胰岛干细胞转分化胰岛样细胞与天然胰岛功能的比较

Comparison between the function of pancreatic stem cells differentiated islet-like cells and normal islets

目的：目前在胰腺干细胞体外转分化为胰岛样细胞的转化效率，转分化胰岛样细胞的成熟度，能否代替天然胰岛的功能等问题上存在争议。实验将分离纯化、扩增的大鼠胰腺导管上皮细胞，并诱导分化为胰岛样细胞团，与天然胰岛功能进行比较。方法：实验于200407／2005-04在暨南大学第二临床医学院暨深圳市人民医院临床医学研究中心实验室完成。实验选用SD大鼠10只．采用差异性贴壁、低血清培养等方法纯化大鼠胰腺导管上皮细胞，以免疫组织化学方法鉴定后，在含exendin-4、角质细胞生长因子、尼克酰胺等细胞因子和葡萄糖的培养基中，将其转分化为胰岛样细胞团，以DTZ染色和胰岛素免疫荧光染色鉴定。分离纯化SD大鼠天然胰岛，对胰岛样细胞团与天然胰岛进行葡萄糖刺激试验，检测培养液上清液中胰岛素和C肽的水平。结果：①免疫组织化学染色显示从大鼠胰腺导管上皮细胞分离纯化的细胞呈CK-19染色阳性，PDX-1染包阴性，表明为胰腺干细胞。诱导4周后，细胞逐渐汇聚成胰岛样细胞团．免疫荧光染色为阳性，证明有胰岛素分泌。②葡萄糖刺激实验显示，在低糖和高糖刺激下，胰岛样细胞团胰岛素的分泌量约为天然胰岛的1／30和1／35，C肽分泌量为天然胰岛的1／32和1／26．结论：目前胰腺干细胞体外转分化为胰岛样细胞的成熟度与天然胰岛仍然存在较大差别，其在功能仅为天然胰岛的1／30—1／35。

AIM： Transformation efficacy, maturity and function of replacing normal islets of in vitro differentiated pancreatic stem cells into islet-like cells are in debate. This study aimed to isolate and purify pancreatic ductal epithelial cells, which are proliferated and differentiated into pancreatic islet ceils, to compare the function between islet-like ceils and normal islets. METHODS： Experiments were performed at the Laboratory of Clinical Medical Research Center, Second Clinical Medical College （Sbenzhen People＇s Hospital）, Jinan University from July 2004 to April 2005. Pancreatic ductal epithelial cells were isolated from ten SD rats by differential adherence and low serum culture. After identified by immunocytochemistry, pancreatic ductal epithelial cells were transdifferentiated into islet-like clusters in glucose medium containing exendin-4, keratinocyte growth factor （KGF） and nicotinanaide （NIC）, and then identified by DTZ （dithizone） staining and insulin immunofluorescence. ARer isolating normal islets from SD rats, glucose stimulation test was performed to measure serum insulin levels and C-peptide secretion in supernatant between islet-like cell mass and normal islets. RESULTS： Immunohistochemistry demonstrated that cells from rat pancreatic ductal epithelial cells were positive for CK-19 and negative for PDX-1. These cells were pancreatic stem cells. Four weeks after induction, cells gradually clustered in islet-like cell mass. Positive results of immunofluorescence showed insulin secretion. Levels of secreted insulin in islet-like cell mass was 1/30 of the normal islets in low glucose and 1/35 in the high glucose. C-peptide secretory volume was 1/32 of the normal islets in low glucose and 1/26 in the high glucose. CONCLUSION： In vitro differentiated islet-like cells from pancreatic stem ceils greatly differ from normal islets. Its function is only 1/30-1/35 of normal islets.