CN112370573A - 一种软骨膜片及其制备方法 - Google Patents
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Abstract
本发明提供一种软骨膜片及其制备方法，软骨膜片通过诱导间充质干细胞和软骨细胞制备而成。制备步骤为将间充质干细胞和软骨细胞混合后，接种于细胞培养板中；待细胞生长密度融合至80%～90%时，加入软骨细胞诱导液进行软骨诱导培养；诱导30天后，形成组织工程类软骨。本发明采用软骨细胞与诱导培养液共同诱导作用，形成类软骨组织，通过细胞‑细胞，细胞因子‑细胞之间的作用，在体外共同作用诱导细胞膜片形成类软骨组织，缩短干细胞分化形成软骨细胞的周期。
Description
技术领域
本发明属于软骨细胞培养领域，尤其涉及一种软骨膜片及其制备方法。
背景技术
关节软骨主要是由软骨细胞和细胞外基质组成的无血管组织，主要依靠关节的运动和挤压吸收营养物质，所以软骨损伤后自我修复能力比较弱，临床研究对患者组织取材有限，此外，由于软骨细胞是终末分化细胞，体外增殖能力有限且易去分化，去分化是指分化细胞失去特有的结构和功能变为具有未分化细胞特性的过程。所以改善与维持软骨细胞表型在软骨细胞的临床应用中起着举足轻重的作用。
目前为了延迟软骨细胞的去分化，维持软骨细胞表型，需要模拟软骨细胞体内生长环境对所述软骨细胞进行培养，但是，随着软骨细胞的增殖都会出现软骨细胞的去分化趋势，与体内生长环境相比，体外培养软骨细胞受外界环境影响较大，对软骨细胞的体外应用造成极大的限制，大量研究表明，骨髓细胞等各种细胞都能够在体外进行细胞膜片构建，模拟体内细胞生长环境对所述骨髓细胞进行培养，然而，不依赖生物材料的软骨细胞膜片的体外构建方法还鲜有报道。
如何构建可注射的，同时具有持续稳定性的软骨表型组织工程软骨，是临床软骨缺损修复所面临的一大挑战。利用软骨细胞构建组织工程软骨中，软骨细胞的来源有限，软骨细胞是终末端分化的细胞，体外培养过程中不易增殖，且软骨细胞容易去分化，失去软骨细胞的生物学特性。申请号为CN104524636 A的专利《通过定向诱导骨髓间充质干细胞获得组织工程软骨的方法》中采用的诱导骨髓间充值干细胞获得组织工程软骨的方法，主要是注射体内形成软骨，且时间为12周，周期较长，应用推广存在一定的局限性。
骨髓间充质干细胞(BMSCs)具有软骨向分化潜能，当BMSCs与软骨细胞共培养时，会促进BMSCs的软骨向分化。目前促进BMSCs软骨向分化的方法在进行在体培养之前，需要大量的外源性生长因子和体外培养操作，但是如何抑制BMSCs在体内肥大化进而骨化的发生，维持持续稳定的软骨向分化仍然是临床应用上的瓶颈问题。
发明内容
针对现有技术存在的问题，本发明提供了一种软骨膜片及其制备方法。
为实现上述发明目的，本发明采用下述技术方案予以实现：
一种软骨膜片，通过诱导间充质干细胞和软骨细胞制备而成。所述软骨膜片含有细胞外基质和软骨细胞，细胞外基质由软骨细胞及间充质干细胞分泌而来，更易于与细胞粘连，促进软骨组织的一致性。
优选的，诱导过程为将间充质干细胞和软骨细胞混合后，采用软骨细胞诱导液进行软骨诱导培养，诱导30天后，形成软骨膜片。
优选的，间充质干细胞与软骨细胞的数量比为1：7～7：1。
优选的，软骨细胞诱导液的组成为：在DMEM培养液含有体积含量为2.5～10%的人血来源的浓缩生长因子、10-3～10-5mol/mL地塞米松、5～30ng/mLTGF-b、0.1～10ug/mL胰岛素、10～80ug/mL维生素C。
本发明还提供了一种软骨膜片及其制备方法，包括如下步骤：
（1）将间充质干细胞和软骨细胞混合后，接种于细胞培养板中；
（2）待细胞生长密度融合至80%～90%时，加入软骨细胞诱导液进行软骨诱导培养；
（3）诱导30天后，形成软骨膜片。
本发明中软骨细胞诱导液可刺激软骨细胞和间充质干细胞分泌大量的细胞外基质，软骨细胞可分泌大量的细胞因子，通过细胞因子-细胞的作用，诱导间充质干细胞向软骨细胞分化，诱导后可形成含有大量细胞外基质及成熟软骨细胞的组织工程类软骨。
优选地，步骤（1）中间充质干细胞与软骨细胞混合的数量比为1：7～7：1，若软骨细胞过多，会耗费大量的软骨细胞，无法达到采用干细胞来替代软骨细胞的目的；若间充质干细胞过多，则软骨细胞与间充质干细胞之间的细胞间接触减少，影响类软骨组织形成，延长类软骨组织形成时间。
优选地，步骤（1）软骨细胞来自来自人、猴、猪、马、兔、大鼠或小鼠，优选自人耳软骨、鼻中隔、肋软骨或关节软骨。
优选地，步骤（1）中间充质干细胞和软骨细胞的接种密度为1E4～1E5/cm2。细胞接种密度主要是形成组织形成的微环境，若细胞接种密度过低，在诱导之前需要进行部分的细胞培养，会增加类软骨组织的形成周期；若细胞密度过高，细胞膜片中在短期分泌大量的ECM与细胞之间的力平衡，容易引起细胞膜片的卷缩，影响类软骨组织的形成。
优选地，步骤（2）中软骨细胞诱导液的组成为：在DMEM培养液含有体积含量为2.5～10%的人血来源的浓缩生长因子、10-3～10-5mol/mL地塞米松、5～30ng/mLTGF-b、0.1～10ug/mL胰岛素、10～80ug/mL维生素C。
本发明添加了人血来源的浓缩生长因子，浓缩生长因子中包含大量的生长因子，如转化生长因子B1，胰岛素样生长因子-1，血小板衍生生长因子AB和血管内皮生长因子等，可促进细胞的增殖及干细胞的分化。细胞膜片构建过程中添加了外源的诱导因子地塞米松、胰岛素、维生素C等，通过混合因子对细胞膜片中细胞的刺激以促进细胞的增殖及分化，可在短期构建细胞膜片。
优选地，步骤（3）为加入软骨细胞诱导液后每三天弃去上清，重新加入软骨细胞诱导液，30天后形成软骨膜片。
本发明提供一种上述软骨膜片在构建组织工程化软骨组织中的应用。
相对于现有技术，本发明的有益效果在于：
本发明提供了一种软骨膜片及其制备方法，采用软骨细胞与间充质干细胞共同接种诱导形成类软骨组织，采用间充质干细胞作为类软骨组织的种子细胞，可扩大种子细胞的来源。细胞膜片构建过程中采用人血来源的浓缩生长因子，还添加了外源的诱导因子地塞米松、胰岛素、维生素C等，诱导液可刺激软骨细胞和间充质干细胞分泌大量的细胞外基质，软骨细胞可分泌大量的细胞因子，通过细胞-细胞和细胞因子-细胞之间的作用，在体外共同作用诱导细胞膜片形成类软骨组织，缩短干细胞分化形成软骨细胞的周期。
附图说明
图1为本发明实施例2中软骨细胞的番红O染色结果；
图2为本发明实施例2中脐带间充质干细胞在显微镜下的照片；
图3A为本发明实施例2中脐带间充质干细胞的细胞外表面分子CD105表达情况的流式检测；
图3B为本发明实施例2中脐带间充质干细胞的细胞外表面分子CD90表达情况的流式检测；
图3C为本发明实施例2中脐带间充质干细胞的细胞外表面分子CD73表达情况的流式检测；
图3D为本发明实施例2中脐带间充质干细胞的细胞外表面分子CD34表达情况的流式检测；
图3E为本发明实施例2中脐带间充质干细胞的细胞外表面分子CD45表达情况的流式检测；
图3F为本发明实施例2中脐带间充质干细胞的细胞外表面分子CD19表达情况的流式检测；
图3G为本发明实施例2中脐带间充质干细胞的细胞外表面分子CD14表达情况的流式检测；
图4为本发明实施例2中软骨膜片的HE染色结果；
图5为本发明实施例2中软骨膜片植入裸鼠皮下30天后取出的类软骨组织的外观；
图6为本发明实施例2中软骨膜片植入裸鼠皮下30天后取出的类软骨组织的HE染色结果。
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。
本发明中软骨细胞的获取方法可参见申请号为CN201510214692.4的专利 《一种软骨细胞膜片的构建方法》，其制备过程为无菌获得人耳软骨组织，对组织进行清洗，去除软骨上附带的肌肉组织等，并剪切成1mm3大小的软骨组织并用0.05-1%II型胶原酶消化，所获得细胞显微镜下拍照，结果如图2所示，可以看出脐带间充质干细胞贴壁生长，呈长梭形，为间充质干细胞的典型形态。图1为番红O染色结果，从结果可以看出通过消化法可获得软骨细胞。
取脐带间充质干细胞传代至P3 代，进行细胞表面分子标记及成骨和成脂检测，结果如图3A-3G所示，脐带间充质干细胞表面分子阳性指标CD105、CD90、CD73大于95%，阴性指标CD14、CD19、CD34、CD45小于2%，说明间充质干细胞符合细胞治疗协会对间充质干细胞的要求，细胞纯度高，干性高，易于分化。
抽取血液100mL，进行变速离心，获取浓缩生长因子。
实施例1
一种软骨膜片，通过诱导间充质干细胞和软骨细胞制备而成。
在一些实施例中，诱导过程为将间充质干细胞和软骨细胞混合后，采用软骨细胞诱导液进行软骨诱导培养，每三天弃去上清，重新加入软骨细胞诱导液，30天后形成软骨膜片。
在一些实施例中，软骨细胞与间充质干细胞的间充质干细胞和软骨细胞混合的数量比为1：7、2:7、5:7、10:7、25:7、40:7或7：1。
在一些实施例中，软骨细胞诱导液的组成为：在DMEM培养液中含有人血来源的浓缩生长因子，人血来源的浓缩生长因子在DMEM培养液中的体积含量为2.5～10%，例如：2.5%、3%、4%、5%、6%、7%、8%、9%或10%；地塞米松10-3～10-5mol/mL，例如：10-3mol/mL、10-4mol/mL或10-5mol/mL；TGF-b 5～30ng/mL，例如：5ng/mL、6ng/mL、10ng/mL、15ng/mL、20ng/mL、25ng/mL或30ng/mL；胰岛素 0.1～10ug/mL，例如：0.1ug/mL、0.2ug/mL、1ug/mL、4ug/mL、6ug/mL、8ug/mL或10ug/mL；维生素C10～80ug/mL，例如：10ug/mL、20ug/mL、30ug/mL、40ug/mL、50ug/mL、60ug/mL、70ug/mL或80ug/mL。
实施例2
一种软骨膜片的制备方法，包括如下步骤：
（1）采用含5%CGF的DMEM培养液悬浮软骨细胞和脐带间充质干细胞，混合比例为1:7，以每孔5E5的细胞量接种细胞与六孔版中，在37℃，5%CO2条件下置于培养箱进行培养；
（2）约2天后，细胞生长融合至90%时，弃掉培养液，加入3mL的诱导液，软骨细胞诱导液的组成为:在5%DMEM培养液中含有，体积含量为2.5%的人血来源的浓缩生长因子、10-3mol/mL地塞米松、5ng/mLTGF-b、0.1ug/mL胰岛素、10ug/mL维生素C；
（3）加入诱导液诱导3天后，弃去上清，加入新鲜诱导液，在37℃，5%CO2条件下继续培养3天后，操作同上，诱导30天后，将细胞膜片植入裸鼠皮下。图4为本发明实施例1中细胞膜片的HE染色结果，由图可知，膜片中含有大量的细胞外基质，软骨细胞均匀分布在细胞外基质中，说明体外诱导的细胞膜片已形成类软骨样组织；
（4）植入裸鼠皮下的膜片30天后，进行取材，以及HE染色。图5为本发明实施例1中植入裸鼠皮下膜片30天后取出的类软骨组织的外观，可以看出皮下移植后的类软骨组织为白色半透明组织，从外形上接近于正常的软骨组织。图6为植入裸鼠皮下膜片30天后取出的类软骨组织的HE染色结果，可以看出，所形成的类软骨组织中为细胞核圆形或卵圆形并可见软骨陷窝，说明已形成成熟的软骨组织。
实施例3
一种软骨膜片的制备方法，包括如下步骤：
（1）采用含5%CGF的DMEM培养液悬浮软骨细胞和脐带间充质干细胞，混合比例为7:1，以每孔5E5的细胞量接种细胞与六孔版中，在37℃，5%CO2条件下置于培养箱进行培养；
（2）约2天后，细胞生长融合至90%时，弃掉培养液，加入3mL的诱导液，软骨细胞诱导液的组成为：在5%DMEM培养液中含有，体积含量为10%的人血来源的浓缩生长因子、10-5mol/mL地塞米松、30ng/mLTGF-b、10ug/mL胰岛素、80ug/mL维生素C；
（3）加入诱导液诱导3天后，弃去上清，加入新鲜诱导液，在37℃，5%CO2条件下继续培养3天后，操作同上，诱导30天后，将细胞膜片植入裸鼠皮下；
（4）植入裸鼠皮下的膜片30天后，进行取材，以及HE染色。
由此可知，本申请所制备的软骨膜片中包含大量的软骨细胞，分泌大量的细胞外基质。诱导液可刺激软骨细胞和间充质干细胞分泌大量的细胞外基质，软骨细胞可分泌大量的细胞因子，可通过细胞因子-细胞的作用，诱导间充质干细胞向软骨细胞分化，缩短干细胞分化形成软骨细胞的周期。
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 (10)
1.一种软骨膜片，其特征在于，通过诱导间充质干细胞和软骨细胞制备而成。
2.如权利要求1所述的软骨膜片，其特征在于，诱导过程为将间充质干细胞和软骨细胞混合后，采用软骨细胞诱导液进行软骨诱导培养，诱导30天后，形成软骨膜片。
3.如权利要求2所述的软骨膜片，其特征在于，间充质干细胞与软骨细胞混合的数量比为1：7～7：1。
4.如权利要求2所述的软骨膜片，其特征在于，软骨细胞诱导液的组成为：在DMEM培养液含有体积含量为2.5～10%的人血来源的浓缩生长因子、10-3～10-5mol/mL地塞米松、5～30ng/mLTGF-b、0.1～10ug/mL胰岛素、10～80ug/mL维生素C。
5.一种软骨膜片的制备方法，其特征在于，包括如下步骤：
（1）将间充质干细胞和软骨细胞混合后，接种于细胞培养板中；
（2）待细胞生长密度融合至80%～90%时，加入软骨细胞诱导液进行软骨诱导培养；
（3）诱导30天后，形成软骨膜片。
6.如权利要求5所述的制备方法，其特征在于，步骤（1）中间充质干细胞与软骨细胞混合的数量比为1：7～7：1。
7.如权利要求5所述的制备方法，其特征在于，步骤（1）软骨细胞来自人、猴、猪、马、兔、大鼠或小鼠，优选自人耳软骨、鼻中隔、肋软骨或关节软骨。
8.如权利要求5所述的制备方法，其特征在于，步骤（1）中间充质干细胞和软骨细胞的接种密度为1E4～1E5/cm2。
9.如权利要求5所述的制备方法，其特征在于，步骤（2）中软骨细胞诱导液的组成为：在DMEM培养液含有体积含量为2.5～10%的人血来源的浓缩生长因子、10-3～10-5mol/mL地塞米松、5～30ng/mLTGF-b、0.1～10ug/mL胰岛素、10～80ug/mL维生素C。
10.如权利要求1所述的软骨膜片在构建组织工程化软骨组织中的应用。
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