CN102552980B - 黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架 - Google Patents

黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架 Download PDF

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CN102552980B
CN102552980B CN 201210007080 CN201210007080A CN102552980B CN 102552980 B CN102552980 B CN 102552980B CN 201210007080 CN201210007080 CN 201210007080 CN 201210007080 A CN201210007080 A CN 201210007080A CN 102552980 B CN102552980 B CN 102552980B
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CN102552980A (zh
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彭浩
周叶方
吴献青
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Third Xiangya Hospital of Central South University
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Abstract

黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架，延缓干细胞衰老，维持干细胞分化潜能的组织工程生物支架。以三维PCL－TCP聚合物陶瓷复合支架[聚乙内酯多元醇（PCL，Polycaprolactone）磷酸三钙(TCP,Tri-calciumphosphate)]生长支架为基础，结合不同特定细胞粘附胶原多肽序列（RGD、DGEA）修饰PCL－TCP支架，将多肽分子以交联方式固定于支架表面，制备延缓干细胞衰老，维持干细胞分化潜能的新型组织工程生物支架。

Description

黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架
技术领域
本发明涉及一种组织工程生物支架，尤其是黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架。
背景技术
成体干细胞具有较强组织再生修复功能，但量少且细胞活性随体外扩增次数增多而下降，限制了干细胞在临床上的应用。培养可用于临床的干细胞于三维类细胞外基质环境中可延缓干细胞衰老。
发现可延缓干细胞衰老，维持干细胞分化潜能的新型生物支架及其培养体系，对干细胞体外扩增及组织工程临床应用有重要意义。以三维聚己内酯多元醇-磷酸三钙(polycaprolactone-tricalcium phosphate PCL-TCP)生长支架为基础，结合不同特定细胞粘附胶原多肽（精氨酸-甘氨酸-天冬氨酸 Arg—Gly—Asp RGD序列、天冬氨酸-甘氨酸-谷氨酸-丙氨酸 Asp—Gly—GIu—Ala DGEA 序列）制备三维类细胞外基质环境新型组织工程生物支架，国内外尚无类似研究报道。
发明内容
本发明所解决的技术问题在于提供黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架，以解决上述背景技术中的缺点。
本发明所解决的技术问题采用以下技术方案来实现：
本发明结合不同特定细胞粘附胶原多肽序列（精氨酸-甘氨酸-天冬氨酸 Arg—Gly—Asp RGD序列、天冬氨酸-甘氨酸-谷氨酸-丙氨酸 Asp—Gly—GIu—Ala DGEA 序列）修饰PCL－TCP支架，将多肽分子以交联方式固定于支架表面，交联比例为比例为 0.2毫克多肽/每平方毫米表面积支架。
黏附特异性多肽序列支架制备方法，包括以下步骤：
第一步：PCL－TCP活化：PCL－TCP支架用10% (w/w) 1,6-hexanediamine/ isopropanol (DEA/IPA己二胺/异丙醇)溶液处理，常温25℃，时间30分钟到90分钟，完成后用0.3%Tween 20（吐温20）和去离子水充分洗涤，干燥；
第二步：0.1M多肽（精氨酸-甘氨酸-天冬氨酸 Arg—Gly—Asp RGD序列及天冬氨酸-甘氨酸-谷氨酸-丙氨酸 Asp—Gly—GIu—Ala DGEA 序列）, EDC （1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐）, N-hydroxysuccinimide (NHS,N-羟基琥珀酰亚胺)分别配制于2-(N-morpholino)-ethanesulfonic acid (MES，2-(N-吗啉)乙磺酸) 缓冲液 (pH=5.5)中；
第三步：精氨酸-甘氨酸-天冬氨酸（Arg—Gly—Asp RGD）序列及天冬氨酸-甘氨酸-谷氨酸-丙氨酸 （Asp—Gly—GIu—Ala DGEA） 序列溶液首先和EDC 溶液以 1:1 摩尔比混合；
第四步：相当于25%EDC摩尔量的NHS 加入到混合液，使得最终溶液中多肽：EDC：NHS摩尔比=4:4:1；
第五步：溶液配好立即处理支架，将已活化的PCL－TCP支架置入多肽：EDC：NHS混合溶液中，比例为 0.2毫克多肽/每平方毫米表面积支架。常温下处理时间3小时左右；
第六步：完成后用磷酸缓冲液冲洗，干燥即形成支架。
有益效果
本发明制成的组织工程支架，对于多次传代（10，20，30）骨髓间充质干细胞及其他来源的干细胞如来源于脂肪组织的干细胞，培养后的形态观察，老化细胞量及增殖速度测定、细胞增殖计数 、3H标胸腺嘧啶摄取率测定、CFU克隆增殖试验、β-半乳糖苷酶试剂盒试剂盒测定衰老程度测定、增殖衰老相关蛋白质(PPARr, stro-1, P53, C-myc, Bcl-2, TERT)表达变化情况、检测不同传代细胞表面抗原及凋亡细胞量，证实: 本发明制成的组织工程支架能够延缓干细胞衰老，维持间充质干细胞及其他类型的干细胞的分化潜能。
附图说明
图1为本发明的实施工艺流程图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体图示，进一步阐述本发明。
参见图1，黏附特异性多肽序列支架的实施工艺流程图，PCL－TCP支架用10% (w/w) 1,6-hexanediamine/ isopropanol (DEA/IPA己二胺/异丙醇)溶液处理活化，常温25℃，时间30分钟到90分钟，完成后用0.3%Tween 20（吐温20）和去离子水充分洗涤，干燥； 0.1M多肽（精氨酸-甘氨酸-天冬氨酸 Arg—Gly—Asp RGD序列及天冬氨酸-甘氨酸-谷氨酸-丙氨酸 Asp—Gly—GIu—Ala DGEA 序列）, EDC （1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐盐）,N-hydroxysuccinimide (NHS,N-羟基琥珀酰亚胺)分别配制于2-(N-morpholino)-ethanesulfonic acid (MES，2-(N-吗啉)乙磺酸) 缓冲液 (pH of 5.5)中。精氨酸-甘氨酸-天冬氨酸（Arg—Gly—Asp RGD）序列及天冬氨酸-甘氨酸-谷氨酸-丙氨酸 （Asp—Gly—GIu—Ala DGEA） 序列溶液首先和EDC 溶液以 1:1 摩尔比混合；相当于25%EDC摩尔量的NHS 溶液加入到混合液，使得最终溶液中多肽：EDC：NHS摩尔比=4:4:1。溶液配好立即处理支架，将已活化PCL－TCP支架置入多肽、EDC、NHS混合溶液中，比例为 0.2毫克多肽/每平方毫米表面积支架。常温下处理时间3小时左右。完成后用磷酸缓冲液冲洗，干燥即形成支架。
以上显示和描述了本发明的基本原理和主要特征及本发明的优点，本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内，本发明要求保护范围由所附的权利要求书及其等效物界定。

Claims (1)

1.黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架的制备方法，其特征在于，包括以下步骤：
第一步：PCL－TCP支架活化：PCL－TCP支架用10%w/w的己二胺/异丙醇溶液处理，常温25℃时间30分钟到90分钟，完成后用0.3%吐温20和去离子水充分洗涤，干燥；
第二步：0.1M多肽，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐，N-羟基琥珀酰亚胺分别配制于pH=5.5的2-(N-吗啉)乙磺酸缓冲液中；所述多肽为精氨酸-甘氨酸-天冬氨酸Arg-Gly-Asp RGD序列及天冬氨酸-甘氨酸-谷氨酸-丙氨酸Asp-Gly-GIu-Ala DGEA序列；
第三步：多肽溶液首先和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐溶液以1:1摩尔比混合；
第四步：25% 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐摩尔量的N-羟基琥珀酰亚胺溶液加入到混合液，使得最终溶液中多肽：1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐：N-羟基琥珀酰亚胺的摩尔比=4:4:1；
第五步：将已活化的PCL－TCP支架置于多肽、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐、N-羟基琥珀酰亚胺溶液中，比例为0.2毫克多肽对每平方毫米表面积支架；常温下处理时间3小时；
第六步：完成后用磷酸缓冲液冲洗，干燥即形成支架。
CN 201210007080 2012-01-11 2012-01-11 黏附特异性多肽序列聚己内酯多元醇-磷酸三钙支架 Expired - Fee Related CN102552980B (zh)

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