Zhang Jue, Zhi Wei, Tan Meiyun, Chen Xiaohe, Li Xiuqun, Deng Li
Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P R China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Jul;24(7):828-33.
To study the biological characteristic of rabbit bone marrow mesenchymal stem cells (BMSCs) double-labeled by PKH26 and BrdU in vitro, and to construct tissue engineered cardiac patch in vitro.
The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensity were observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. The characteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkaline phosphatase (ALP) staining, Alizarin red staining, Oil red O staining, immunocytochemical technique of collagen type I, and osteocalcin expression. The labeled BMSCs were seeded on the small intestinal submucosa (SIS) and co-cultured for 5-7 days to construct tissue engineered cardiac patch. The patches were tested by inverted phase contrast microscope, fluorescent microscope, scanning electron microscope, and HE staining to observe the cell proliferation.
The double-labeled cells grew well and showed red fluorescence. There was no significant difference in the growth characteristic between the labeled and unlabeled cells. There was no significant difference in the expression of stem cell specific surface antigen between before labeling and after labeling. After osteogenic induction of labeled BMSCs, ALP staining and Alizarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, lipid droplets could be observed in cytoplasm by Oil red O staining. After the co-culture in vitro for 5-7 days, the double-labeled cells grew well, showing a multi-layer cellular structure on the surface of SIS.
Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal ability and multi potent differentiation ability; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.
研究体外经PKH26和BrdU双标记的兔骨髓间充质干细胞（BMSCs）的生物学特性，并构建体外组织工程心脏补片。
从6月龄新西兰兔中获取BMSCs，并用PKH26和BrdU进行标记。通过倒置相差显微镜、荧光显微镜、流式细胞术和MTT检测观察细胞的生长及荧光强度。采用碱性磷酸酶（ALP）染色、茜素红染色、油红O染色、Ⅰ型胶原免疫细胞化学技术及骨钙素表达鉴定双标记BMSCs体外分别向成骨细胞和脂肪细胞分化的特性。将标记的BMSCs接种于小肠黏膜下层（SIS）上，共培养5 - 7天构建组织工程心脏补片。通过倒置相差显微镜、荧光显微镜、扫描电子显微镜及HE染色检测补片，观察细胞增殖情况。
双标记细胞生长良好，呈现红色荧光。标记细胞与未标记细胞的生长特性无显著差异。标记前后干细胞特异性表面抗原的表达无显著差异。标记的BMSCs经成骨诱导后，ALP染色和茜素红染色呈阳性，细胞表达Ⅰ型胶原和骨钙素。经脂肪细胞诱导后，油红O染色可见细胞质内有脂滴。体外共培养5 - 7天后，双标记细胞生长良好，在SIS表面呈现多层细胞结构。
兔BMSCs可被PKH26和BrdU稳定双标记。标记后的细胞仍具有自我更新能力和多向分化潜能；通过体外将标记的BMSCs与SIS共培养可构建组织工程心脏补片。
