人胚胎成纤维细胞作为组织工程皮肤种子细胞的可行性研究(1)
[摘要]目的：评估人胚胎成纤维细胞(human embryonic fibroblasts，HEF)作为组织工程皮肤种子细胞的可行性和优越性。方法：取人流产胚胎皮肤、正常儿童皮肤，相同条件下分离培养成纤维细胞，比较两组细胞镜下、超微结构以及增殖特性，异体淋巴细胞混合实验对比其抗原性。以第三代细胞复合鼠尾胶原构建三维培养，ELASA法分别测定两组三维构建培养液中IL-6，TGF-β1含量。结果：与普通成纤维细胞相比，胚胎成纤维细胞扩增后具有更好的细胞形态和功能，生长速度快，分裂指数高，几乎不刺激异体淋巴细胞增殖。在鼠尾胶原支架中成纤维细胞生长状态良好，并且具有一定的组织强度。胎儿成纤维细胞组培养液中的TGF-β1和IL-6在各个时相上分别显著低于和高于普通成纤维细胞组。结论：胚胎成纤维细胞是组织工程皮肤较理想的种子细胞。
[关键词]】组织工程；皮肤；无斑痕愈合；胚胎；成纤维细胞
[中图分类号]Q813.1[文献标识码]A[文章编号]1008-6455(2011)04-0605-04
, http://www.100md.com
Investigation on feasibility of constructing engineered skin substitutes with human embryonic fibroblasts
LIU Liu1,LI Wu-de1,CAI Guo-bin2
(1.Cleft lip and Palate Treatment Center,China Meitan General Hospital,Beijing 100028,China;2.Scar Integrated Treatment Centre,Plastic Surgery Hospital,Chinese Academy of Medical Sciences, Peking Union Medical College,Beijing 100041,China)
Abstract:ObjectiveTo assess the biological and functional properties oftissue-engineered three-dimensional (3D) composites seeded with human embryonic fibroblasts (HEF).MethodsFibroblast cultures were developed from biopsies of human fetus and children. To determine the differences between fetal and adult fibroblasts, the cell shape, ultrastructure, growth cycle were observed. In addition, mixed lymphocyte culture was examined to evaluate the antigenicity. Three-dimensional skin constructs were obtained by seeding cultured cells in rat tail collagen scaffold. The content of IL-6 and TGF-β1 in the dermis culturing supernatant was measured by ELISA method.ResultsIn monolayer culture, HEF displayed more typical pattern and faster growing rate. HEF was poor stimulators of lymphocyte proliferation. In 3D culture, fetal constructs appeared more developed based on gross examination with significantly higher level secretion of IL-6 and lower of TGF-β than adults'(P<0.05).Conclusion Fetal dermal fibroblasts may be an effective source of cells for fabricating skin equivalents.
, 百拇医药
Key words: tissue engineering; skin; scarless wound healing; fetal fibroblast
寻找理想的种子细胞一直是组织工程皮肤领域的热点和难点，由于种子细胞增殖能力有限、抗原性、与基质相容性差等问题，存在着皮肤产量低，移植后排斥反应等缺陷。前2/3妊娠期的胚胎在受到创伤后具有无瘢痕愈合的能力，而这种愈合能力完全是由胎儿自身组织细胞的内在特性决定的[1]。我们取材于人胎儿皮肤、儿童正常皮肤进行成纤维细胞培养，比较其生长特性、形态学、生长动力学以及免疫原性差异，并复合鼠尾胶原构建人工真皮，观察种子细胞在三维构建中的增殖和功能,以探讨人胚胎成纤维细胞(human embryonic fibroblasts，HEF)作为组织工程皮肤种子细胞的可行性和优越性,为组织工程种子细胞的选择及将细胞移植用于临床提供实验依据。
1材料和方法
, 百拇医药 1.1主要仪器和试剂：MCO-15A型C02培养箱，倒置生物显微镜，超净工作台，台式高速离心机，恒温水浴箱，常规医用手术器械，日本H-600透射电镜；全自动蒸汽消毒器，针式滤器；培养基(DMEM:F12为1:1，含10%胎牛血清)，中性蛋白酶。
1.2 二维培养以及相关实验
1.2.1 成纤维细胞取材：①实验组：5例，取人孕中期(20～28周)引产胎儿，性别不限；②对照组：12例，取自12岁以下瘢痕患者手术改形时修剪下的正常皮肤。
1.2.2 成纤维细胞的培养和传代：采用组织块法培养：将标本消毒后无菌条件下取中厚皮，切成1～1.5mm3的小块，将组织块贴到培养瓶内，加入培养基孵育，待成纤维细胞达60%～70%左右，传代培养。取两组第三代细胞进行光镜、透射电镜观察，绘制细胞生长曲线。
1.2.3 淋巴细胞体外增殖实验，参考文献[2]中毕建军等的方法，计量数据用均数士标准差(x±s)表示。P<0.05有显著性差异，并绘制不同代数细胞cpm值变化趋势图。
, 百拇医药
1.3 三维培养以及相关实验
1.3.1 制作三维构建：取第三代HEFs和普通FBs，制成1×106个/cm3细胞悬液。迅速与鼠尾胶原液、重组缓冲液(2.756g碳酸钠、4.766gHEPES、0.35gNaOH溶于100ml蒸馏水中)以1200:4200:300的比例加入6孔培养板中，在孵箱内孵育30min后即形成胶原凝胶。加入DMEM:F12为1:1的含10%胎牛血清的培养基在孵箱内继续培养。
1.3.2 测定构建培养液中TGF-β1和IL-6含量：分别在1天、3天、5天、7天、9天、11天、14天每天收集两组各6个孔培养液，每次换液两组加液量均为5ml，用人IL-6、TGF-β1 ELISA试剂盒检测细胞因子含量。, 百拇医药(刘柳 李武德 蔡国斌)
[关键词]】组织工程；皮肤；无斑痕愈合；胚胎；成纤维细胞
[中图分类号]Q813.1[文献标识码]A[文章编号]1008-6455(2011)04-0605-04
, http://www.100md.com
Investigation on feasibility of constructing engineered skin substitutes with human embryonic fibroblasts
LIU Liu1,LI Wu-de1,CAI Guo-bin2
(1.Cleft lip and Palate Treatment Center,China Meitan General Hospital,Beijing 100028,China;2.Scar Integrated Treatment Centre,Plastic Surgery Hospital,Chinese Academy of Medical Sciences, Peking Union Medical College,Beijing 100041,China)
Abstract:ObjectiveTo assess the biological and functional properties oftissue-engineered three-dimensional (3D) composites seeded with human embryonic fibroblasts (HEF).MethodsFibroblast cultures were developed from biopsies of human fetus and children. To determine the differences between fetal and adult fibroblasts, the cell shape, ultrastructure, growth cycle were observed. In addition, mixed lymphocyte culture was examined to evaluate the antigenicity. Three-dimensional skin constructs were obtained by seeding cultured cells in rat tail collagen scaffold. The content of IL-6 and TGF-β1 in the dermis culturing supernatant was measured by ELISA method.ResultsIn monolayer culture, HEF displayed more typical pattern and faster growing rate. HEF was poor stimulators of lymphocyte proliferation. In 3D culture, fetal constructs appeared more developed based on gross examination with significantly higher level secretion of IL-6 and lower of TGF-β than adults'(P<0.05).Conclusion Fetal dermal fibroblasts may be an effective source of cells for fabricating skin equivalents.
, 百拇医药
Key words: tissue engineering; skin; scarless wound healing; fetal fibroblast
寻找理想的种子细胞一直是组织工程皮肤领域的热点和难点，由于种子细胞增殖能力有限、抗原性、与基质相容性差等问题，存在着皮肤产量低，移植后排斥反应等缺陷。前2/3妊娠期的胚胎在受到创伤后具有无瘢痕愈合的能力，而这种愈合能力完全是由胎儿自身组织细胞的内在特性决定的[1]。我们取材于人胎儿皮肤、儿童正常皮肤进行成纤维细胞培养，比较其生长特性、形态学、生长动力学以及免疫原性差异，并复合鼠尾胶原构建人工真皮，观察种子细胞在三维构建中的增殖和功能,以探讨人胚胎成纤维细胞(human embryonic fibroblasts，HEF)作为组织工程皮肤种子细胞的可行性和优越性,为组织工程种子细胞的选择及将细胞移植用于临床提供实验依据。
1材料和方法
, 百拇医药 1.1主要仪器和试剂：MCO-15A型C02培养箱，倒置生物显微镜，超净工作台，台式高速离心机，恒温水浴箱，常规医用手术器械，日本H-600透射电镜；全自动蒸汽消毒器，针式滤器；培养基(DMEM:F12为1:1，含10%胎牛血清)，中性蛋白酶。
1.2 二维培养以及相关实验
1.2.1 成纤维细胞取材：①实验组：5例，取人孕中期(20～28周)引产胎儿，性别不限；②对照组：12例，取自12岁以下瘢痕患者手术改形时修剪下的正常皮肤。
1.2.2 成纤维细胞的培养和传代：采用组织块法培养：将标本消毒后无菌条件下取中厚皮，切成1～1.5mm3的小块，将组织块贴到培养瓶内，加入培养基孵育，待成纤维细胞达60%～70%左右，传代培养。取两组第三代细胞进行光镜、透射电镜观察，绘制细胞生长曲线。
1.2.3 淋巴细胞体外增殖实验，参考文献[2]中毕建军等的方法，计量数据用均数士标准差(x±s)表示。P<0.05有显著性差异，并绘制不同代数细胞cpm值变化趋势图。
, 百拇医药
1.3 三维培养以及相关实验
1.3.1 制作三维构建：取第三代HEFs和普通FBs，制成1×106个/cm3细胞悬液。迅速与鼠尾胶原液、重组缓冲液(2.756g碳酸钠、4.766gHEPES、0.35gNaOH溶于100ml蒸馏水中)以1200:4200:300的比例加入6孔培养板中，在孵箱内孵育30min后即形成胶原凝胶。加入DMEM:F12为1:1的含10%胎牛血清的培养基在孵箱内继续培养。
1.3.2 测定构建培养液中TGF-β1和IL-6含量：分别在1天、3天、5天、7天、9天、11天、14天每天收集两组各6个孔培养液，每次换液两组加液量均为5ml，用人IL-6、TGF-β1 ELISA试剂盒检测细胞因子含量。, 百拇医药(刘柳 李武德 蔡国斌)