CN112094844A - miRNA激动剂及应用、人源间充质干细胞培养基及培养方法 - Google Patents

miRNA激动剂及应用、人源间充质干细胞培养基及培养方法 Download PDF

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CN112094844A
CN112094844A CN202011023824.2A CN202011023824A CN112094844A CN 112094844 A CN112094844 A CN 112094844A CN 202011023824 A CN202011023824 A CN 202011023824A CN 112094844 A CN112094844 A CN 112094844A
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魏亮
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Chengdu S&km Biotechnology Co ltd
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Abstract

本发明公开一种miRNA激动剂及应用、人源间充质干细胞培养基及培养方法，所述miRNA激动剂由miRNA‑378 agomir和miRNA‑126 agomir组成；所述人源间充质干细胞培养基包括：所述miRNA激动剂；所述miRNA激动剂、人源间充质干细胞培养基及培养方法可有效提高原代脂肪间充质干细胞的扩增速度，并可稳定传代至15代以上，保持其分化潜能，满足间充质干细胞在科学研究与应用上的需求。

Description

miRNA激动剂及应用、人源间充质干细胞培养基及培养方法
技术领域
本发明涉及生物工程技术领域，具体涉及一种miRNA激动剂及应用、人源间充质干细胞培养基及培养方法。
背景技术
干细胞(stem cells)不同于成熟细胞首先是因为它能够在长时间内保持自我更新和扩增的能力，其次干细胞能够向多种细胞系分化。干细胞的这些特点使其成为良好的种子细胞来源。其中，间充质干细胞(mesenchymal stem cells,MSC)是干细胞家族的重要成员，来源于发育早期的中胚层和外胚层。间充质干细胞最初在骨髓中发现，因其具有多向分化潜能、造血支持和促进干细胞植入、免疫调控和自我复制等特点而日益受到人们的关注。间充质干细胞在体内或体外特定的诱导条件下，可分化为脂肪、骨、软骨、肌肉、肌腱、韧带、神经、肝、心肌、内皮等多种组织细胞，连续传代培养和冷冻保存后仍具有多向分化潜能，可作为理想的种子细胞用于衰老和病变引起的组织器官损伤修复。
脂肪间充质干细胞(ADSC)是存在于脂肪组织中的干细胞，具有多项分化潜能，来源充足，且含量更高(100g的脂肪组织中MSCs数量约是100mL骨髓中的300倍)，可以分泌更多的细胞因子，在代谢调节方面有其他来源细胞无法比拟的优势，因此更能满足未来组织工程学的相关应用。
目前，人脂肪肝细胞的体外分离培养常用方法是采用机械法去除血管、胰蛋白酶加胶原酶消化法分离，但是此种方法分离效率低，脂肪干细胞无法充分释放，分离周期长，脂肪组织需要量大，且大部分方法不可避免的需要使用胎牛血清，容易引起外源性污染，细胞形态变异等，特别是动物血清内潜在动物源性内毒素或病毒将对人体健康构成极大的风险，这样培养出的干细胞不适于直接应用于临床。干细胞无血清培养基的出现，为解决这些问题提供了希望，但是现有市售无血清培养基存在细胞增殖不够理想的缺陷，不能满足大量培养需求。
发明内容
有鉴于此，本申请提供一种miRNA激动剂及应用、人源间充质干细胞培养基及培养方法,可有效提高原代脂肪间充质干细胞的扩增速度，并可稳定传代至15代以上，保持其分化潜能，满足间充质干细胞在科学研究与应用上的需求。
为解决以上技术问题，本申请提供的技术方案是一种miRNA激动剂，所述miRNA激动剂由miRNA-378 agomir和miRNA-126 agomir组成。
优选的，所述miRNA-78 agomir为双链结构，含正义链和反义链；其中，所述正义链的核苷酸序列如SEQ ID NO：1所示，所述反义链序列的核苷酸序列如SEQ ID NO：2所示；
所述miRNA-126 agomir为双链结构，含正义链和反义链；其中，所述正义链的核苷酸序列如SEQ ID NO：3所示，所述反义链序列的核苷酸序列如SEQ ID NO：4所示。
优选的，所述miRNA-378 agomir和所述miRNA-126 agomir经化学修饰，所述的化学修饰包括：反义链3’端进行胆固醇修饰，5’端进行两个硫代骨架修饰，3’端进行四个硫代骨架修饰，全链进行甲氧基修饰。
本发明还提供了上述miRNA激动剂在培养间充质干细胞中的应用或在制备培养间充质干细胞的产品中的应用。
优选的，所述产品为间充质干细胞培养基。
本发明还提供了一种人源间充质干细胞培养基，包括：上述miRNA激动剂。
优选的，所述培养基为无血清培养基。
优选的，所述人源间充质干细胞选自人脂肪间充质干细胞、人脐带间充质干细胞和骨髓间充质干细胞中任意一种。
优选的，所述人源间充质干细胞为人脂肪间充质干细胞。
优选的，所述miRNA-378 agomir的浓度为10～130nM和所述miRNA-126 agomir的浓度为10～130nM。
优选的，所述miRNA-378 agomir的浓度为30～110nM和所述miRNA-126 agomir的浓度为30～110nM。
优选的，所述miRNA-378 agomir的浓度为50～90nM和所述miRNA-126 agomir的浓度为50～90nM。
优选的，所述miRNA-378 agomir的浓度为50nM和所述miRNA-126 agomir的浓度为50nM。
优选的，所述培养基还包括：氨基酸、生长因子、抗生素。
优选的，所述氨基酸为胱氨酸、半胱氨酸、天冬酰胺酸、天门冬氨酸、丙氨酸、苯丙氨酸、精氨酸、亮氨酸、异亮氨酸、赖氨酸、蛋氨酸、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸。
优选的，所述氨基酸为胱氨酸100mg/L、半胱氨酸400mg/L、天冬酰胺酸30mg/L、天门冬氨酸50mg/L、丙氨酸40mg/L、苯丙氨酸60mg/L、精氨酸80mg/L、亮氨酸150mg/L、异亮氨酸200mg/L、赖氨酸300mg/L、蛋氨酸50mg/L、谷氨酸70mg/L、谷氨酰胺1200mg/L、甘氨酸30mg/L、脯氨酸40mg/L、丝氨酸50mg/L、苏氨酸100mg/L、色氨酸20mg/L、酪氨酸100mg/L、缬氨酸50mg/L。
优选的，所述生长因子为干细胞生长因子、成纤维细胞生长因子、血小板衍生生长因子、表皮生长因子、胰岛素样生长因子、转化生长因子、内皮细胞生长因子。
优选的，所述生长因子为干细胞生长因子15mg/L、成纤维细胞生长因子15mg/L、血小板衍生生长因子15mg/L、表皮生长因子15mg/L、胰岛素样生长因子15mg/L、转化生长因子15mg/L、内皮细胞生长因子15mg/L。
优选的，所述抗生素为青霉素、链霉素。
优选的，所述抗生素为青霉素80U/mL、链霉素80ug/mL。
优选的，所述培养基还包括：葡萄糖、亚硒酸钠、黄体酮、人胰岛素、转铁蛋白、胎球蛋白、纤连蛋白、L-抗坏血酸、非必须氨基酸、维生素C、维生素E、氯化钠、氯化铁、烟酰胺。
优选的，所述培养基还包括：葡萄糖6g/L、亚硒酸钠15ug/L、黄体酮7ug/L、人胰岛素6mg/L、转铁蛋白6mg/L、胎球蛋白1g/L、纤连蛋白55mg/L、L-抗坏血酸40mg/L、非必须氨基酸3mM、维生素C7mg/L、维生素E5mg/L、氯化钠2g/L、氯化铁2g/L、烟酰胺0.01g/L。
优选的，非必须氨基酸包括：甘氨酸、丙氨酸、脯氨酸、酪氨酸、丝氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸。
优选的，所述培养基由所述miRNA激动剂、所述氨基酸、所述生长因子、所述抗生素、葡萄糖、亚硒酸钠、黄体酮、人胰岛素、转铁蛋白、胎球蛋白、纤连蛋白、L-抗坏血酸、非必须氨基酸、维生素C、维生素E、氯化钠、氯化铁、烟酰胺组成。
本发明还提供了一种间充质干细胞的培养方法，包括：采用上述间充质上述人源间充质干细胞培养基进行培养。
优选的，所述间充质干细胞的培养方法包括：间充质干细胞分离、间充质干细胞原代培养和间充质干细胞传代培养。
优选的，所述间充质干细胞原代培养和间充质干细胞传代培养均采用所述人源间充质干细胞培养基进行培养
本申请与现有技术相比，其详细说明如下：
现有文献研究表明：miRNA-378可增强兔脐带间充质细胞的增殖活性，在黑色素瘤中，miRNA-378可通过Wnt信号通路抑制FOXN3的表达，来促进肿瘤细胞的增殖、迁移和侵袭性。
此外，miRNA-126可有效抑制肺动脉内皮细胞的凋亡和增强细胞增殖活力。
本发明提供了由miRNA-378 agomir和miRNA-126 agomir组成的miRNA激动剂，本发明中，申请人发现在培养基中添加miRNA-378 agomir和miRNA-126 agomir组成物，可有效提高脂肪间充质干细胞的增殖活性。本发明特异性的microRNA激动剂针对间充质干细胞培养过程中的microRNA表达，不需要转染试剂和载体，直接进入细胞，从而安全高效的调控细胞表达，上调miRNA-378和miRNA-126，从而可促进细胞增殖和活化，本发明miRNA激动剂可应用于培养间充质干细胞中的应用或在制备培养间充质干细胞的产品。
本发明提供的miRNA激动剂及间充质干细胞培养基，可有效提高原代脂肪间充质干细胞的扩增速度，并可稳定传代至15代以上，保持其分化潜能，满足间充质干细胞在科学研究与应用上的需求。
本发明人源脂肪间充质干细胞培养基，人离体脂肪细胞易于获得，便于自体移植、给患者带来的痛苦较小，分化后诱导所得的成脂细胞具有足够存货量和存活期，为糖尿病、肥胖等代谢性疾病细胞治疗提供理想的种子细胞，为糖尿病细胞移植治疗将提供新途径，具有广阔的临床应用前景。骨髓来源的间充质干细胞存在以下问题：随着年龄的老化，干细胞数目显著降低、增殖分化能力大幅度衰退；制备过程不容易质控；移植给异体可能引起免疫反应；取材时对患者有损伤，患者有骨髓疾病时不能采集，即使是健康供者，亦不能抽取太多的骨髓。本发明培养得到的脂肪间充质干细胞可替代骨髓间充质干细胞作为间充质干细胞来源。
本发明人源间充质干细胞培养基，为无血清，避免对细胞的具有潜在毒性作用和血清源性污染。
本发明提供了快捷、高效分离人源脂肪间充质干细胞的方法，本发明能够在较短时间得到脂肪充质干细胞，而且对细胞的损伤更小；本发明的方法能发明能够利用小量的脂肪组织获得大量的状态良好、保持很好的多向分化能力的脂肪干细胞，并且操作方法简单易行、可重复性强。最大可能的保护间充质干细胞，具有更高活率及活性。
附图说明
图1为效果例1中采用培养基3、商用培养基A和商用培养基B培养过程原代培养生长曲线图；
图2为效果例1中采用培养基1-3培养过程原代培养生长曲线图；
图3为效果例1中采用培养基3-9人源脂肪间充质干细胞增殖结果数据图；
图4为效果例2中本发明分充质干细胞细胞表面标志物的CD13表达情况图；
图5为效果例2中本发明分充质干细胞细胞表面标志物的CD34表达情况图；
图6为效果例2中本发明分充质干细胞细胞表面标志物的CD105表达情况图。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实施例对本发明作进一步的详细说明。
实施例1
人脂肪间充质干细胞的无血清培养基
本实施例人脂肪间充质干细胞的无血清培养基由表1组分如下：
表1
Figure BDA0002701531010000061
Figure BDA0002701531010000071
Figure BDA0002701531010000081
培养基4-9：和培养基3的区别仅在于，miRNA-378 agomir和miRNA-126 agomir浓度不同，培养基4-9按miRNA-378 agomir和miRNA-126 agomir均为10、30、70、90、110、130nM的浓度配置培养基。
表1中，非必须氨基酸包括：甘氨酸、丙氨酸、脯氨酸、酪氨酸、丝氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸。
培养基1中miRNA激动剂为miRNA-378 agomir，培养基2中miRNA激动剂为miRNA-126 agomir，培养基3中miRNA激动剂由miRNA-378 agomir和miRNA-126 agomir组成。
所述miRNA-378 agomir(miRNA-378激动剂)为双链结构，含正义链和反义链；其中，所述正义链的核苷酸序列为5’-CUCCUGACUCCAGGUCCUGUGU-3’，如SEQ ID NO：1所示，所述反义链序列的核苷酸序列为5’-ACACAGGACCUGGAGUCAGGAG-3’，如SEQ ID NO：2所示。所述miRNA-378 agomir经化学修饰，包括：反义链3’端进行胆固醇修饰，5’端进行两个硫代骨架修饰，3’端进行四个硫代骨架修饰，全链进行甲氧基修饰。
所述miRNA-126 agomir(miRNA-126激动剂)为双链结构，含正义链和反义链；其中，所述正义链的核苷酸序列为5’-UCGUACCGUGAGUAAUAAUGCG-3’，如SEQ ID NO：3所示，所述反义链序列的核苷酸序列为5’-CGCAUUAUUACUCACGGUACGA-3’，如SEQ ID NO：4所示。所述miRNA-126 agomir经化学修饰，包括：反义链3’端进行胆固醇修饰，5’端进行两个硫代骨架修饰，3’端进行四个硫代骨架修饰，全链进行甲氧基修饰。
实施例2
本实施例材料人间充质干细胞培养基：为实施例1中人间充质干细胞培养基(培养基3)。
人脂肪间充质干细胞培养方法：
1、人脂肪间充质干细胞分离和原代培养：
(1)采取人脂肪组织20mg，送入细胞操作室。
用4℃含2wt％—5wt％双抗的PBS缓冲液(灭菌，含青霉素200-500U/mL，链霉素200-500ug/mL)清洗人脂肪组织三次以上。
(3)用灭菌过的手术剪将人脂肪组织剪碎至糊状，转移至离心管。
(4)加入2-5倍体积的0.1wt％(1mg/ml)的I型胶原酶(用0.22微米滤器过滤过)，封口膜封好，37℃恒温水浴锅振荡消化1h，振荡频率约100r/min，注意水浴锅液面没过人脂肪组织。
(5)1500rpm/min离心10min，弃上清，加入人间充质干细胞培养基，重悬细胞及组织。
(6)用70目细胞筛过滤，收集过滤液加入人间充质干细胞培养基，1800rpm/min离心5min，得到细胞悬液。
(7)调整细胞悬液重悬细胞浓度为1*104/ml，接种于培养瓶内，加入人间充质干细胞培养基，37℃，5％CO2细胞培养箱内进行原代培养。
(8)原代培养72h后首次进行观察(贴壁细胞数量)并半换液，以后每48h进行半换液一次，直至细胞融合度达到50％以上后每48h进行全换液一次。
2、人脂肪间充质干细胞传代培养
(1)当细胞融合度85％-90％融合后进行传代培养，0.25wt％胰蛋白酶消化1min，吹打培养瓶瓶底，使细胞脱离瓶壁后呈单细胞悬液，1800rpm/min离心5min，重悬细胞后，传代分3-4瓶。
(2)重复上述步骤(1)至传代到第15代。
对照例1
人脂肪间充质干细胞培养方法
本对照例和实施例2的区别仅在于：
对照例1采用商用培养基A替代实施例2中的人间充质干细胞培养基，商用培养基A为Solarbio间充质干细胞无血清培养基(货号N6010)。
对照例2
人脂肪间充质干细胞培养方法
本对照例和实施例2的区别仅在于：
对照例2采用商用培养基B替代实施例2中的人间充质干细胞培养基，商用培养基B为友康生物脂肪干细胞无血清培养基(货号NC0103+NC0104.S)。
对照例3
人脂肪间充质干细胞培养方法
本对照例和实施例2的区别仅在于：
本对照例材料人间充质干细胞培养基：为实施例1中人间充质干细胞培养基(培养基1)。
对照例4
人脂肪间充质干细胞培养方法
本对照例和实施例2的区别仅在于：
本对照例材料人间充质干细胞培养基：为实施例1中人间充质干细胞培养基(培养基2)。
效果例1
细胞增殖能力检测
在实施例2、对照例1-4原代培养后特定时间(原代培养72h、120h、144h、168h)CCK-8测定细胞增殖。
实施例2(培养基3，miRNA-378 agomir+miRNA-126 agomir)、对照例1(商用培养基A)和对照例2(商用培养基B)细胞增殖结果见图1生长曲线图。
图1结果表明原代培养72h～168h，采用本发明实施例2间充质干细胞培养基培养细胞增殖速度比对照组1、2快，对照组1、对照组2细胞增殖速度相近。实施例2间充质干细胞培养基原代培养168h间充质干细胞培养基培养细胞增殖速率最大，且明显优于对照组1、2。
实施例2(培养基3，miRNA-378 agomir+miRNA-126 agomir)、对照例3(培养基1，miRNA-378 agomir)和对照例4(培养基2，miRNA-126 agomir)细胞增殖结果见图2生长曲线图。
图2结果表明原代培养72h时，实施例2、对照例3、对照例4间充质干细胞培养基培养细胞增殖速度相近，对照例4细胞增殖速度略高于实施例2和对照例3。但实施例2间充质干细胞培养基培养细胞增殖速度增长率明显高于对照例3和对照例4，随着原代培养时间增加，实施例2间充质干细胞培养基培养细胞增殖速度快于对照组3、4，原代培养12h～168h，采用本发明实施例2间充质干细胞培养基培养细胞增殖速度比对照组3、4快。实施例2间充质干细胞培养基原代培养168h间充质干细胞培养基培养细胞增殖速率最大，且明显优于对照组3、4。
效果例2
细胞增殖能力检测
按miRNA-378 agomir和miRNA-126 agomir均为10、30、50、70、90、110、130nM的浓度配置培养基(实施例1中培养基4、5、3、6、7、8、9)，按实施例2步骤培养至第7天(原代培养168h)后，检测细胞增殖量。
CCK-8测定细胞增殖结果见图3，其中，miRNA-378 agomir和miRNA-126 agomir浓度均为50nM时，采用效果例1中培养基3原代培养168h的数据。
图3结果显示，培养基从10nM到70nM时明显增殖，90nM时相比于70nM已经增殖不明显，110nM-130nM和90nM相比，变化不明显。因此，本发明人脂肪间充质干细胞培养基中miRNA-378 agomir的浓度为10～130nM和miRNA-126 agomir的浓度为10～130nM；优选的，miRNA-378 agomir的浓度为30～110nM和miRNA-126 agomir的浓度为30～110nM；更优选的，miRNA-378 agomir的浓度为50～90nM和miRNA-126 agomir的浓度为50～90nM；最优选的，miRNA-378 agomir的浓度为50nM和miRNA-126 agomir的浓度为50nM。
效果例3
流式细胞仪检测
将实施例2人脂肪间充质干细胞培养传代到第15代后，使用流式细胞仪进行表面分子CD13,CD44,CD105的鉴定。
鉴定结果见图4～6。
结果显示，本发明分离培养的人脂肪间充质干细胞，CD13,CD44,CD105三种特异性marker的阳性率均大于90％，本发明获得细胞表型稳定，培养至15代的间充质干细胞表型正常。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 成都仕康美生物科技有限公司
<120> miRNA激动剂及应用、人源间充质干细胞培养基及培养方法
<160> 4
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
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<212> RNA
<213> 人工序列(Artificial Sequence)
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<212> RNA
<213> 人工序列(Artificial Sequence)
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cgcauuauua cucacgguac ga 22

Claims (10)

1.一种miRNA激动剂，其特征在于，所述miRNA激动剂由miRNA-378agomir和miRNA-126agomir组成。
2.根据权利要求1所述的miRNA激动剂，其特征在于，所述miRNA-78agomir为双链结构，含正义链和反义链；其中，所述正义链的核苷酸序列如SEQ ID NO：1所示，所述反义链序列的核苷酸序列如SEQ ID NO：2所示；
所述miRNA-126agomir为双链结构，含正义链和反义链；其中，所述正义链的核苷酸序列如SEQ ID NO：3所示，所述反义链序列的核苷酸序列如SEQ ID NO：4所示。
3.根据权利要求2所述的miRNA激动剂，其特征在于，所述miRNA-378agomir和所述miRNA-126agomir经化学修饰，所述的化学修饰包括：反义链3’端进行胆固醇修饰，5’端进行两个硫代骨架修饰，3’端进行四个硫代骨架修饰，全链进行甲氧基修饰。
4.权利要求1～3任意一项所述的miRNA激动剂在培养间充质干细胞中的应用或在制备培养间充质干细胞的产品中的应用。
5.一种人源间充质干细胞培养基，其特征在于，包括：权利要求1～3任意一项所述的miRNA激动剂。
6.根据权利要求5所述的培养基，其特征在于，所述培养基为无血清培养基。
7.根据权利要求5所述的培养基，其特征在于，所述人源间充质干细胞为人脂肪间充质干细胞。
8.根据权利要求5所述的培养基，其特征在于，所述miRNA-378agomir的浓度为10～130nM和所述miRNA-126agomir的浓度为10～130nM。
9.根据权利要求5所述的培养基，其特征在于，所述培养基还包括：氨基酸、生长因子、抗生素。
10.一种间充质干细胞的培养方法，其特征在于，包括：采用权利要求5所述的培养基进行培养。
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