CAJ | 학술논문

背景：三维(3D)组织化培养模型的体外构建是现代组织工程与再生医学工程技术的重要核心。如何实现所培养模型的微血管化以改善培养体系内部的营养传递并最终提高细胞的活性是组织工程研究领域所亟待解决的关键。 　　目的：尝试探索在3D体系内采用细胞共培养技术促进类前微血管结构发生的可行性。 　　方法：以蚕丝蛋白为多孔材料支架，将人骨髓间充质干细胞与血管内皮细胞进行体外共培养。通过DNA含量测定定量检测细胞的增殖；扫描电镜和激光共聚焦显微镜的图像分析表征细胞的生长形态学特征；实时定量RT-PCR方法对内皮细胞功能性标志基因的表达水平进行定量分析。 　　结果与结论：丝蛋白支架和人骨髓间充质干细胞能够提供理想的3D生长微环境，利于血管内皮细胞的体外增殖。微环境还能够显著提高内皮细胞功能性标志基因CD31和vWF的表达水平，促进类前微血管结构的发生。提示共培养体系有利于内皮细胞在体外的进一步分化和自组织化，可能为微血管化组织工程研究提供一定的技术基础。
배경：삼유(3D)조직화배양모형적체외구건시현대조직공정여재생의학공정기술적중요핵심。여하실현소배양모형적미혈관화이개선배양체계내부적영양전체병최종제고세포적활성시조직공정연구영역소극대해결적관건。 　　목적：상시탐색재3D체계내채용세포공배양기술촉진류전미혈관결구발생적가행성。 　　방법：이잠사단백위다공재료지가，장인골수간충질간세포여혈관내피세포진행체외공배양。통과DNA함량측정정량검측세포적증식；소묘전경화격광공취초현미경적도상분석표정세포적생장형태학특정；실시정량RT-PCR방법대내피세포공능성표지기인적표체수평진행정량분석。 　　결과여결론：사단백지가화인골수간충질간세포능구제공이상적3D생장미배경，리우혈관내피세포적체외증식。미배경환능구현저제고내피세포공능성표지기인CD31화vWF적표체수평，촉진류전미혈관결구적발생。제시공배양체계유리우내피세포재체외적진일보분화화자조직화，가능위미혈관화조직공정연구제공일정적기술기출。
BACKGROUND:Constructing a three-dimensional tissue-like structure in vitro plays a critical role in modern tissue engineering and regenerative medicine. Several advances have been made in the past decade. However, it is stil a chal enge to promote microvascular-like structure formation and improve limited nutritional transportation, thereby promoting cel viability. OBJECTIVE:To explore the feasibility of constructing a three-dimensional microvascular-like structure through the co-culture technique. METHODS:Human bone marrow mesenchymal stem cel s and human endothelial cel s were co-cultured on a three-dimensional porous silk scaffold. Cel proliferation was analyzed by Pico-green DNA assay. Their growth profiles were evaluated by scanning electron microscope and laser scanning confocal microscopy, respectively. The mRNA levels of von Wil ebrand factor and CD31, two key functional markers of endothelial cel s, in the co-cultured endothelial cel s was assayed by real-time quantitative reverse transcription-PCR. RESULTS AND CONCLUSION:The three-dimensional culture system constructed by the silk scaffold and bone marrow mesenchymal stem cel s provided an ideal microenvironment for cel growth and proliferation in vitro. Moreover, this microenvironment was capable of promoting endothelial cel differentiation evidenced by their significantly improved mRNA levels of von Wil ebrand factor and CD31. Premicrovascular-like structure was also observed in the co-cultures under the confocal microscope. Thus, al the data supported that the unique co-culture system could promote endothelial cel differentiation and self-assembling in vitro. This culture system provides a robust tool for the studies addressing microvessel-based tissue engineering.