大鼠脂肪间充质干细胞诱导为类肝细胞及其细胞片的制备

Cell sheet fabrication of hepatocyte-like cells differentiated from adipose tissue mesenchymal stem cells

摘要:

成体多能干细胞, 如来自骨髓和脂肪组织的间充质干细胞等具有多向分化的潜能。虽然自体干细胞移植已经发展成为器官移植的有效代替疗法之一, 但是由于移植位点细胞的流失和分化条件的限制等问题使得这种疗法的效率大大降低。本研究目的是将由脂肪干细胞分化而来的类肝细胞制备成具有稳定细胞性状的可移植的肝细胞片。首先在体外分离扩增脂肪干细胞, 并通过控制严格地分化条件获得类肝细胞。然后将此细胞接种到聚N-异丙基丙烯酰胺(PNIPAAm)结合的细胞培养皿表面, 通过调节培养温度到20oC, 使细胞成片脱离培养皿形成细胞片。对细胞片进行了常规HE染色和免疫组化观察, 结果显示: 这类细胞片中平均含有2~3层细胞, 并且保持了细胞外基质的完整。同传统的胰酶消化收集移植用细胞相比, 细胞片方法极大地减少了对移植用细胞的细胞膜和细胞外基质的损伤, 这将大大促进细胞片和原位组织的相互作用, 增加细胞利用效率, 从而有望提高治疗效果。

Abstract:

Adult pluripotent stem cells, such as mesenchymal stem cells derived from bone marrow and adipose tissue are capable of multilineage differentiation. Although autologous stem cell transplantation is an effective alternative to organ transplantation, the loss of cell viability and differentiation confinement of implanted cells has largely impaired the therapeutic efficacy. To produce biomaterial-free liver construct to integrate into living tissue, we isolated adipose mesenchymal stem cells and subjected them to a delicate culture configuration to mediate the hepatocyte differentiation. The differentiated hepatocyte-like cells were then inoculated onto poly (N-isopropylacrylamide) (PNIPAAm) grafted cell culture dish. By lowering the culture temperature to 20oC, cells detached from the dish surface into a complete cell sheet. Hematoxylin and eosin staining and immunohistochemistry results showed that cell sheet was composed of 2–3 layers of cells and extracellular matrix was maintained intact. As compared with traditional cell harvest using trypsin digestion, cell sheet fabrication causes no damage to cell membrane and extracellular matrix. Hence, cell sheets would form a better interaction with tissues in situ, and a higher cell viability and therapeutic efficiency would be expected.