CN111849878A - 一种提高间充质干细胞成骨能力的方法 - Google Patents
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Abstract
本发明提供一种提高间充质干细胞成骨能力的方法，包括将间充质干细胞接种于含有10%胎牛血清的α‑MEM培养基中培养，间充质干细胞融合度为80%～85%时采用0.2%胰酶消化传代；将上述得到的间充质干细胞接种于含有10%胎牛血清的α‑MEM培养基中培养，间充质干细胞密度大于90%时加入成骨诱导液和二甲双胍水溶液进行诱导，诱导时间为14～21天。将二甲双胍作用于间充质干细胞，茜素红染色结果发现二甲双胍可以显著提高细胞钙矿化结节程度，表明二甲双胍对间充质干细胞成骨分化具有显著的诱导作用；二甲双胍可显著提高间充质干细胞成骨分化相关基因SIRT1、ALP、COL1A1、RUNX2、BGLAP的表达；间充质干细胞成骨分化能力的提高，对于干细胞移植及骨生物学研究有十分重要的意义。
Description
技术领域
本发明属于生物领域，具体涉及一种提高间充质干细胞成骨能力的方法。
背景技术
骨组织工程是指将分离的自体高浓度成骨细胞、骨髓基质干细胞或软骨细胞，经体外培养扩增后种植于一种天然或人工合成的、具有良好生物相容性、可被人体逐步降解吸收的细胞支架或称细胞外基质上，这种生物材料支架可为细胞提供生存的三维空间，有利于细胞获得足够的营养物质，进行气体交换，排除废料，使细胞在预制形态的三维支架上生长，然后将这种细胞杂化材料植入骨缺损部位，在生物材料逐步降解的同时，种植的骨细胞不断增殖，从而达到修复骨组织缺损的目的，因此足够量的成骨细胞在骨组织工程中成为关键因素。如何通过诱导获得足够量的成骨种子细胞，并在诱导过程中维持种子细胞的表型特征成为骨组织工程研究中的热点。
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，属于多能干细胞，MSC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。如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。MSC由于其自我更新、高增值性和中胚层分化潜力，能够在临床实际应用中修复受损组织，促进组织功能修复。但是，随着对间充质干细胞研究的深入，越来越多的未知领域展现出来，等待深入研究。目前，促进间充质干细胞向成骨细胞分化，改善骨再生修复的效果，是相关领域科学研究和临床应用的重要问题，现有的间充质干细胞分化为成骨细胞的方法效率较低，故探究并发现一种新的成骨分化方法变得尤为重要。
发明内容
本发明的目的在于，针对现有技术存在的问题，提供一种提高间充质干细胞成骨分化能力的方法。
本发明的目的是通过以下技术方案来实现的：
一种提高间充质干细胞成骨分化能力的方法，包括如下步骤：
（1）将间充质干细胞接种于含有10%胎牛血清的α-MEM培养基中，置于CO2培养箱中培养，间充质干细胞融合度为80%～85%时采用0.2%胰酶消化传代；
（2）将步骤（1）得到的间充质干细胞接种于含有10%胎牛血清的α-MEM培养基中，置于CO2培养箱中培养，间充质干细胞密度大于90%时加入成骨诱导液和二甲双胍水溶液进行诱导，诱导时间为14～21天，诱导时间太短，影响间充质干细胞成骨分化的诱导效果，诱导时间太长，会导致间充质干细胞的大量死亡。
二甲双胍是一种对抗2型糖尿病的常用药物，通过降低肝脏生产葡萄糖，增加外周组织对胰岛素的敏感性，从而实现葡萄糖的利用，其临床药效已经受到充分肯定。此外，二甲双胍能促进成骨细胞活性，增加矿物质沉淀，同时抑制破骨细胞活性，防止骨量减少。二甲双胍可以诱导成骨样细胞系（UMR106,MC3T3）的生长和分化，同时具有增加细胞外基质矿化的能力。通过采用二甲双胍提高间充质干细胞成骨分化能力，对于干细胞移植及骨生物学研究有十分重要的意义。
优选地，还包括对步骤（2）得到的间充质干细胞进行茜素红染色、钙结节定量和测定细胞内成骨分化相关基因表达。
优选地，测定细胞内成骨分化相关基因包括SIRT1、ALP、COL1A1、RUNX2和BGLAP。
优选地，步骤（1）中间充质干细胞包括骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞或脐带间充质干细胞。所述间充质干细胞为原代培养的间充质干细胞或传代培养的间充质干细胞。
优选地，步骤（2）中步骤（2）中成骨诱导液为包含50 ug/mL抗坏血酸、100 nM***、10 mMβ甘油磷酸钠的α-MEM培养基。
优选地，步骤（2）中二甲双胍的浓度为50～400ummol/L；优选地，步骤（2）中二甲双胍的浓度为100～200ummol/L。二甲双胍浓度低于50 ummol/L时不能很好的起到诱导作用，二甲双胍浓度高于400 ummol/L时会影响细胞活性。
优选地，步骤（1）和步骤（2）中CO2培养箱的温度为37℃，CO2浓度为5 %。
优选地，步骤（1）和步骤（2）中CO2培养箱中的培养液更换频率为2～3天/次。
本发明提供了一种提高间充质干细胞成骨分化能力的方法制备的产品，所述产品包括提高间充质干细胞成骨分化能力的二甲双胍。
本发明提供了所述产品在提高间充质干细胞成骨分化能力方面的应用。
相对于现有技术，本发明的有益效果在于：
本发明提供一种提高间充质干细胞成骨能力的方法，将二甲双胍作用于间充质干细胞，二甲双胍能促进成骨细胞活性，增加矿物质沉淀，同时抑制破骨细胞活性，防止骨量减少，诱导成骨样细胞系（UMR106，MC3T3）的生长和分化，同时具有增加细胞外基质矿化的能力。通过茜素红染色结果发现二甲双胍可以显著提高细胞钙矿化结节程度，表明二甲双胍对间充质干细胞成骨分化具有显著的诱导作用；二甲双胍可显著提高间充质干细胞成骨分化相关基因SIRT1、ALP、COL1A1、RUNX2、BGLAP的表达。间充质干细胞成骨分化能力的提高，对于干细胞移植及骨生物学研究有十分重要的意义。
附图说明
图1A为本发明实施例中脐带间充质干细胞CD90阳性表面标志物的流式检测结果；
图1 B为本发明实施例中脐带间充质干细胞CD105阳性表面标志物的流式检测结果；
图1 C为本发明实施例中脐带间充质干细胞CD73阳性表面标志物的流式检测结果；
图1 D为本发明实施例中脐带间充质干细胞CD34阴性表面标志物的流式检测结果；
图1E为本发明实施例中脐带间充质干细胞CD45阴性表面标志物的流式检测结果；
图1F为本发明实施例中脐带间充质干细胞CD14阴性表面标志物的流式检测结果；
图1G为本发明实施例中脐带间充质干细胞CD19阴性表面标志物的流式检测结果；
图1H为本发明实施例中脐带间充质干细胞HLA-DR阴性表面标志物的流式检测结果；
图2A为本发明实施例中对照组脐带间充质干细胞成骨分化的茜素红染色结果；
图2B为本发明实施例中二甲双胍组脐带间充质干细胞成骨分化的茜素红染色结果；
图3为本发明实施例中脐带间充质干细胞成骨分化的钙结节定量值比较图；
图4为本发明实施例中SIRT1基因相对表达图；
图5为本发明实施例中ALP基因相对表达图；
图6为本发明实施例中COL1A1基因相对表达图；
图7为本发明实施例中RUNX2基因相对表达图；
图8为本发明实施例中BGLAP基因相对表达图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
试剂说明：
α-MEM培养基购自Sigma公司；
二甲双胍购自Sigma公司；
人脐带间充质干细胞成骨诱导液可以按如下方法配制：
在α-MEM培养基中加入50 ug/mL抗坏血酸、100 nM***、10 mMβ甘油磷酸钠、5%胎牛血清。
实施例1
一、人脐带间充质干细胞的分离培养
1．在生物安全柜内用磷酸盐缓冲液(phosphate buffered solution，PBS)充分冲洗脐带表面，将脐带组织的表层膜剥除，然后将脐带剖开，除去两条脐动脉和一条脐静脉内的残存血并剔除动静脉血管。把处理好的脐带组织剪成大小约为1～2mm3的组织块，然后置于含10%FBS 的α-MEM培养基中，摇匀组织块后铺在培养平面上，接着放入含有5％CO2、37℃的饱和湿度培养箱中培养，培养液更换频率为两天一次，密切关注细胞的生长状态，换液时间可根据细胞生长情况调整。
2．观察细胞大约铺满培养瓶底的80％～85％，将其按照1:3的比例进行传代。用PBS将细胞冲洗两次之后，把2ml 0. 20％的胰蛋白酶加入培养瓶中，消化1min。倒置显微镜观察酶消化过程中细胞形态，如果看到细胞的间隙变大，并且细胞质出现回缩现象，则立即吸出消化液，将含10%FBS 的α-MEM培养基加入，终止消化。对细胞进行反复吹打，直至将细胞吹落。把液体收集起来倒入离心管，放在转速1000rprn离心机中，离心8min，倒掉上清液，把含10%FBS的α-MEM培养基加入进去重悬，接种培养，得到P4代人脐带间充质干细胞。
二、人脐带间充质干细胞的鉴定
取上述传代培养得到的P4代人脐带间充质干细胞用0.20％胰蛋白酶消化，并收集稳定增殖细胞。将106个收集得到的人脐带间充质干细胞悬浮在100ml含1％牛血清白蛋白的PBS缓冲溶液中，参考图1A-H，图1A-H分别为间充质干细胞CD90、CD73、CD34、CD45、CD14、CD19、HLA-DR表面标志物的流失检测结果，由结果可知，阳性表面标志物CD90，CD105，CD73均大于等于95%，阴性表面标志物CD45，CD34，CD11b， CD19和HLA-DR均小于2%，所以本实施例培养所得到的人脐带间充质干细胞符合干细胞标准。
三、人脐带间充质干细胞成骨分化诱导
取上述传代培养得到的P4代人脐带间充质干细胞用0.20％胰蛋白酶消化，制成浓度为1×106/mL的细胞悬液，接种于12孔板，每三天换液，观察细胞的密度变化，待细胞密度大于90%后随机分为对照组和二甲双胍组，对照组中加入成骨诱导液（10%FBS，50ug/mL抗坏血酸，100nM***，10mMβ甘油磷酸钠的α-MEM），二甲双胍组在成骨诱导液基础上增加100ummol/L二甲双胍，在诱导第14天后进行茜素红染色和钙结节定量及测定细胞内成骨分化相关基因的表达。
1.茜素红染色和钙结节定量
去除孔板内培养液，PBS清洗待测细胞3次。12孔板内加入4%多聚甲醛，室温固定15min后吸尽，PBS清洗3次。而后孔板内每孔加入300uL茜素红染液，室温染色15min后吸尽，PBS清洗3次。使用显微镜采集图像，图像采集完毕后，12孔板每孔加入200uL 5%高氯酸，室温静置10min。溶解液转移至96孔板内，分组后酶标仪检测420nm波长吸光值，每组检测2个复孔取平均值。
2.RT-PCR检测成骨标志基因表达
吸尽培养板内培养液，PBS清洗待测细胞3次。6孔板每孔加入500μL TRIzol试剂（总RNA抽提试剂，Invitrogen公司），室温下吹打混匀后放入1 .5mL EP管。各样品加入100μL氯仿，充分震荡，室温静置15min，12000g 4℃离心12min。吸取上层无色透明清液至另一EP管，各样品加入200μL异丙醇，充分震荡，室温静置10min，12000g 4℃离心8min，弃上清液，获得管底微量羽毛状RNA沉淀。加入适量75%乙醇溶液，震荡EP管，混匀上述RNA沉淀，8000g 4℃离心5min，弃上清液，室温下风干30min。使用无RNA酶水混匀RNA沉淀，60℃放置10min。取少量样品，用酶标仪测RNA浓度。
使用PrimeScriptTM RT Master Mix (TaKaRa)试剂盒逆转录RNA。反应体系：（下述按500μg RNA计算）待测RNA、5μL 5×PrimeScript RT Master Mix，后加入无RNA酶水至10μL体系。设定PCR仪反应条件为42℃ 60min，70℃ 5min，4℃ 60min。分别使用SIRT1、ALP、COL1A1、RUNX2、BGLAP基因相对应的引物，用SYBR Green PCR master mix（ThermoFisher）试剂盒进行RT-PCR检测。反应体系10ul：cDNA稀释4倍后，按1μL、5μL、0 .4μL、0 .4μL、3.2μL体积混合待测cDNA、SYBR Green、上游引物、下游引物、无RNA酶水。反应条件：设定PCR仪反应条件为95℃ 3min，95℃ 10s，56℃ 30s，60℃ 5s，进行40个循环。基因相对表达量计算公式为χ = 2−ΔΔCt， ΔΔCt = ΔE – ΔC， ΔE = Ctexp – Ct内参，ΔC=Ctct1 –Ct内参（Ctexp为目标基因的循环值，Ctct1为各测试样品对照组的循环值）。
参考图2A-图2B，图2A为对照组脐带间充质干细胞成骨分化的茜素红染色结果，图2B为二甲双胍组脐带间充质干细胞成骨分化的茜素红染色结果，可以看出，使用UCMSC进行成骨诱导时加入二甲双胍14天后，二甲双胍组相比于对照组的茜素红染色加深，图3为脐带间充质干细胞成骨分化的钙结节定量值比较图，二甲双胍组相比于对照组的钙结节定量提高，即二甲双胍可以显著提高细胞钙矿化结节程度，表明二甲双胍对间充质干细胞成骨分化具有显著的诱导作用。参考图4-图8，分别为SIRT1、ALP、COL1A1、RUNX2、BGLAP五种基因相对表达图，RT-PCR结果显示成骨诱导时加入二甲双胍可以提高基因SIRT1、ALP、COL1A1、RUNX2、BGLAP的表达，这说明二甲双胍诱导可以提高脐带间充质干细胞的成骨能力。
以上所述，仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，任何在本申请揭露的技术范围内的变化或替换，都应涵盖在本申请的保护范围之内。因此，本申请的保护范围应以所述权利要求的保护范围为准。
Claims (6)
1.一种提高间充质干细胞成骨分化能力的方法，其特征在于，包括如下步骤：
（1）将间充质干细胞接种于含有10%胎牛血清的α-MEM培养基中，置于CO2培养箱中培养，间充质干细胞融合度为80%～85%时采用0.2%胰酶消化传代；
（2）将步骤（1）得到的间充质干细胞接种于含有10%胎牛血清的α-MEM培养基中，置于CO2培养箱中培养，间充质干细胞密度大于90%时加入成骨诱导液和二甲双胍水溶液进行诱导，诱导时间为14～21天。
2.根据权利要求1所述的提高间充质干细胞成骨分化能力的方法，其特征在于，步骤（1）中间充质干细胞包括骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞或脐带间充质干细胞。
3.根据权利要求1所述的提高间充质干细胞成骨分化能力的方法，其特征在于，步骤（2）中成骨诱导液为包含50 ug/mL抗坏血酸、100 nM***、10 mMβ甘油磷酸钠的α-MEM培养基。
4.根据权利要求1所述的提高间充质干细胞成骨分化能力的方法，其特征在于，步骤（2）中二甲双胍的浓度为50～400ummol/L。
5.用于提高间充质干细胞成骨能力的产品，其特征在于，所述产品包括二甲双胍。
6.一种根据权利要求5所述的产品在提高间充质干细胞成骨分化能力方面的应用。
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| CN114317421B (zh) * | 2021-12-16 | 2024-05-03 | 北京科技大学 | 强化间充质干细胞促进血管生成的方法、组合物及应用 |
| CN114836377A (zh) * | 2022-06-08 | 2022-08-02 | 海南济民博鳌国际医院有限公司 | 一种干细胞体外成骨诱导分化方法 |
| CN114836377B (zh) * | 2022-06-08 | 2023-09-08 | 海南济民博鳌国际医院有限公司 | 一种干细胞体外成骨诱导分化方法 |
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