摘要
目的建立一种简便高效的定向诱导小鼠胚胎干细胞向Ⅱ型肺泡细胞分化的方法。方法①通过细胞培养，获得充足小鼠胚胎干细胞，接种于小鼠胚胎成纤维细胞滋养层上，通过滋养层细胞以及白血病抑制因子来保持其多向分化能力。②STLV型生物反应器中培养的拟胚体在细胞大小、形态和数量方面均明显优于传统的悬浮培养法。③将STLV型生物反应器中制备并扩增得到的拟胚体，用ESC分化培养基连续贴壁诱导14d后，进行细胞染色，聚合酶链式反应检测。结果通过3个部分的实验，最终得到表达Ⅱ型肺泡细胞特异性标志物的细胞。前两部分中，得到了大量状态良好的未分化ESC，经扩增获得了高质量和大数量的EBS。在第三部分，免疫荧光及RT—PCR证明出现了Ⅱ型肺泡细胞特异性标志物-SPC的表达。结论通过以胚胎成纤维为种子细胞，利用组织工程的技术可形成Ⅱ型肺泡细胞。
Objective To establish a simple and sufficient way to directional derive type II alveolar cells from murine embryonic stem cells through mESC cultivation and differentiation. Methods Firstly, we get sufficient murine embryonic stem cells （ESC） obtained through the cells cultivation, and then were inoculated onto the trophoblast of the mouse embryo fibroblasts. The multi-directional differentiation was maintained by tropho- blastic cell and leukemia inhibitory factor. Secondly, the traditional respectively suspension culture method and STLV type biological reactor were trained for the cultivation of EBS carrier, the later method was better at the shape, size and quality. Thirdly, EBS trained by STLV type biological reactor were cultured continuously in the ESC differential meium and the cultured ESC were detected by cell staining and PCR. Results Through the three parts of the experiment, finally the expression of specific markers of the II type alveolar cells with tumor were obtained. The undifferentiated ESC in good condition from the prevenient two parts were gotten and the high-quality ESC were harvested from the undifferentiated ESC by PCR. the expression of SPC, the specific markers of the type II alveolar cells, were deteeted by immune fluorescence and PCR. Conclusion The embryo fibroblas used as the seed cells, type II alveolar cells could be obtained by tissue engineering technology.
出处
《中国美容整形外科杂志》
CAS
2012年第8期506-510,共5页
Chinese Journal of Aesthetic and Plastic Surgery
关键词
组织工程
胚胎干细胞
体外培养
拟胚体
Tissue engineering
Embryonic stem cell
In vitro cuhure
Embryoid body
