同一供者及不同供者的人胎盘MSCs与脐静脉内皮细胞复合培养的比较研究_《中国修复重建外科杂志》

作者：

杨婧 , 项舟 , 魏代清 , 黄潇 , 高宇 , 高博 , 舒涛 , 段鑫 , 张兴栋

四川大学华西医院骨科（成都，610041）;

关键词：

组织工程骨血管化人胎盘MSCs 人脐静脉内皮细胞复合培养

DOI：

10.7507/1002-1892.20130202

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的比较同一供者及不同供者的人胎盘MSCs（human placenta-derived MSCs，HPMSCs）与人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）三维复合培养效果，为体外构建三维血管化组织工程骨提供实验依据。方法取健康足月产妇自愿捐赠的胎盘及脐带组织，采用Ⅳ型胶原酶消化和人淋巴细胞分层液密度梯度离心法分离培养HPMSCs，通过流式细胞学检测细胞表型，取第2代细胞向成骨细胞诱导培养鉴定；以Ⅰ型胶原酶消化分离培养HUVECs，通过Ⅷ因子相关抗原因子免疫组织化学染色进行鉴定。实验分为两组，A组取不同供者的第3代HUVECs和成骨诱导培养3周的第3代HPMSCs以1∶1比例复合种植于胶原水凝胶，制备细胞-胶原水凝胶复合物；B组将同一供者的以上两种细胞同法复合制备复合物。复合培养第1、3、5、7天取材，行CD31免疫荧光染色，于激光共聚焦显微镜下观察其成血管倾向，测量复合物中的索道长度和节点数，并进行统计学分析。结果经鉴定，成功分离培养HPMSCs及HUVECs。CD31免疫荧光染色示，与A组相比，B组细胞-胶原水凝胶复合物中的HUVECs可在三维空间上较早、较好地伸展，在水凝胶内部相互交织能力较强，形成的索道更长，节点更多，网络更密集。第7天时B组索道长度和节点数分别为（8.11 ± 0.62）mm/mm2及（21.30 ± 1.41）个/mm2，显著优于A组的（6.68 ± 0.35） mm/mm2及（17.10 ± 1.10）个/mm2，差异均有统计学意义（t=0.894，P=0.000；t=0.732，P=0.000）。结论将同一供者的HPMSCs与HUVECs体外复合种植于胶原水凝胶支架上，比不同供者来源的细胞形成的网络成血管倾向更明显，交织连续性好，层次丰富。

引用本文： 杨婧,项舟,魏代清,黄潇,高宇,高博,舒涛,段鑫,张兴栋. 同一供者及不同供者的人胎盘MSCs与脐静脉内皮细胞复合培养的比较研究. 中国修复重建外科杂志, 2013, 27(8): 916-922. doi: 10.7507/1002-1892.20130202 复制

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1. Lovett M, Lee K, Edwards A, et al. Vascularization strategies for tissue engineering. Tissue Eng Part B Rev, 2009, 15(3): 353-370.
2. Dohle E, Fuchs S, Kolbe M, et al. Sonic hedgehog promotes angiogenesis and osteogenesis in a coculture system consisting of primary osteoblasts and outgrowth endothelial cells. Tissue Eng Part A, 2010, 16(4): 1235-1237.
3. Carmeliet P. Angiogenesis in life, disease and medicine. Nature, 2005, 438(7070): 932-936.
4. 参考文献.
5. 杨志明. 组织工程基础与临床. 成都: 四川科学技术出版社, 2000: 1-11.
6. Kolbe M, Xiang Z, Dohle E, et al. Paracrine effects influenced by cell culture medium and consequences on microvessel-like structures in cocultures of mesenchymal stem cells and outgrowth endothelial cells. Tissue Eng Part A, 2011, 17(17-18): 2199-2212.
7. Orkin SH, Morrison SJ. Stem-cell competition. Nature, 2002, 418(6893): 25-27.
8. Fukuchi Y, Nakajima H, Sugiyama D, et al. Human placenta-derived cells have mesenchymal stem/progenitor cell potential. Stem Cells, 2004, 22(5): 649-658.
9. Longo UG, Loppini M, Berton A, et al. Stem cells from umbilical cord and placenta for musculoskeletal tissue engineering. Curr Stem Cell Res Ther, 2012, 7(4): 272-281.
10. Maruyama Y. The human endothelial cell in tissue culture. Z Zellforsch Mikrosk Anat, 1963, 60: 69-79.
11. Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest, 1973, 52(11): 2745-2756.
12. Gimbrone MA Jr, Cotran RS, Folkman J. Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Biol, 1974, 60(3): 673-684.
13. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery, 1978, 84(4): 498-504.
14. Baudin B, Bruneel A, Bosselut N, et al. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc, 2007, 2(3): 481-485.
15. Marin V, Kaplanski G, Grès S, et al. Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells. J Immunol Methods, 2001, 254(1-2): 183-190.
16. Sumita Y, Honda MJ, Ohara T, et al. Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering. Biomaterials, 2006, 27(17): 3238-3248.
17. Liu C, Xia Z, Czernuszka JT. Design and development of three-dimensional scaffolds for tissue engineering. Chemical Engineering Research and Design, 2007, 85(7): 1051-1064.
18. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell, 1982, 30(1): 215-224.
19. Schindler OS. Current concepts of articular cartilage repair. Acta Orthop Belg, 2011, 77(6): 709-726.
20. Lee SH, Shin H. Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Adv Drug Deliv Rev, 2007, 59(4-5): 339-359.
21. Balakrishnan B, Banerjee R. Biopolymer-based hydrogels for cartilage tissue engineering. Chem Rev, 2011, 111(8): 4453-4474.
22. Wenger A, Stahl A, Weber H, et al. Modulation of in vitro angiogenesis in a three-dimensional spheroidal coculture model for bone tissue engineering. Tissue Eng, 2004, 10(9-10): 1536-1547.
23. Borges J, Tegtmeier FT, Padron NT, et al. Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model. Tissue Eng, 2003, 9(3): 441-450.
24. Unger RE, Sartoris A, Peters K, et al. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials. Biomaterials, 2007, 28(27): 3965-3976.
25. Lutolf MP. Biomaterials: Spotlight on hydrogels. Nat Mater, 2009, 8(6): 451-453.
26. Sharma MB, Limaye LS, Kale VP, et al. Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells. Haematologica, 2012, 97(5): 651-660.
27. Kanga Y, Kima S, Fahrenholtzb M, et al. Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold. Acta Biomaterialia, 2013, 9(1): 4906-4915.
28. Li H, Daculsi R, Grellier M, et al. The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network. PLoS One, 2011, 6(2): e16767.
29. Tsigkou O, Pomerantseva I, Spencerc JA, et al. Engineered vascularized bone grafts. Proc Natl Acad Sci U S A, 2010, 107(8): 3311-3316.

《中国修复重建外科杂志》

同一供者及不同供者的人胎盘MSCs与脐静脉内皮细胞复合培养的比较研究

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《中国修复重建外科杂志》

同一供者及不同供者的人胎盘MSCs与脐静脉内皮细胞复合培养的比较研究

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