CN107308497B - 髓核细胞源性活性微载体的构建 - Google Patents
髓核细胞源性活性微载体的构建 Download PDFInfo
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Abstract
本发明提供一种髓核细胞源性活性微载体构建方法，将大鼠髓核细胞扩增后利用离心法构建出髓核细胞微球，在特定环境中培养12天，使髓核细胞分泌足够的细胞外基质成分，通过联合使用脱细胞剂将细胞微球中原有的髓核细胞组分除去，从而获得一种由髓核细胞自身分泌的基质构成的髓核细胞源性活性微载体。本发明得到具有高效诱导干细胞成髓核细胞定向分化的载体材料；获得的细胞载体所含成分由髓核细胞自身合成，符合椎间盘原生态微环境；避免载体构建过程中所引入的化学成分对载体生物活性的破坏，不含其他高分子材料，避免载体降解产物对椎间盘环境的影响，能搭载干细胞且能通过注射进入生物体内，可用于负载干细胞从而进行退变椎间盘体内修复。
Description
技术领域
本发明属于生物组织工程细胞载体材料，涉及一种髓核细胞源性活性微载体的构建方法，是一种新型细胞载体。
背景技术
椎间盘退行性病变是目前社会的常见病，但当前却无有效手段从病因上修复退变。基于组织工程的椎间盘修复是目前治疗椎间盘退变的新思路，适当的细胞载体搭载间充质干细胞后，可诱导间充质干细胞向髓核细胞分化，从而修复退变的椎间盘。目前细胞载体的研究处于瓶颈期，以高分子聚合物为原料的细胞载体在体内椎间盘环境中的降解产物易加剧椎间盘的恶劣环境，不利于椎间盘修复。网状或纤维网状的生物大分子材料在植入体内时需要有开放性切口，破坏了椎间盘天然密闭微环境，同样缺陷明显。凝胶状的生物大分子材料虽然具有可注射特性，植入过程中避免了椎间盘结构的破坏，但水凝胶的弹性模量远低于天然椎间盘，无法为搭载于其中的干细胞提供原生态微环境，导致干细胞定向分化能力减弱，不利于退变椎间盘的修复。而为了优化材料的理化特性，往往需要加入许多化学试剂，容易导致生物材料活性的降低，影响最后载体的促干细胞分化的效果。在椎间盘的主要结构髓核中，主要细胞成分为髓核细胞，其分泌的细胞外基质成分构成了正常髓核环境，利用髓核细胞这一特性，可一改原先通过物理或化学方法构建细胞载体的传统思路，通过生物的方法在体外构建出一种髓核细胞源性的具有生物活性的原生态干细胞载体。
发明内容
本发明的目的是提供一种髓核细胞源性活性微载体构建方法，是一种为了得到能搭载干细胞且能通过注射进入生物体内的细胞载体，该载体是用于负载干细胞从而进行退变椎间盘体内修复的。
本发明提供的细胞载体利用髓核细胞自身分泌能力构建，通过以下步骤实现：首先从大鼠体内获得髓核细胞，体外扩增至一定数量后利用离心法构建出髓核细胞微球，在含有特定的生长因子的环境中培养12天，使髓核细胞分泌足够的细胞外基质成分。通过联合使用脱细胞剂将细胞微球中原有的髓核细胞组分除去，从而获得一种由髓核细胞自身分泌的基质构成的髓核细胞源性活性微载体。
具体构建方法如下：
(1)髓核细胞微球制备
取SD大鼠髓核细胞，体外使用F12培养基扩增至第3代，取4×10^5个髓核细胞使用1mL F12培养基(含3151mg/L D-葡萄糖，365mg/L L-谷氨酰胺，55mg/L丙酮酸钠，2438mg/L碳酸氢钠，100U/mL青霉素，100ug/mL链霉素，pH 7.0-7.4)重悬，并置于15mL离心管，通过离心富集法在1000rpm下离心5分钟，在F12培养基中静置24h，利用髓核细胞自卷曲能力获得髓核细胞微球；
(2)髓核细胞微球基质合成
更换髓核细胞微球的培养环境：DMEM高糖培养基(含4500mg/L D-葡萄糖，584mg/LL-谷氨酰胺，110mg/L丙酮酸钠，1.5g/L碳酸氢钠，100U/mL青霉素，100ug/mL链霉素，pH7.0-7.2)中添加特定生长因子(10ng/mL的TGF-β3，10％胎牛血清，50nM维生素C)，于37℃，5％CO2的环境中培养12天，每3天换一次液，促进髓核细胞微球中基质产生和沉积；(3)获得髓核细胞源性活性微载体
首先，联合使用脱细胞剂2％Triton-100和50mM的SB-10对培养后的髓核细胞微球进行脱细胞处理，37℃恒温摇床中以100转/分钟的速率处理1小时，在脱去髓核细胞后利用720mU/mL脱氧核糖核酸酶和720mU/mL核糖核酸酶对残留核酸成分进行灭活，最后使用磷酸盐缓冲液在先前摇床环境下对获得的微载体清洗3次，每次5分钟，从而获得一种由髓核细胞自身分泌的基质构成的髓核细胞源性活性微载体。
本发明的另一个目的是提供所述的活性微载体在负载干细胞中应用。
本发明方法能得到具有高效诱导干细胞成髓核细胞定向分化的载体材料；获得的细胞载体所含成分由髓核细胞自身合成，符合椎间盘原生态微环境；避免了载体构建过程中所引入的化学成分对载体生物活性的破坏，不含其他高分子材料，避免了载体降解产物对椎间盘环境的影响。
附图说明
图1是髓核细胞源性活性微载体构建流程示意图。
图2髓核细胞源性活性微载体对载荷干细胞增殖能力和细胞毒性的效应。
图3基质相关基因检测。其中A：ACAN，B：COL2，C：SOX9为髓核细胞功能基因标记物，与干细胞细胞外基质分泌能力相关，高表达说明细胞外基质合成能力增强。
图4分化相关基因检测。其中A：KRT19为髓核细胞特异性基因标记物，表达量高说明髓核细胞分化效率高，B：COL1为软骨细胞基因标记物，表达量低表明干细胞向软骨细胞表型分化较少。
图5髓核细胞源性活性微载体的扫描电子显微镜照片。A：低倍镜下微球微观结构全态(×400)；B：高倍镜下表面形貌(×1000)。
图6髓核细胞源性活性微载体载荷间充质干细胞后对SD大鼠退变椎间盘的修复作用。
具体实施方式
下面结合附图和实施例对本发明作出进一步的具体说明，但本发明不局限于这些实施例。实施例1：欲以4×10^5个髓核细胞构建髓核细胞源性活性微载体。
参见图1，取体外扩增的4×10^5个第3代SD大鼠髓核细胞，使用1mL F12培养基重悬后置于15mL离心管，通过离心富集法在1000rpm下离心5分钟，在F12培养基中静置24h，利用髓核细胞自卷曲能力获得髓核细胞微球。更换髓核细胞微球的培养环境：DMEM高糖培养基中添加10ng/mL的TGF-β3，10％胎牛血清，50nM维生素C。于37℃，5％CO2的环境中培养12天，每3天换一次液，促进髓核细胞微球中基质产生和沉积。联合使用脱细胞剂2％Triton-100和50mM的SB-10对培养后的髓核细胞微球进行脱细胞处理，37℃恒温摇床中以100转/分钟的速率处理1小时。在脱去髓核细胞后利用720mU/mL脱氧核糖核酸酶和720mU/mL核糖核酸酶对残留核酸成分进行灭活。最后使用磷酸盐缓冲液在先前摇床环境下对获得的微载体清洗3次，每次5分钟，从而获得一种由髓核细胞自身分泌的基质构成的髓核细胞源性活性微载体。
实施例2：欲以8×10^5个髓核细胞构建髓核细胞源性活性微载体。
参见图1，取体外扩增的8×10^5个第3代SD大鼠髓核细胞，使用2mL F12培养基重悬，并平均分装至2管15mL离心管，使每管离心管中细胞数量为4×10^5个。通过离心富集法在1000rpm下离心5分钟，在F12培养基中静置24h，利用髓核细胞自卷曲能力获得髓核细胞微球。更换髓核细胞微球的培养环境：DMEM高糖培养基中添加10ng/mL的TGF-β3，10％胎牛血清，50nM维生素C。于37℃，5％CO2的环境中培养12天，每3天换一次液，促进髓核细胞微球中基质产生和沉积。联合使用脱细胞剂2％Triton-100和50mM的SB-10对培养后的髓核细胞微球进行脱细胞处理，37℃恒温摇床中以100转/分钟的速率处理1小时。在脱去髓核细胞后利用720mU/mL脱氧核糖核酸酶和720mU/mL核糖核酸酶对残留核酸成分进行灭活。最后使用磷酸盐缓冲液在先前摇床环境下对获得的微载体清洗3次，每次5分钟，从而获得一种由髓核细胞自身分泌的基质构成的髓核细胞源性活性微载体。
实施例3：髓核细胞源性活性微载体体外活性验证。
通过如前所述步骤获得髓核细胞源性活性微载体后，在体外载荷间充质干细胞，将所获得的髓核细胞源性活性微载体置于培养皿中，滴加2uL含有5*10^4间充质干细胞的培养基，37℃静置2h后使间充质干细胞充分负载至髓核细胞源性活性微载体表面，转移微球至DMEM低糖培养基中，培养14天后检测干细胞增殖活性，并进行基因水平检测，发现间充质干细胞种植于髓核细胞源性活性微载体后其增殖能力较平面培养和壳聚糖水凝胶载体培养增强(见图2A)，髓核细胞源性活性微载体对载荷的干细胞不产生细胞毒性(见图2B)，基质合成能力增强(见图3)，髓核细胞表型分化能力增强(见图4)。
实施例4：髓核细胞源性活性微载体大鼠体内活性验证。
对获得的髓核细胞源性活性微载体进行扫描电子显微镜观察，测定微载体直径。在已构建的椎间盘退变大鼠模型中通过注射器将髓核细胞源性活性微载体连同间充质干细胞注射入退变椎间盘，8周后对大鼠进行MRI拍摄，明确椎间盘含水量，处死后取材，通过HE染色，SO染色明确椎间盘内基质含量，评估退变椎间盘修复效果。结果显示微载体直径约为200uL(见图5)，复合可注射标准，MRI结果提示大鼠椎间盘水含量较退变对照组有所提高，椎间盘得到修复。HE染色与SO染色结果提示椎间盘基质含量较退变组明显提升，椎间盘髓核形态得到恢复(见图6)，证明该髓核细胞源性活性微载体搭载间充质干细胞后具有体内修复效果。
Claims (4)
1.一种髓核细胞源性活性微载体构建方法，其特征在于，具体构建方法如下：
（1）髓核细胞微球制备
取SD大鼠髓核细胞，使用F12培养基扩增至第3代，取4×10^5个髓核细胞使用1mL F12培养基重悬，并置于15mL离心管，通过离心富集法在1000rpm下离心5分钟，在F12培养基中静置24h，利用髓核细胞自卷曲能力获得髓核细胞微球；
（2）髓核细胞微球基质合成
更换髓核细胞微球的培养环境：在DMEM高糖培养基中添加10ng/mL的TGF-β3，10%胎牛血清，50nM维生素C，于37℃，5%CO2的环境中培养12天，每3天换一次液，促进髓核细胞微球中基质产生和沉积；
（3）获得髓核细胞源性活性微载体
首先，联合使用脱细胞剂2% Triton-100和50mM的SB-10对培养后的髓核细胞微球进行脱细胞处理，37℃恒温摇床中以100转/分钟的速率处理1小时，在脱去髓核细胞后利用720mU/mL脱氧核糖核酸酶和720mU/mL核糖核酸酶对残留核酸成分进行灭活，最后使用磷酸盐缓冲液在先前摇床环境下对获得的微载体清洗3次，每次5分钟，从而获得一种由髓核细胞自身分泌的基质构成的髓核细胞源性活性微载体。
2.根据权利要求1所述的一种髓核细胞源性活性微载体构建方法，其特征在于，步骤（1）所述的F12培养基为：3151 mg/L D-葡萄糖，365mg/L L-谷氨酰胺，55mg/L丙酮酸钠，2438mg/L碳酸氢钠，100U/mL青霉素，100ug/mL链霉素，pH 7.0-7.4。
3.根据权利要求2所述的一种髓核细胞源性活性微载体构建方法，其特征在于，步骤（2）所述的DMEM高糖培养基为：4500mg/L D-葡萄糖，584mg/L L-谷氨酰胺，110mg/L丙酮酸钠，1.5g/L碳酸氢钠，100U/mL青霉素，100ug/mL链霉素，pH 7.0-7.2。
4.根据权利要求1所述方法构建的活性微载体在负载干细胞中的应用。
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