基于细胞膜片技术构建三维真皮样组织的体外研究  

Construction of three-dimensional dermoid tissue based on cell sheets technology in vitro

作　　者：项桦 陈锐 武姗 席大力 龙思琪 沈圆 范增杰[1] 任利玲[1] XIANG Hua;CHEN Rui;WU Shan;XI Dali;LONG Siqi;SHEN Yuan;FAN Zengjie;REN Liling(School of Stomatology,Lanzhou University,Lanzhou Gansu,730000,P.R.China;Department of Pathology,the First Affiliated Hospital of Lanzhou University,Lanzhou Gansu,730000,P.R.China)

机构地区：[1]兰州大学口腔医学院,兰州730000 [2]兰州大学第一附属医院病理科,兰州730000

出　　处：《中国修复重建外科杂志》2020年第1期116-123,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81571829、81670969、81300860);甘肃省自然科学基金资助项目(17JR5RA202)~~

摘　　要：目的探讨应用细胞膜片技术构建三维真皮样组织的新策略。方法取2~3周龄新西兰大白兔骨髓,采用全骨髓贴壁法分离培养兔BMSCs(rabbit BMSCs,rBMSCs)并传代,取人皮肤成纤维细胞(human dermal fibroblasts,HDFs)传代培养。取第2代rBMSCs、第3代HDFs分别于培养皿采用膜片条件培养基连续培养2周,获得单层细胞膜片。取人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)接种于rBMSCs膜片上,构建预血管化膜片。培养期间于倒置相差显微镜下观察膜片上细胞形态变化;共培养1、3、7、14 d后取材行HE染色、CD31免疫荧光染色,观察细胞分布及微血管网络生成情况,以rBMSCs膜片作为对照。分别将培养7 d的预血管化膜片(实验组)及rBMSCs膜片(对照组)放于2层HDFs膜片中间,制备三维真皮样组织,培养24 h后行CD31免疫荧光染色及Ⅰ、Ⅲ型胶原免疫组织化学染色,评估细胞分布及胶原表达情况。结果细胞连续培养2周后成功制备HDFs膜片及rBMSCs膜片。HUVECs接种于rBMSCs膜片3 d后重新排列,7 d后形成网状结构,14 d后网状结构更明显;HE染色见膜片间有空泡形成,且随时间延长空泡日趋明显、膜片厚度显著增加;CD31免疫荧光染色可见微血管管腔形成。而rBMSCs膜片仅见细胞膜片增厚,未见细胞形态改变、空泡结构形成。三维真皮样组织观测显示,实验组内皮细胞CD31免疫荧光染色呈阳性,rBMSCs、HDFs及HUVECs细胞排列整齐;而对照组染色呈阴性,细胞随机排列。实验组和对照组Ⅰ、Ⅲ型胶原均呈阳性表达,与对照组比较,实验组细胞排列紧密,细胞基质分布规律,呈"蜂巢状"结构;两组Ⅰ、Ⅲ型胶原表达量差异无统计学意义(P>0.05)。结论应用细胞膜片技术可以构建三维真皮样组织,其中采用HUVECs联合rBMSCs膜片构建预血管化膜片后细胞基质分布较规律,具有形成组织工程真皮的潜力。Objective To explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology. Methods Rabbit bone marrow mesenchymal stem cells(rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts(HDFs) were cultured and passaged in vitro. The 2 nd generation rBMSCs and the 3 rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells(HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet.During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet(experimental group) and rBMSCs sheet(control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression. Results HDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no ch

关 键 词：组织工程真皮 细胞膜片技术 BMSCS 内皮细胞 预血管化 

分 类 号：R622[医药卫生—整形外科]