CN115197906A - 利用骨髓间充质干细胞构建组织工程软骨的方法 - Google Patents

利用骨髓间充质干细胞构建组织工程软骨的方法 Download PDF

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CN115197906A
CN115197906A CN202210988282.5A CN202210988282A CN115197906A CN 115197906 A CN115197906 A CN 115197906A CN 202210988282 A CN202210988282 A CN 202210988282A CN 115197906 A CN115197906 A CN 115197906A
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stem cells
mesenchymal stem
cartilage
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李浪
万莎
林科夫
田臻
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Tibet Autonomous Region People's Government In Chengdu Office Hospital
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Abstract

本发明公开了利用骨髓间充质干细胞构建组织工程软骨的方法，涉及组织工程软骨技术领域，其技术方案要点是：包括(1)获取骨髓间充值干细胞，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片；(3)获取软骨细胞砖(4)获取富血小板血浆；(5)获取细胞复合体，将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，然后向溶液中加入含有凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，将细胞复合体注射于裸鼠背部皮下，进行体内培养。能够获取质量良好的组织工程软骨，且能够保持骨髓间充质干细胞软骨向分化的稳定；同时，能够确定骨髓间充质干细胞与软骨细胞共同培养构建组织工程软骨的最佳比例。

Description

利用骨髓间充质干细胞构建组织工程软骨的方法
技术领域
本发明涉及组织工程软骨术领域，更具体地说，它涉及利用骨髓间充质干细胞构建组织工程软骨的方法。
背景技术
组织工程软骨的构建需要具备三个主要条件：足够数量、功能正常的种子细胞；合适的细胞支架；调节细胞增殖、保持细胞表型特征的细胞因子。
目前，关节软骨细胞可以分泌Ⅱ型胶原和蛋白聚糖等软骨细胞外基质关键成分，不需要体外诱导培养，是软骨组织工程理论上最理想的种子细胞；然而软骨细胞的组织取材有限，并且也因为软骨细胞是终末分化细胞，体外增殖能力有限且易去分化，因此，在一些构建组织工程软骨的技术中，利用了骨髓间充质干细胞。骨髓间充质干细胞来源广泛，取材容易，对供体损伤小，且具有软骨分化潜能，它们与软骨细胞共培养时，软骨细胞及其分泌的细胞因子构成的微环境有利于BMSC的软骨向分化。
现有技术中，通常在在体培养之前需要使用大量的外源性生长因子和大量的BMSC体外培养操作，同时，无法确定骨髓间充质干细胞与软骨细胞共同培养构建组织工程软骨的最佳比例。
发明内容
本发明的目的是提供利用骨髓间充质干细胞构建组织工程软骨的方法，能够获取质量良好的组织工程软骨，且能够保持骨髓间充质干细胞软骨向分化的稳定；同时，能够确定骨髓间充质干细胞与软骨细胞共同培养构建组织工程软骨的最佳比例。
本发明的上述技术目的是通过以下技术方案得以实现的：利用骨髓间充质干细胞构建组织工程软骨的方法，包括(1)获取骨髓间充值干细胞，包括取小鼠骨髓，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片，包括取小鼠软骨细胞，将软骨细胞接种于6孔板中培养，所述软骨细胞在培养基中培养 3-5周，期间每3天更换一次培养基；所述软骨细胞分泌软骨细胞外基质包裹软骨细胞，获得所述软骨细胞膜片；(3)获取软骨细胞砖，包括将所述软骨细胞膜片浸入海藻酸钠溶液中，在所述海藻酸钠溶液中加入氯化钙溶液，直至海藻酸钠溶液成为凝胶；将所述凝胶切片后浸入海藻酸钠溶液，获取软骨细胞砖；(4)获取富血小板血浆；(5) 获取细胞复合体，包括将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，然后向混合溶液中加入含有凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，包括将细胞复合体注射于裸鼠背部皮下，在体内进行培养，获得组织工程软骨。
本发明进一步设置为：获取所述富血小板血浆，包括取小鼠全血，加入抗凝剂，以1500rpm离心15分钟，取最上层得到富血小板血浆。
本发明进一步设置为：所述骨髓间充质干细胞培养至第2代。
本发明进一步设置为：所述获取组织工程软骨包括在体内培养8 周。
本发明进一步设置为：所述抗凝剂为3.8wt％的柠檬酸钠抗凝剂，且所述抗凝剂与小鼠全血为1:9。
综上所述，本发明具有以下有益效果：能够获取质量良好的组织工程软骨，且能够保持骨髓间充质干细胞软骨向分化的稳定；同时，能够确定骨髓间充质干细胞与软骨细胞共同培养构建组织工程软骨的最佳比例。
附图说明
图1是本发明实施例中的流程图。
具体实施方式
以下结合附图1对本发明作进一步详细说明。
实施例一：利用骨髓间充质干细胞构建组织工程软骨的方法，包括(1)获取骨髓间充值干细胞，包括取小鼠骨髓，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片，包括取小鼠软骨细胞，将软骨细胞接种于6孔板中培养，软骨细胞在培养基中培养3-5周，培养基包括基础培养基、骨形成蛋白 BMP-2、多西环素和地塞米松，期间每3天更换一次培养基；软骨细胞分泌软骨细胞外基质包裹软骨细胞，获得软骨细胞膜片；(3)获取软骨细胞砖，包括将软骨细胞膜片浸入1.2wt％海藻酸钠溶液中，在海藻酸钠溶液中加入102mM的氯化钙溶液，直至海藻酸钠溶液成为凝胶；将凝胶切片后浸入海藻酸钠溶液，获取软骨细胞砖；(4)获取富血小板血浆；(5)获取细胞复合体，包括将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，骨髓间充质干细胞的个数为2.5×107个，软骨细胞的个数为2.5×107个，然后向混合溶液中加入含有50uL凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，包括将细胞复合体注射于裸鼠背部皮下，在体内进行培养，获得组织工程软骨。
获取富血小板血浆，包括取小鼠全血，加入抗凝剂，以1500rpm 离心15分钟，取最上层得到富血小板血浆。
骨髓间充质干细胞培养至第2代。
获取组织工程软骨包括在体内培养8周。
抗凝剂为3.8wt％的柠檬酸钠抗凝剂，且抗凝剂与小鼠全血为 1:9。
实施例二：利用骨髓间充质干细胞构建组织工程软骨的方法，包括(1)获取骨髓间充值干细胞，包括取小鼠骨髓，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片，包括取小鼠软骨细胞，将软骨细胞接种于6孔板中培养，软骨细胞在培养基中培养3-5周，培养基包括基础培养基、骨形成蛋白 BMP-2、多西环素和地塞米松，期间每3天更换一次培养基；软骨细胞分泌软骨细胞外基质包裹软骨细胞，获得软骨细胞膜片；(3)获取软骨细胞砖，包括将软骨细胞膜片浸入1.2wt％海藻酸钠溶液中，在海藻酸钠溶液中加入102mM的氯化钙溶液，直至海藻酸钠溶液成为凝胶；将凝胶切片后浸入海藻酸钠溶液，获取软骨细胞砖；(4)获取富血小板血浆；(5)获取细胞复合体，包括将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，骨髓间充质干细胞的个数为2.5×107个，软骨细胞的个数为5×107个，然后向混合溶液中加入含有50uL凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，包括将细胞复合体注射于裸鼠背部皮下，在体内进行培养，获得组织工程软骨。
获取富血小板血浆，包括取小鼠全血，加入抗凝剂，以1500rpm 离心15分钟，取最上层得到富血小板血浆。
骨髓间充质干细胞培养至第2代。
获取组织工程软骨包括在体内培养8周。
抗凝剂为3.8wt％的柠檬酸钠抗凝剂，且抗凝剂与小鼠全血为 1:9。
实施例三：利用骨髓间充质干细胞构建组织工程软骨的方法，包括(1)获取骨髓间充值干细胞，包括取小鼠骨髓，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片，包括取小鼠软骨细胞，将软骨细胞接种于6孔板中培养，软骨细胞在培养基中培养3-5周，培养基包括基础培养基、骨形成蛋白 BMP-2、多西环素和地塞米松，期间每3天更换一次培养基；软骨细胞分泌软骨细胞外基质包裹软骨细胞，获得软骨细胞膜片；(3)获取软骨细胞砖，包括将软骨细胞膜片浸入1.2wt％海藻酸钠溶液中，在海藻酸钠溶液中加入102mM的氯化钙溶液，直至海藻酸钠溶液成为凝胶；将凝胶切片后浸入海藻酸钠溶液，获取软骨细胞砖；(4)获取富血小板血浆；(5)获取细胞复合体，包括将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，骨髓间充质干细胞的个数为 5×107个，软骨细胞的个数为2.5×107个，然后向混合溶液中加入含有50uL凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，包括将细胞复合体注射于裸鼠背部皮下，在体内进行培养，获得组织工程软骨。
获取富血小板血浆，包括取小鼠全血，加入抗凝剂，以1500rpm 离心15分钟，取最上层得到富血小板血浆。
骨髓间充质干细胞培养至第2代。
获取组织工程软骨包括在体内培养8周。
抗凝剂为3.8wt％的柠檬酸钠抗凝剂，且抗凝剂与小鼠全血为 1:9。
实施例四：利用骨髓间充质干细胞构建组织工程软骨的方法，包括(1)获取骨髓间充值干细胞，包括取小鼠骨髓，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片，包括取小鼠软骨细胞，将软骨细胞接种于6孔板中培养，软骨细胞在培养基中培养3-5周，培养基包括基础培养基、骨形成蛋白 BMP-2、多西环素和地塞米松，期间每3天更换一次培养基；软骨细胞分泌软骨细胞外基质包裹软骨细胞，获得软骨细胞膜片；(3)获取软骨细胞砖，包括将软骨细胞膜片浸入1.2wt％海藻酸钠溶液中，在海藻酸钠溶液中加入102mM的氯化钙溶液，直至海藻酸钠溶液成为凝胶；将凝胶切片后浸入海藻酸钠溶液，获取软骨细胞砖；(4)获取富血小板血浆；(5)获取细胞复合体，包括将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，骨髓间充质干细胞的个数为2.5×107个，软骨细胞的个数为10×107个，然后向混合溶液中加入含有50uL凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，包括将细胞复合体注射于裸鼠背部皮下，在体内进行培养，获得组织工程软骨。
获取富血小板血浆，包括取小鼠全血，加入抗凝剂，以1500rpm 离心15分钟，取最上层得到富血小板血浆。
骨髓间充质干细胞培养至第2代。
获取组织工程软骨包括在体内培养8周。
抗凝剂为3.8wt％的柠檬酸钠抗凝剂，且抗凝剂与小鼠全血为 1:9。
实施例五：利用骨髓间充质干细胞构建组织工程软骨的方法，包括(1)获取骨髓间充值干细胞，包括取小鼠骨髓，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片，包括取小鼠软骨细胞，将软骨细胞接种于6孔板中培养，软骨细胞在培养基中培养3-5周，培养基包括基础培养基、骨形成蛋白 BMP-2、多西环素和地塞米松，期间每3天更换一次培养基；软骨细胞分泌软骨细胞外基质包裹软骨细胞，获得软骨细胞膜片；(3)获取软骨细胞砖，包括将软骨细胞膜片浸入1.2wt％海藻酸钠溶液中，在海藻酸钠溶液中加入102mM的氯化钙溶液，直至海藻酸钠溶液成为凝胶；将凝胶切片后浸入海藻酸钠溶液，获取软骨细胞砖；(4)获取富血小板血浆；(5)获取细胞复合体，包括将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，骨髓间充质干细胞的个数为10×107个，软骨细胞的个数为2.5×107个，然后向混合溶液中加入含有50uL凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，包括将细胞复合体注射于裸鼠背部皮下，在体内进行培养，获得组织工程软骨。
获取富血小板血浆，包括取小鼠全血，加入抗凝剂，以1500rpm 离心15分钟，取最上层得到富血小板血浆。
骨髓间充质干细胞培养至第2代。
获取组织工程软骨包括在体内培养8周。
抗凝剂为3.8wt％的柠檬酸钠抗凝剂，且抗凝剂与小鼠全血为1:9
结论：将获取的细胞复合体在体内培养8周后，取出样本，进行观察，测量其体积、质量和厚度。经观察对比得知，骨髓间充值干细胞与软骨细胞比例为1：2组所形成的软骨相较于其它两组体积最大，厚度厚，形态饱满，表面光滑有光泽，弹性好，边缘未有明显的骨化现象，且当骨髓间充质干细胞的占比越高，质量和体积越小，表面不平整，质地较硬。通过HE染色、番红O染色、甲苯胺蓝染色和Ⅱ型胶原免疫荧光组织化学染色来检验所形成的软骨组织的特性，结果显示，骨髓间充值干细胞与软骨细胞比例为1：2组所形成软骨组织质量最好。
本具体实施例仅仅是对本发明的解释，其并不是对本发明的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本发明的权利要求范围内都受到专利法的保护。

Claims (5)

1.利用骨髓间充质干细胞构建组织工程软骨的方法，其特征是：包括(1)获取骨髓间充值干细胞，包括取小鼠骨髓，采用差速贴壁培养法结合密度梯度离心法获取骨髓间充质干细胞；(2)获取软骨细胞膜片，包括取小鼠软骨细胞，将软骨细胞接种于6孔板中培养，所述软骨细胞在培养基中培养3-5周，期间每3天更换一次培养基；所述软骨细胞分泌软骨细胞外基质包裹软骨细胞，获得所述软骨细胞膜片；(3)获取软骨细胞砖，包括将所述软骨细胞膜片浸入海藻酸钠溶液中，在所述海藻酸钠溶液中加入氯化钙溶液，直至海藻酸钠溶液成为凝胶；将所述凝胶切片后浸入海藻酸钠溶液，获取软骨细胞砖；(4)获取富血小板血浆；(5)获取细胞复合体，包括将骨髓间充质干细胞和软骨细胞砖混合浸于富血小板血浆的溶液中，然后向混合溶液中加入含有凝血酶的氯化钙溶液，获得细胞复合体；(6)获取组织工程软骨，包括将细胞复合体注射于裸鼠背部皮下，在体内进行培养，获得组织工程软骨。
2.根据权利要求1所述的利用骨髓间充质干细胞构建组织工程软骨的方法，其特征是：获取所述富血小板血浆，包括取小鼠全血，加入抗凝剂，以1500rpm离心15分钟，取最上层得到富血小板血浆。
3.根据权利要求1所述的利用骨髓间充质干细胞构建组织工程软骨的方法，其特征是：所述骨髓间充质干细胞培养至第2代。
4.根据权利要求1所述的利用骨髓间充质干细胞构建组织工程软骨的方法，其特征是：所述获取组织工程软骨包括在体内培养8周。
5.根据权利要求2所述的利用骨髓间充质干细胞构建组织工程软骨的方法，其特征是：所述抗凝剂为3.8wt％的柠檬酸钠抗凝剂，且所述抗凝剂与小鼠全血为1:9。
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