CN106190962B - 一种猪骨骼肌卫星细胞的培养基pm+及其应用 - Google Patents
一种猪骨骼肌卫星细胞的培养基pm+及其应用 Download PDFInfo
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Abstract
本发明属于动物细胞培养技术领域，具体涉及一种猪骨骼肌卫星细胞的培养PM+及其应用。所述的猪骨骼肌卫星细胞培养的培养基PM+，按重量/体积计的成分如下所述：培养基PM+总体积为1L，添加胎牛血清(FBS)20％；细胞培养添加剂(GlutaMax)1％；非必需氨基酸(NEAA)1％；抗生素‑抗真菌剂(AA)1％；鸡胚培养物0.5％；庆大霉素50mg/L；碱性成纤维细胞生长因子终浓度为25μL/L，用RPMI 1640液体基础培养基补足至培养基PM+总体积的全量。本发明还公开了该培养基的应用。
Description
技术领域
本发明属于动物细胞培养技术领域，具体涉及一种猪骨骼肌卫星细胞的培养基PM+及其应用。
背景技术
猪肉是我国主要消费肉类之一，猪骨骼肌的生长发育研究，直接与猪肉的经济效益相关。研究结果表明，猪骨骼肌的生长发育与其卫星细胞(porcine satellite cells,简称PSC)的功能密切相关。PSC属于骨骼肌干细胞，通常以静息状态存在于基膜和肌细胞膜之间，在骨骼肌受刺激后会从静息状态激活、增殖、分化，并融合形成肌管，引起肌纤维肥大，进而引起肌纤维类型转化[1]。近年来，体外分离、培养PSC已有较多研究，同时PSC也成为研究肌肉生长和发育机制的重要细胞模型。
由于PSC的成肌分化能力强，对培养条件要求苛刻，不稳定的培养条件会促进PSC分化、融合形成形成肌管，因此体外培养PSC难度较大。目前并没有统一的PSC培养基，大部分PSC培养基培养情况并不理想，细胞增殖速度较慢，且难以保持其骨骼肌干细胞的特性，无法为进一步的实验提供实验材料。因此有必要研制一种配制便利，能够保持PSC的肌肉干细胞特性，同时使细胞拥有较强自我更新能力的猪骨骼肌卫星细胞培养新方案。
发明内容
本发明的目的在于克服现有技术的缺陷，提供一种猪骨骼肌卫星细胞的培养基PM+及其应用，该培养基既能保持被培养的骨骼肌干细胞的特性，又能实现良好骨骼肌干细胞的自我更新能力，得到一种专用型猪骨骼肌卫星细胞的培养基。
本发明是专门针对猪骨骼肌卫星细胞培养所设计的一种专用培养基，该专用培养基以RPMI 1640液体培养基为基础培养基，通过添加适量的胎牛血清(FBS)、鸡胚提取物(CEE)、非必需氨基酸(MEM-NEAA)、细胞培养添加剂GlutaMax、碱性成纤维细胞生长因子(bFGF)、抗生素和抗真菌剂(Antibiotic-Antimycotic,AA)以及庆大霉素(Gentamycin)等成分得到一种性能更加优良的适合猪骨骼肌卫星细胞培养的专用培养基。
根据文献报道，部分已有的培养基培养的骨骼肌卫星细胞在4-6天达到生长高峰，7天左右能够长满培养板[2、3]，本发明的PM+培养基能够促进卫星细胞增殖速度，使用PM+培养基在一个直径为100mm培养皿中培养的PSC，2-3天即能增长至60-70％，且细胞传代后仍会保持相应的增殖速度。培养的PSC在低浓度马血清诱导2天后会出现明显的肌管，表现出了较强的分化成肌能力，可以最大限度保持PSC的肌肉干细胞特性，同时拥有较强的自我更新能力。
本发明的技术方案如下所述：
一种适用于猪骨骼肌卫星细胞培养的培养基PM+，按重量/体积计的成分如下所述：
以培养基PM+总体积为1L为基准计：
胎牛血清(FBS)20％；细胞培养添加剂(GlutaMax)1％；非必需氨基酸(NEAA)1％；抗生素-抗真菌剂(AA)1％；鸡胚培养物0.5％；庆大霉素50mg/L；碱性成纤维细胞生长因子终浓度为25μL/L；用RPMI 1640液体基础培养基补足培养基PM+至总体积的全量(即，1L)。
该培养基按照下列步骤制备：
先用无核酸酶水溶解鸡胚培养物，然后将碱性成纤维细胞生长因子、庆大霉素分别预配成储藏液；按重量/体积比加入胎牛血清，细胞添加剂GlutaMax，非必需氨基酸，抗生素-抗真菌剂，以及鸡胚培养物，碱性成纤维细胞生长因子和庆大霉素储藏液，最后用RPMI1640液体基础培养基补足至培养基PM+的总体积的全量，然后用0.22μm微孔滤膜过滤除菌，保存在4℃下，备用。
本发明将RPMI 1640液体培养基和胎牛血清作为培养基的基础成分。
其中，FBS含有细胞生长所必须的生长因子，且高浓度的优质血清能够提高细胞的增殖能力[4]。
细胞培养添加剂GlutaMax含有L-丙氨酰-L-谷氨酰胺二肽，在溶液中更稳定，可替代细胞培养基中的L-谷氨酰胺，是细胞进行能量生成及蛋白质、核酸合成所必需的营养素。
非必需氨基酸，为细胞生长提供非必需氨基酸。
抗生素-抗真菌剂和庆大霉素为抗生素，可以抑制培养过程中可能出现的细菌和真菌污染。
碱性成纤维细胞生长因子和鸡胚提取物作为培养基中的特殊生长因子，可以促进细胞增殖，抑制细胞分化，是培养基中的必须添加物[5、6、7]。
附图说明
图1：应用本发明制备的PM+培养基培养猪骨骼肌卫星细胞增殖期结果(100X)。附图标记说明：细胞形态正常饱满，增殖状态良好。
图2：应用本发明制备的PM+培养基培养的猪骨骼肌卫星细胞诱导分化2天后的效果图(100X)。附图标记说明，诱导分化2d后猪骨骼肌卫星细胞分化能力强，有明显肌管生成。
图3：使用Myosin和Hoechst33342对猪骨骼肌卫星细胞诱导分化2d的肌管的免疫荧光染色结果图(100X)。附图标记说明，图3中的A图是猪骨骼肌卫星细胞诱导分化2天的肌管结果图；图3中的B图是Myosin免疫荧光染色结果图；图3中的C图是Hoechst33342免疫荧光染色结果图；图3中的D图是Myosin和Hoechst33342免疫荧光染色Merge结果图。
图4：不同培养条件下卫星细胞的生长曲线图。附图标记说明，使用本发明制备的PM+培养基培养的卫星细胞增殖速度更快。
具体实施方案
实施例1
培养基PM+成分及其配制：
1、庆大霉素、碱性成纤维生长因子、鸡胚提取物储藏液的配制：
(1)庆大霉素溶液的配制：准确称取1g庆大霉素粉末溶于20mL无核酸酶水中，配制成50mg/mL的庆大霉素溶液，分装后于-20℃下保存备用；
(2)碱性成纤维生长因子溶液的配制：准确称取10μg碱性成纤维生长因子粉末溶于100μL无核酸酶水中，配制成100ng/μL的bFGF溶液，即为碱性成纤维生长因子储藏液；分装后于-20℃下保存备用；
(3)鸡胚提取物溶液的配制：向鸡胚提取物粉末中加入10mL的无核酸酶水，充分溶解得到储藏液，分装后于-20℃下保存备用；
2、抗生素-抗真菌剂、胎牛血清分装后于-20℃下保存备用。
3、培养基PM+配制：在1L RPMI 1640液体培养基中添加下列成分：200mL胎牛血清；5mL鸡胚提取物储液；10mL非必需氨基酸；10mL细胞培养添加剂GlutaMax；10mL抗生素-抗真菌剂；1mL庆大霉素储藏液，25μL碱性成纤维生长因子储藏液；培养基配齐后用0.22μm微孔滤膜过滤除菌，在4℃下保存备用。
本发明培养培养猪骨骼肌卫星细胞(PSC)的效果检测：
增殖培养效果鉴定：
使用重悬分离得到的PSC，纯化后接种于预先包被的100mm培养皿中；每24h更换新鲜培养基一次，观察细胞生长状况，得到如图1所示的结果。
卫星细胞分化效果鉴定：将传代培养的卫星细胞接种于6孔板中，培养1-2d；待细胞长至60％-70％，去除PM+培养基，加入杜氏磷酸缓冲液(DPBS)清洗一遍细胞，更换含5％马血清的分化培养基(马血清(HS)5％；抗生素-抗真菌剂(AA)1％；庆大霉素50mg/L以及常用的DMEM基础培养基)；每24h换一次新鲜培养基液，观察并拍照，记录细胞的形态变化(结果见图2)。
附录
培养基中成分来源公司、货号及原产国：
(1)无核酸酶水(Nuclease-Free Water)(cat#AM9932,Ambion,USA)；
(2)鸡胚提取物(CEE)(cat#100-163P,GEMINI,USA)；
(3)RPMI 1640液体培养基(cat#A10491-01,Life,USA)；
(4)抗生素-抗真菌剂(Antibiotic-Antimycotic,AA)(cat#15240-096,Life,USA)；
(5)庆大霉素(Gentamycin)(cat#15750-060,Life,USA)；
(6)碱性成纤维细胞生长因子(bFGF)(cat#13256-029,Life,USA)；
(7)胎牛血清(FBS)(cat#10099-141,Gbico,USA)；
(8)非必需氨基酸(MEM-NEAA)(cat#11140-050,Gbico,USA)；
(9)细胞培养添加剂GlutaMax(cat#35050-061,Gbico,USA)；
(10)杜氏磷酸缓冲液(DPBS)(cat#14190-25,Gbico,USA)；
(11)DMEM基础培养基(cat#10569-010,Life,USA)。
参考文献
1.Rhoads,et al.Extrinsic regulation of domestic animal-derivedmyogenic satellite cells II.Domest.Anim.Endocrinol,2009,36:111-126.
2.罗桂芬等,猪肌卫星细胞的分离培养及鉴定[J].细胞与分子免疫学杂志,2006,22(06):823-825.
3.吕晓等,猪肌肉卫星细胞的分离培养及生物学特性鉴定[J].中国兽医学报,2011,31(10):1480-1484.
4.Doumit M,et al.,Conditions for isolation and culture of porcineembryogenic satellite cells.Tissue Cell,1992,24(2)：253～262.
5.FRIESEL R,et al.,Fibroblast growth factor prototype release andfibroblast growth factor signaling[J].Thromb Haemostas,1999,82(2):748-754.
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Claims (2)
1.一种猪骨骼肌卫星细胞的培养基PM+，其特征在于，按照以下比例配制：
以培养基PM+总体积1L计：
胎牛血清20v/v％；美国Gbico公司出品的货号为cat#35050-061的细胞培养添加剂1v/v％；非必需氨基酸1v/v％；抗生素-抗真菌剂1v/v％；美国GEMINI公司出品的货号为cat#100-163P的鸡胚提取物0.5v/v％；庆大霉素50mg/L，100ng/L的碱性成纤维细胞生长因子25μL，用RPMI 1640液体基础培养基补足至全量。
2.权利要求1所述的猪骨骼肌卫星细胞的培养基PM+在培养猪骨骼肌卫星细胞中的应用。
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| 猪肌肉卫星细胞的分离培养及生物学特性鉴定;吕晓等;《中国兽医学报》;20111031;第31卷(第10期);1480-1484 * |
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