论文部分内容阅读
目的观察PKH26与BrdU体外双重标记兔BMSCs的生物学特性,并体外构建组织工程心肌补片。方法取6月龄新西兰大白兔分离培养BMSCs,并用细胞膜荧光染料PKH26和细胞核标记物BrdU对BMSCs进行标记,通过倒置相差显微镜、荧光显微镜、流式细胞仪、MTT法观察细胞生长状态和荧光标记前后细胞生物学特性的变化;ALP、茜素红、油红O染色及骨钙素、Ⅰ型胶原蛋白等免疫细胞化学技术检测,观察和评价标记前后BMSCs体外分化为成骨细胞和成脂细胞的能力。将标记细胞与小肠黏膜下层(small intestinal submucosa,SIS)复合后共培养5～7d构建组织工程心肌补片,在倒置相差显微镜、荧光显微镜和扫描电镜下观察细胞在SIS上的生长情况,并做活体及石蜡包埋HE染色观察。结果标记后细胞生长状态良好,基本生长特性无明显改变;荧光显微镜下可见标记后细胞膜上红色荧光呈颗粒状分布;细胞表面干细胞标志性抗原表达与标记前无明显差异。标记后的BMSCs成骨诱导后,ALP、茜素红染色阳性,细胞表达骨钙素及Ⅰ型胶原蛋白;成脂诱导后,细胞胞浆内出现明显的脂滴。标记细胞与SIS共培养5～7d后,标记细胞生长状态良好,在材料上呈现出多层细胞结构。活体及石蜡包埋后HE染色可见细胞生长状态良好。结论兔BMSCs能被PKH26和BrdU稳定标记;标记的细胞在体外具有自我更新和多向分化的能力;标记的BMSCs与SIS体外复合培养可以构建组织工程心肌补片。
Objective To observe the biological characteristics of dual labeled rabbit BMSCs with PKH26 and BrdU in vitro and to construct tissue engineered myocardium patch in vitro. Methods BMSCs were isolated from 6-month-old New Zealand white rabbits and labeled with fluorescent dye PKH26 and BrdU. The morphological changes of BMSCs were observed by inverted phase contrast microscope, fluorescence microscope, flow cytometry and MTT assay before and after fluorescent labeling The changes of biological characteristics of cells were detected by immunocytochemical techniques such as ALP, alizarin red, oil red O staining and osteocalcin and collagen type Ⅰ. The differentiation of BMSCs into osteoblasts and adipocytes was observed and evaluated before and after labeling ability. The labeled cells were co-cultured with small intestinal submucosa (SIS) for 5 to 7 days to construct tissue engineered cardiac muscle patch. The growth of SIS cells was observed under inverted phase contrast microscope, fluorescence microscope and scanning electron microscope. Living and paraffin embedded HE staining observed. Results After labeling, the cell growth was good and the basic growth characteristics did not change significantly. The red fluorescence of the cell membrane showed a granular distribution under the fluorescence microscope. The expression of the labeled antigen on the cell surface was not significantly different from that before labeling. After labeled osteoblast-induced BMSCs were stained with ALP and Alizarin Red, the cells expressed osteocalcin and collagen Ⅰ. After adipogenic induction, obvious lipid droplets appeared in cytoplasm. After the labeled cells were co-cultured with SIS for 5 to 7 days, the labeled cells grew well and showed multi-layered cell structure on the material. After in vivo and paraffin embedding, HE staining showed that the cells grew well. Conclusion Rabbit BMSCs can be stably labeled with PKH26 and BrdU. The labeled cells have the ability of self-renewal and multidirectional differentiation in vitro. The labeled BMSCs and SIS in vitro can be used to construct tissue-engineered cardiac muscle patch.