课题背景：气管重建是气管外科学中的一个重要课题。许多学者尝试应用同种异体移植、自体组织移植以及人工材料替代物等方法，效果均不够理想。近年来，随着组织工程学的发展，有学者开始了组织工程化气管的研究工作。解放军第四军医大学第二附属医院呼吸科立足组织工程气管替代物的实验，为气管重建的研究开拓了一条新路。

应用要点：常规分离培养法的培养需要2次取材，所以过程相对繁琐，加之上皮细胞之间以及与成纤维细胞间连接紧密，很难严格把握酶浓度及消化时间。消化不到位则分离下来的细胞数量较少，消化过度则会将黏膜层的成纤维细胞一同消化下来，同时还会破坏已经消化下来细胞膜的完整性，影响上皮细胞的贴壁、增殖与使用。联合培养方法简化了操作过程，同时减少了污染几率。

相关链接：组织工程化气管的构建过程是：首先选取一定数量的经体外培养扩增种子细胞，把它们种植在生物相容性好、可降解的支架材料上；然后将此细胞材料复合物置于生物反应器当中或自体皮下，经过一段时间的诱导分化和培养即形成组织工程化气管。然而，要将组织工程化气管真正应用到临床，还有很长的路要走，首要做的是先从种子细胞的培养方面进行初步研究。

摘要
目的：构建体外组织工程化气管种子细胞的共培养模式，并与常规培养相比较，为细胞复合材料构建组织工程化气管奠定研究基础。
方法：实验于2006-05/2007-05在解放军第四军医大学唐都医院呼吸科实验室完成。①实验材料：1个月龄雄性健康新西兰兔3只，体质量（250.00±0.75）g。②实验过程：联合培养模式：分离气管上皮细胞和成纤维细胞，联合培养7~10 d后，根据两种细胞对胰酶浓度耐受性不同的特性，A孔中加入0.5 g/L胰酶消化，用体积分数为0.05的胎牛血清的培养液DMEM重悬细胞后移入培养板B孔中(成纤维细胞)；A孔中再次加入2.5 g/L胰酶继续消化后，用K-FSM培养液重悬细胞后移入培养板C孔中(气管上皮细胞)。常规培养法：分别进行气管上皮细胞分离培养和成纤维细胞分离纯化。③实验评估：观察细胞生长曲线与四甲基偶氮唑盐检测细胞的生长状态，并与常规培养的细胞进行比较。
结果：①细胞生长状态：联合培养的两种细胞生长状态良好，A孔加入胰酶消化后，细胞间隙增大；B孔细胞呈现长梭形，未见铺路石状细胞；C孔出现片状生长的铺路石状细胞，未见长梭形细胞。②细胞生长曲线：分离后的细胞与常规方法培养的细胞生长曲线一致。③细胞增殖情况：四甲基偶氮唑盐检测联合培养组细胞增殖与常规方法培养差异无显著性（P < 0.05）。
结论：联合培养模式较常规方法更加简便易行，可以作为一种较好的气管组织工程种子细胞体外培养模式得到应用。
关键词：组织工程；气管；种子细胞；联合培养

王琳，姜威. 构建组织工程气管上皮细胞和成纤维细胞联合培养的模式：与常规培养的比较[J].中国组织工程研究与临床康复，2008，12(15):2825-2828 [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2825(ps).pdf]

Co-culture mode of tissue-engineered tracheal epithelial cells and fibroblasts: Comparison with routine culture

Abstract

AIM: To establish the co-culture mode of tissue-engineered tracheal seeding cells, and compare with the routine culture, so as to provide the fundament of tissue-engineered trachea construction by cell compound materials.
METHODS: The experiment was completed at the laboratory, Department of Respiratory Diseases, Tangdu Hospital, the Fourth Military Medical University of Chinese PLA from May 2006 to May 2007.①Three healthy New Zealand male rabbits, weighed (250.00±0.75) g, were used in this study.②Co-culture mode: The tracheal epithelial cells and fibroblasts were isolated and then co-cultured for 7-10 days. The two kinds of cells were distinguished according to their different tolerances to trypsin. Subsequently, A hole received 0.5 g/L trypsinization, cells were suspended in DMEM medium containing 0.05 volume fraction of CO2 and then transplanted into B hole (fibroblasts); again, A hole was added with 2.5 g/L trypsin for digestion, afterwards cells were suspended with K-FSM medium and transplanted into C hole (tracheal epithelial cells). Routine culture: after isolation, tracheal epithelial cells were cultured and fibroblasts were purified, respectively.③The co-cultured cells and the traditionally cultured cells were compared via cell growth curve and cell proliferation detected by 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.
RESULTS: ①Cell growth: Co-cultured cells grew well, those in A hole increased intercellular apace after trypsinization, while cells in B hole were shaped as fusiform, without the manifestation of typical road stone-like cells, and cells in C hole were apposite, showing lamellar road stone-like morphology.②Cell growth curve: The growth curve of co-cultured cells was identical with that of cells by routine culture method.③Cell proliferation: MTT detection revealed no significant difference in the proliferation of co-cultured cells and cells cultured by traditional method (P < 0.05).
CONCLUSION: Compared with routine culture method, co-culture model is an effective method to establish tissue-engineered tracheal seeding cells culture in vitro.

Wang L, Jiang W. Co-culture mode of tissue-engineered tracheal epithelial cells and fibroblasts: Comparison with routine culture.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(15):2825-2828(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2825(ps).pdf]