疗中避免损伤正常唾液腺，并利用唾液腺干细胞修复唾液腺组织及其功能。
唾液腺上皮细胞是研究唾液腺的重要材料，体外培养唾液腺上皮细胞和唾液腺干细胞是本研究领域的重要环节。以往的唾液腺上皮细胞培养液中添加有血清。血清中的复杂成分掩盖了唾液腺上皮细胞的真实特性，并加速了唾液腺上皮干细胞的分化，细胞无法长期培养和继代。近几年市面上出售了进口的角化上皮细胞无血清培养液，有少数报道提示，这种培养液可以培养唾液腺上皮细胞，并可继代3-4次。2008年Lombaert等人报道了用无血清细胞培养液培养小鼠颌下腺上皮细胞(I.M.Lombaert，J.F.Brunsting，P.K.Wierenga，H.H.Kampinga，G.de Haan and R.P.Coppes，Keratinocyte growth factor preventsradiation damage to salivary glands by expansion of the stem/progenitor pool，StemCells 26(2008)，2595-2601)，他们在DMEM/F12基础培养基中添加了表皮细胞生长因子EGF(20ng/mL)，成纤维细胞生长因子FGF2(20ng/mL)，商品化产品N2(1/100)，胰岛素(10μg/mL)和地塞米松(1μM)。N2是一种商品化产品，其组分不明确，而且价格昂贵。EGF和FGF2也是高价格商品，因此本领域迫切需要一种成本低廉、应用性强、适用于唾液腺上皮细胞和干细胞的无血清细胞培养液。一方面能够保证实验过程中唾液腺上皮细胞的稳定生长，另一方面，能够获得唾液腺干细胞。
发明内容
本发明的目的就是提供一种用于哺乳动物唾液腺上皮细胞和唾液腺干细胞培养的无血清培养液及其配制方法和使用方法。
为实现上述目的，本发明提供了一种无血清培养液，是在基础培养基中添加氨基酸、维生素、盐类、脂类、微量元素、缓冲液、激素类化合物、转金属蛋白、抗氧化剂、血清白蛋白、糖类、嘌呤、嘧啶碱类物质以及pH值指示剂。
基础培养基是MCDB153HAA；或三种培养基MCDB153HAA培养基、高糖DMEM和RPMI1640三者混合的培养基；具体的，可以是在MCDB153HAA基础培养基中，添加1/20(v/v)的高糖DMEM和1/20(v/v)的RPMI1640培养基调配而成，上述该培养基是一种适合上皮细胞生长的培养基。
氨基酸选自丙氨酸、精氨酸、天冬酰胺酸、天冬氨酸、半胱氨酸、胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、羟基脯氨酸中的一种或几种；添加含量优选谷氨酰胺600～900mg/L，精氨酸100～300mg/L，亮氨酸、丝氨酸、半胱氨酸、缬氨酸、脯氨酸或赖氨酸20～100mg/L，组氨酸、天冬酰胺酸、苏氨酸、谷氨酸或甘氨酸含量为10～20mg/L，异亮氨酸、酪氨酸二钠、丙氨酸、甲硫氨酸或苯基丙氨酸的含量为5～10mg/L，天冬氨酸、色氨酸或羟基脯氨酸的含量为0.5～5mg/L；
更优选氨基酸及含量如下：
维生素选自生物素、氯化胆碱、叶酸、肌醇、腐胺、泛酸钙、核黄素、维生素B1、维生素B6、维生素B12、吡多素HCL、盐酸硫胺、烟酰胺、胺基安息香酸中的一种或几种；添加含量优选如下，肌醇或氯化胆碱含量为10～30mg/L，叶酸或腐胺1～5mg/L，胺基安息香酸、泛酸、维生素B1、维生素B6、维生素B-12、烟酰胺或核黄素的含量是0.1～1mg/L，生物素0.01～0.1mg/L；
更优选维生素及含量如下：
盐类选自氯化钙、氯化钠、氯化钾、磷酸二氢钠、磷酸氢二钠、丙酮酸钠、无水醋酸钠、硅酸钠、硝酸钙、中的一种或几种；优选含量如下氯化钠5000～7000mg/L，碳酸氢钠1000～1500mg/L，无水磷酸二氢钠、丙酮酸钠或无水醋酸钠100～400mg/L，硅酸钠0.1～1mg/L，氯化钾200～500mg/L，氯化钙或硝酸钙1～20mg/L；
更优选盐类及含量如下：
脂类选自油酸、亚油酸、氨基乙醇、硫辛酸中的一种或几种；其中优选油酸70～140mg/L，氨基乙醇0.1～1.5mg/L，硫辛酸0.1～1.0mg/L；更优选油酸88～126mg/L，氨基乙醇0.48～1.1mg/L，硫辛酸0.15～0.2mg/L。
微量元素选自硫酸铜、硝酸铁、硫酸亚铁、氯化镁、硫酸镁、硫酸锌、偏钒酸铵、亚硒酸钠、钼酸、氯化镍、氯化锡、硫酸铁、硫酸锰中的一种或几种；优选含量如下，氯化镁90～140mg/L，硫酸锰、硫酸铁1～10mg/L，硫酸锌0.1～1mg/L，硫酸铜、硝酸铁或亚硒酸钠0.001～0.01mg/L，氯化镍、偏钒酸铵、钼酸0.0001～0.0015mg/L，氯化锡0.00001～0.00015mg/L；
更优选微量元素及含量如下：
缓冲液为碳酸氢钠和/或HEPES；优选含量为碳酸氢钠1000～1500mg/L，HEPES 6000～7000mg/L；更优选含量为碳酸氢钠1180～1210mg/L，HEPES6600～6700mg/L。
激素类化合物为胰岛素，优选1～20mg/L，更优选1.6～12.8mg/L；
转金属蛋白为转铁蛋白，优选3～8mg/L，更优选3.6～5.8mg/L；
血清白蛋白为牛血清白蛋白，优选400～700mg/L，更优选450～600mg/L；
糖类为葡萄糖和丙酮酸钠，优选葡萄糖3000～4000mg/L、丙酮酸45～57mg/L；
抗氧化剂为β-巯基乙醇和/或谷胱甘肽，优选β-巯基乙醇0.52～0.88mg/L，谷胱甘肽0.04～0.06mg/L；
嘌呤类为腺嘌呤，优选27～29mg/L；
所述嘧啶碱类物质为胸腺嘧啶，优选0.6～0.75mg/L；
pH值指示剂酚红，优选8～9mg/L。
更优选的，上述培养液的添加组合如下：
上述无血清细胞培养液可以用于哺乳动物唾液腺上皮细胞培养，也可以用于其他正常上皮组织的原代细胞培养，优选乳腺上皮、胰腺上皮，皮肤角化上皮，粘膜上皮。
进一步的，上述培养液中还可以添加12～15mg/L牛脑提取物(BPE)，BPE是一种多种生长因子的混合物，从牛的垂体组中提取，可以有效地促进细胞增殖。上述获得的培养液用于更好的培养富集哺乳动物唾液腺上皮细胞，也可以用于其他正常上皮组织的原代细胞培养，正常上皮组织优选乳腺上皮、胰腺上皮，皮肤角化上皮，粘膜上皮等。
本发明无血清细胞培养液的使用方法是将哺乳动物唾液腺上皮细胞置于前述无血清培养液中培养；或将哺乳动物唾液腺上皮组织的组织块剪碎或消化成单细胞接种到前述的无血清培养液中进行原代培养，在37℃，5％CO2环境下培养48-72h后，即可获得上皮细胞。
或者是使哺乳动物正常上皮组织细胞置于所述的无血清培养液中培养，或将哺乳动物上皮组织的组织块剪碎或消化成单细胞接种到所述的无血清培养液中进行原代培养，在37℃，5％CO2环境下培养48-72h后，即可获得上皮细胞。正常上皮组织包括：乳腺上皮细胞、胰腺上皮，皮肤角化上皮，粘膜上皮等。
可以选择的，上述培养液中不添加BPE，进一步添加人FGF2、肝素钠、L-抗坏血酸-2-磷酸三钠盐和谷丙氨酸二肽，用于培养富集哺乳动物唾液腺干细胞。
其中，将谷丙氨酸二肽应用于唾液腺上皮干细胞的培养时，谷丙氨酸二肽在培养条件下比较稳定，不仅可以持续为干细胞提供能量，而且可以避免因自然代谢而造成的有毒代谢物的积累；添加的L-抗坏血酸-2-磷酸三钠盐是一种比维生素C更稳定的抗氧化剂，它在培养液中可以长时间保持活性，大大延缓唾液腺上皮干细胞衰老的速度，且有助于维持干细胞的特性；进一步添加了肝素钠，以促进细胞内FGF2的信号转导。
上述无血清细胞培养液用于培养唾液腺上皮干细胞，具体的使用方法是将哺乳动物正常唾液腺上皮干细胞置于上述获得的无血清培养液中在37℃，5％CO2环境下培养。
本发明另一方面提供了上述无血清培养液的配制方法，包括以下步骤：
(1)以MCDB153HAA为基础培养基，添加氨基酸、维生素、盐类、脂类、微量元素、缓冲液、激素类化合物、转金属蛋白、抗氧化剂、血清白蛋白、糖类、嘌呤、嘧啶碱类物质以及pH值指示剂；
(2)加入12～15mg/L的牛脑提取物(BPE)在步骤(1)所得到的培养液中；或分别添加9.5～12.5mg/L的L-抗坏血酸-2-磷酸三钠盐，0.3～1mg/L的肝素钠，5～20μg/L的FGF2和400-460mg/L的谷丙氨酸二肽至步骤(1)所得到的培养液中。
具体的，可以是直接选用MCDB153HAA为基础培养基，或是在MCDB153HAA基础培养基中，添加1/20(v/v)的高糖DMEM和1/20(v/v)的RPMI1640培养基的成分调配而成的。该培养基是一种适合上皮细胞生长的培养基。
接下来，在基础培养基中添加氨基酸、维生素、盐类、脂类、微量元素、缓冲液、血清白蛋白制备基础培养液，将酸碱度调至7.0～7.2，经过滤灭菌处理；再添加转铁蛋白、胰岛素、氨基乙醇、亚硒酸钠、β-巯基乙醇得到进一步的培养液；
可以向培养液中加入12～15mg/L的牛脑提取物(BPE)；或分别添加9.5～12.5mg/L的L-抗坏血酸-2-磷酸三钠盐，0.3～1mg/L的肝素钠，5～20μg/L的FGF2和400-460mg/L的谷丙氨酸二肽至上述得到的培养液中。
其中，上述组分溶解于无热源超纯水中(电阻率为18.2Ω)配置而成。
本发明的无血清培养液可以使培养的细胞保持良好的增殖能力和生物学特性。本发明的无血清培养液的积极效果是：
(1)不含血清，实验体系清晰、成份确定，有利于实验结果的分析，避免了不可知成份所造成的实验背景不清对实验结果所带来的影响；
(2)本发明的无血清培养液中如添加BPE，可极大地促进上皮细胞的增殖和细胞成活率，为实验提供充足的细胞数量；
(3)适用范围广，适用于多种类型的哺乳动物上皮细胞的培养，如乳腺上皮细胞、胰腺上皮细胞和角化上皮细胞等；
(4)适用于唾液腺干细胞培养，在3-D Matrigel培养体系中可以分化成为唾液腺导管或腺泡。
本发明的无血清培养液成分明确，添加物成分简单易配制，成本更低廉，应用范围更广范。本发明用上述成分代替传统有血清培养液，避免了血清组分对实验研究的影响。与现有方法相比，本发明的无血清培养液可以有效地抑制原代细胞培养过程中成纤维细胞的生长，因而可以得到纯度较高的上皮细胞。
附图说明
图1所示为根据本发明实施例2制备的无血清细胞培养液培养人原代颌下腺导管上皮细胞图。
图2所示为根据本发明实施例2制备的无血清细胞培养液培养人原代颌下腺腺泡上皮细胞图。
图3所示为根据本发明实施例2制备的无血清细胞培养液培养人原代腮腺上皮细胞图。
图4所示为根据本发明实施例2制备的无血清细胞培养液培养人原代舌下腺上皮细胞图。
图5所示为根据本发明实施例2制备的无血清细胞培养液培养大鼠颌下腺上皮细胞图。
图6所示为根据本发明实施例2制备的无血清细胞培养液培养小鼠原代胰腺上皮细胞图。
图7所示为根据本发明实施例2制备的无血清细胞培养液培养小鼠原代颌下腺上皮细胞图。
图8所示为根据本发明实施例2制备的无血清细胞培养液培养小鼠颌下腺上皮细胞系第42代细胞图。
图9所示为本发明不含BPE的无血清细胞培养液培养人颌下腺导管干细胞图。
图10(a)-10(d)所示为根据本发明制备的无血清细胞培养液三维立体培养人颌下腺导管干细胞图。
图11所示为根据本发明实施例2制备的无血清细胞培养液培养小鼠颌下腺上皮细胞系第42代细胞的核型分析。
图12(a)和12(b)所示为本发明不含BPE的无血清细胞培养液培养小鼠颌下腺上皮细胞系第42代对表皮细胞生长因子EGF和成纤维细胞生长因子FGF1的反应性。
图13所示为本发明不含BPE的无血清细胞培养液培养小鼠颌下腺上皮细胞对表皮细胞生长因子EGF、成纤维细胞生长因子FGF1和牛脑提取物BPE的反应性。
具体实施方式
下面结合具体实施例进一步阐述本发明，应理解，这些实施例仅用于说明本发明而不用于限制本发明的应用范围。
本发明用于哺乳动物唾液腺上皮细胞培养的无血清培养液，是在基础培养基MCDB153HAA中添加如下成分：氨基酸、维生素、盐类、脂类、微量元素、激素、缓冲剂及葡萄糖、非必须氨基酸、胸苷、次黄嘌呤、酚红、β-巯基乙醇、转铁蛋白、牛血清白蛋白，其各组分的含量见如下实施例1～6所示，将各实施例中组分溶解于无热源超纯水中(电阻率为18.2Ω)配置而成。
实施例1
实施例2
实施例3
实施例4
与实施例1相比，该实施例中不添加BPE，另外添加9.5mg/L的L-抗坏血酸-2-磷酸三钠盐，0.3mg/L的肝素钠，5μg/L的FGF2和400mg/L的谷丙氨酸二肽，其他添加成分及含量与实施例1相同。
实施例5
与实施例2相比，该实施例中不添加BPE，另外添加11.5mg/L的L-抗坏血酸-2-磷酸三钠盐，0.6mg/L的肝素钠，15μg/L的FGF2和420mg/L的谷丙氨酸二肽，其他添加成分及含量与实施例2相同。
实施例6
与实施例3相比，该实施例中不添加BPE，另外添加12.5mg/L的L-抗坏血酸-2-磷酸三钠盐，1mg/L的肝素钠，20μg/L的FGF2和460mg/L的谷丙氨酸二肽，其他添加成分及含量与实施例3相同。
实施例7
如图9所示，为根据本发明实施例5制备的无血清细胞培养液培养人颌下腺导管上皮干细胞图。在该实施例中，为培养5-7天获得的唾液腺上皮干细胞。
实施例8
应用三维立体培养的方法，观察导管形成情况。培养10天后，利用免疫荧光染色的分析方法，检测实施例7的唾液腺干细胞中α-amylase的表达。如图10(a)-10(d)所示，在Matrigel中干细胞形成导管状，蓝色荧光是细胞核染料DAPI，绿色荧光是α-amylase(唾液腺上皮细胞特异性分子标志物)抗体染色。
实施例9
利用核型分析方法，检测小鼠颌下腺上皮细胞系。如图11所示，为本发明无血清细胞培养液培养小鼠颌下腺上皮细胞系第42代细胞的核型分析，结果显示核型正常，为二倍体，表明经过长期培养和连续传代后，细胞仍保持健康的状态。
实施例10
图12所示为本发明不含BPE的无血清细胞培养液培养小鼠颌下腺上皮细胞系第42代对表皮细胞生长因子EGF和成纤维细胞生长因子FGF1的反应性。将处于对数生长期的细胞播种至24孔板，每孔10000个细胞；24小时后分别加入不同浓度的EGF或FGF1。如图12(a)和(b)所示，生长因子添加5天后计数。结果表明，第42代细胞仍然对两种生长因子有明显反应，与第7代细胞没有明显差异。圆形标记代表第7代细胞，三角形标记代表第42代细胞。
图13所示为本发明不含BPE的无血清细胞培养液培养小鼠颌下腺上皮细胞对表皮细胞生长因子EGF、成纤维细胞生长因子FGF1和牛脑提取物BPE的反应性。白色圆形标记代表同时添加EGF(1nM)和FGF(100pM)，正方形标记代表只添加FGF(100pM)，三角形标记代表只添加EGF(1nM)，黑色圆形标记代表添加BPE(15mg/mL)。如图13所示，一定量的生长因子添加后，每天计算细胞数量，共计6天。结果表明，在培养液中添加BPE可以显著促进细胞增殖。
Claims (2)
1.用于哺乳动物唾液腺上皮细胞培养的无血清培养液，其特征在于，是在基础培养基中添加氨基酸、维生素、盐类、脂类、微量元素、缓冲液、激素类化合物、转金属蛋白、抗氧化剂、血清白蛋白、糖类、嘌呤、嘧啶碱类物质以及pH值指示剂；
其中，所述基础培养基是MCDB153HAA培养基；所添加的组分及添加量如下
所述培养液中还添加12～15mg/L牛脑提取物。
2.一种如权利要求1所述的无血清培养液的配制方法，其特征在于包括以下步骤
（1）以MCDB153HAA为基础培养基，添加氨基酸、维生素、盐类、脂类、微量元素、缓冲液、激素类化合物、转金属蛋白、抗氧化剂、血清白蛋白、糖类、嘌呤、嘧啶碱类物质以及pH值指示剂；
（2）加入12～15mg/L的牛脑提取物在步骤（1）所得到的培养液中。
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| CN103060264B (zh) * | 2012-12-20 | 2015-01-21 | 上海市第十人民医院 | 一种干细胞培养基及其应用和干细胞培养方法 |
| US9321995B2 (en) | 2012-12-20 | 2016-04-26 | Suzhou Biowisetech Co., Ltd. | Stem cell culture medium and its applications as well as a stem cell culture method |
| CN105647850A (zh) * | 2016-03-01 | 2016-06-08 | 赛百慷（上海）生物技术股份有限公司 | 一种上皮培养体系 |
| CN105543174A (zh) * | 2016-03-01 | 2016-05-04 | 赛百慷（上海）生物技术股份有限公司 | 一种肿瘤细胞生长促进添加剂及其使用方法 |
| CN106591219A (zh) * | 2016-12-22 | 2017-04-26 | 叶宗耀 | 一种表皮细胞培养基 |
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| CN109749997B (zh) * | 2018-05-11 | 2020-03-17 | 中山大学中山眼科中心 | 一种角膜缘干细胞无血清培养基及其培养方法 |
| WO2020203850A1 (ja) * | 2019-03-29 | 2020-10-08 | 国立大学法人長崎大学 | 培養組織及びその製造方法 |
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