猪胰腺干细胞分离与培养

Isolation and Culture of Porcine Fetus Islet Stem Cell

【目的】探寻一种简单的胰腺干细胞的分离方法,以便为糖尿病的细胞治疗研究提供丰富的种子细胞。【方法】采用胰酶消化法分离获得细胞,利用仅含有5%的血清的RPMI1640培养基培养细胞,低浓度的血清使得细胞大量死亡漂浮,只留下少数贴壁的小圆细胞。而后将血清浓度提高到15%以上继续培养;采用免疫组化方法对获得的细胞是否具有胰腺干细胞特征予以检测,并测定了细胞的生长曲线。【结果】在血清浓度提高后每个小圆细胞能快速增殖并形成一个克隆,经过这样增殖后的细胞可以连续传代,目前已传至60代。经免疫组化检测,细胞表达PDX-1、GLUT-2、vimentin等胰腺干细胞的标志;细胞也表达Glucagon、insulin、somatostatin、polypeptide等胰腺内分泌细胞的标志.【结论】通过本方法获得的细胞经检测确定为胰腺干细胞,在体外可自发分化形成胰胰内分泌部4种主要细胞。

[Objective] To find an easy method for islet stem cell isolation. [Method] Cells derived from porcine fetus pancreas have been cultured in RPMI 1640 with only 5% serum. This method made most of those cells die. Only a few little cells were survived. These cells proliferated quickly when medium was changed to RPMI 1640 with 15% serum and could form cells＇ clone. Islet stem cell＇s marker was noticed and detected by immunochemical method. [Result]Cells possess strong proliferation ability and were sub cultured over 60 passages till now. It expressed islet stem cell＇s marker such as PDX-1, GLUT-2, and vimentin, etc. Also, it expressed insulin, Glucagon, somatostatin and polypeptide. [ Conclusion ] Cells derived by this method were determined as islet stem cells. It can differentiate into islet endocrine cell in vitro. This study has provided a reference for isolation and expansion of islet stem cell. Also, it has provided materials for diabetes therapy.