以脱细胞脐动脉为支架构建组织工程血管的研究
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摘要
组织工程血管的构建是组织工程研究领域的重点和难点，其成功构建不仅为可供移植的材料提供了新的来源，更为临床上诸多心血管疾病的患者带来治愈的希望。本研究拟以人毛囊间充质干细胞为种子细胞，以脱细胞胎儿脐动脉为支架材料，体外构建组织工程血管，所得结果如下：
（一）应用拔取成人毛发的方法，从中分离获得人毛囊间充质干细胞，通过显微镜观察、流式细胞分析的方法，从形态学、分子标记物方面检测一般特性；通过诱导成骨、成脂肪及成软骨检测其多向分化潜能的生物学行为；通过流式细胞分析、免疫荧光染色及实时定量PCR对人毛囊间充质干细胞来源的平滑肌细胞进行鉴定，发现其表达成熟平滑肌细胞的特异性标志物。从而证明人毛囊间充质干细胞可以作为构建组织工程血管的种子细胞。
（二）获取在临床上废弃的胎儿脐动脉，经胰蛋白酶和十二烷基硫酸钠联合处理去掉脐动脉的细胞成分作为组织工程血管的支架。组织学观察发现该动脉细胞成分已经完全去除，但胶原和弹力纤维的结构仍保持完好。扫描电镜进一步确定纤维互相交织，为以后种子细胞的铺展、迁徙、分化提供合适的生存环境。细胞化学及生物化学分析还发现脱细胞脐动脉细胞外基质成分依然存在，胶原蛋白及弹力纤维的含量并未发生明显变化。将脱细胞前后的脐动脉进行生物力学的比较发现脱细胞的脐动脉具有较好的生物力学性质。可见利用临床废弃组织，经脱细胞处理后作为组织工程血管的支架，具有很好的应用前景。
（三）将人毛囊间充质干细胞接种在脱细胞脐动脉支架上构建组织工程血管，组织学确定种子细胞黏附并迁移至血管的中膜，免疫组织化学染色检测平滑肌标志物α-actin和calponin呈阳性表达。重要的是构建的组织工程血管已经表现出对血管活性物质如氯化钾、去甲肾上腺素及硝普钠的反应活性，从而成功构建了在组织结构和生理功能与天然血管极为接近的组织工程血管。
综上所述，我们建立了高效的分离、纯化、培养及扩增人毛囊间充质干细胞的技术体系；制备了具有合理的空间结构及组织相容性的脱细胞动脉支架；成功构建了具有生物活性的组织工程血管，为开展无免疫排斥的个性化血管移植治疗打下坚实的实验基础。
Cardiovascular disease is the leading cause of mortality and morbidity in thewest. Autologous tissues are presently preferred for surgical grafts of cardiovasculardisease but have the disadvantage of being insufficient in quantity or of poor quality.In contrast， the use of allografts or xenografts for cardiovascular repair carries therisks of severe immune rejection and disease transmission， which limit their clinicalapplication. Tissue engineering is an emerging multidisciplinary frontierscience involving molecular biology， cell biology， bioengineering， andclinical medicine that is likely to revolutionize the ways to improvethe health and quality of life for millions of people worldwide byrestoring， maintaining or enhancing tissue and organ function. This experiment aimsto determine if hair follicle mesenchymal stem cells can be used as seed cells incombination with an acellular umbilical artery as scaffold material to construct asmall tissue-engineered vascular graft.
Materials&Methods: We isolated mesenchymal stem cells from human hairfollicle (HF-MSC). The HF-MSC display CD44， CD90， CD73and CD105andexhibit multipotency toward to adipocytes， osteoblasts and condrocytes underappropriate culture conditions. Very promisingly HF-MSC expressed vascular smoothmuscle specific markers: ɑ-actin， caloponin and myosin heavy chain. The expressionof ɑ-actin and caloponin of HF-MSC significantly increased when cultured in thepresence of TGF-β， shift from30%to90%by flow cytometry analysis， whichprovides potential smooth muscle cells (HF-SMC) for cardiovascular tissueengineering. Cord blood vessel is a rich source of native human biomaterials，providing unlimited material sources for engineering artificial organs， in particularfor engineering cardio vasculature as their components， histological structure andmechanical properties are very similar to cardio vasculature. We created decellularsmall diameter blood vessel scaffolds by digesting the arterial cord blood vessels withtrypsin and SDS. The decellular arterial scaffolds (De-AS) remained the main extracellular matrix， such as， collagen， proteoglycans and elastin and mechanicalproperties by histochemistry and biochemistry， compared to their native counterparts.We seeded HF-SMC into the De-AS.
Results: The HF-SMC in the De-AS migrated approximately to the2/3of theDe-AS from the lumen， distributed circumferentially and still expressed ɑ-actin andcalponin， laying the base for the generation of functional vasoreactivity in responseto constrictors.
Conclusion: In this study we have allowed hair follicle mesenchymal stem cellsto grow on an acellular umbilical artery. With this approach we have successfullyconstructed a tissue-engineered blood vessel.
（一）应用拔取成人毛发的方法，从中分离获得人毛囊间充质干细胞，通过显微镜观察、流式细胞分析的方法，从形态学、分子标记物方面检测一般特性；通过诱导成骨、成脂肪及成软骨检测其多向分化潜能的生物学行为；通过流式细胞分析、免疫荧光染色及实时定量PCR对人毛囊间充质干细胞来源的平滑肌细胞进行鉴定，发现其表达成熟平滑肌细胞的特异性标志物。从而证明人毛囊间充质干细胞可以作为构建组织工程血管的种子细胞。
（二）获取在临床上废弃的胎儿脐动脉，经胰蛋白酶和十二烷基硫酸钠联合处理去掉脐动脉的细胞成分作为组织工程血管的支架。组织学观察发现该动脉细胞成分已经完全去除，但胶原和弹力纤维的结构仍保持完好。扫描电镜进一步确定纤维互相交织，为以后种子细胞的铺展、迁徙、分化提供合适的生存环境。细胞化学及生物化学分析还发现脱细胞脐动脉细胞外基质成分依然存在，胶原蛋白及弹力纤维的含量并未发生明显变化。将脱细胞前后的脐动脉进行生物力学的比较发现脱细胞的脐动脉具有较好的生物力学性质。可见利用临床废弃组织，经脱细胞处理后作为组织工程血管的支架，具有很好的应用前景。
（三）将人毛囊间充质干细胞接种在脱细胞脐动脉支架上构建组织工程血管，组织学确定种子细胞黏附并迁移至血管的中膜，免疫组织化学染色检测平滑肌标志物α-actin和calponin呈阳性表达。重要的是构建的组织工程血管已经表现出对血管活性物质如氯化钾、去甲肾上腺素及硝普钠的反应活性，从而成功构建了在组织结构和生理功能与天然血管极为接近的组织工程血管。
综上所述，我们建立了高效的分离、纯化、培养及扩增人毛囊间充质干细胞的技术体系；制备了具有合理的空间结构及组织相容性的脱细胞动脉支架；成功构建了具有生物活性的组织工程血管，为开展无免疫排斥的个性化血管移植治疗打下坚实的实验基础。
Cardiovascular disease is the leading cause of mortality and morbidity in thewest. Autologous tissues are presently preferred for surgical grafts of cardiovasculardisease but have the disadvantage of being insufficient in quantity or of poor quality.In contrast， the use of allografts or xenografts for cardiovascular repair carries therisks of severe immune rejection and disease transmission， which limit their clinicalapplication. Tissue engineering is an emerging multidisciplinary frontierscience involving molecular biology， cell biology， bioengineering， andclinical medicine that is likely to revolutionize the ways to improvethe health and quality of life for millions of people worldwide byrestoring， maintaining or enhancing tissue and organ function. This experiment aimsto determine if hair follicle mesenchymal stem cells can be used as seed cells incombination with an acellular umbilical artery as scaffold material to construct asmall tissue-engineered vascular graft.
Materials&Methods: We isolated mesenchymal stem cells from human hairfollicle (HF-MSC). The HF-MSC display CD44， CD90， CD73and CD105andexhibit multipotency toward to adipocytes， osteoblasts and condrocytes underappropriate culture conditions. Very promisingly HF-MSC expressed vascular smoothmuscle specific markers: ɑ-actin， caloponin and myosin heavy chain. The expressionof ɑ-actin and caloponin of HF-MSC significantly increased when cultured in thepresence of TGF-β， shift from30%to90%by flow cytometry analysis， whichprovides potential smooth muscle cells (HF-SMC) for cardiovascular tissueengineering. Cord blood vessel is a rich source of native human biomaterials，providing unlimited material sources for engineering artificial organs， in particularfor engineering cardio vasculature as their components， histological structure andmechanical properties are very similar to cardio vasculature. We created decellularsmall diameter blood vessel scaffolds by digesting the arterial cord blood vessels withtrypsin and SDS. The decellular arterial scaffolds (De-AS) remained the main extracellular matrix， such as， collagen， proteoglycans and elastin and mechanicalproperties by histochemistry and biochemistry， compared to their native counterparts.We seeded HF-SMC into the De-AS.
Results: The HF-SMC in the De-AS migrated approximately to the2/3of theDe-AS from the lumen， distributed circumferentially and still expressed ɑ-actin andcalponin， laying the base for the generation of functional vasoreactivity in responseto constrictors.
Conclusion: In this study we have allowed hair follicle mesenchymal stem cellsto grow on an acellular umbilical artery. With this approach we have successfullyconstructed a tissue-engineered blood vessel.
引文
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