摘要目的:探讨大鼠骨髓间充质干细胞(BMSCs)与胰岛细胞共培养体系下分化为胰岛样细胞团的可能性。方法:无菌条件下从Wistar大鼠的股骨及胫骨分离纯化BMSCs，流式细胞仪检测BMSCs表面标志物CD 44和CD 90表达；行成骨方向及成脂肪方向诱导分化，采用茜素红染色和油红O染色对诱导后的细胞进行鉴定。分离纯化大鼠胰腺的胰岛细胞，采用双硫腙染色鉴定。使用ELISA法检测胰岛细胞培养液的基础胰岛素水平；分别加入含葡萄糖5.6 mmol/L(低糖)和25.0 mmol/L(高糖)的培养液，ELISA法检测胰岛素释放量。取第5代BMSCs和胰岛细胞，随机分为干细胞单独培养组(干细胞组)、干细胞-胰岛细胞共培养组(共培养组)及胰岛细胞单独培养组(胰岛组)，使用倒置相差显微镜观察各组BMSCs的形态学变化；使用ELISA法检测各组基础胰岛素分泌量及高、低葡萄糖刺激后胰岛素释放量；免疫细胞化学染色法检测共培养组诱导形成的胰岛样细胞团的胰岛素蛋白表达；透射电镜观察胰岛样细胞超微结构。 结果:BMSCs表面标志物CD 44和CD 90的阳性率分别为99.48%、99.50%，经成骨方向诱导分化形成多个钙结节，经成脂肪方向诱导分化胞质内形成较多脂滴。双硫腙染色显示胰岛中β细胞呈棕红色，每个胰腺可获得胰岛约450个，平均纯度达80%。低糖、高糖组刺激后胰岛的胰岛素释放量分别为(7.105±1.551)mIU/ml和(20.231±1.592)mIU/ml，差异有统计学意义( P<0.05)。共培养组培养7 d后，见局部干细胞开始聚拢并以出芽的方式向上生长成小团块，直至最后形成球形的胰岛样细胞团结构。干细胞组基础胰岛素分泌量<0.5 mIU/L；胰岛组培养5 d胰岛素分泌量达峰值，随后逐渐下降，13 d时降至最高值的20%；共培养组胰岛素水平在5 d时达到峰值，13 d时仍维持在峰值水平的40%左右，胰岛组与共培养组8、10、13 d基础胰岛素分泌量差异有统计学意义( P值均<0.05)，但高糖和低糖刺激后胰岛组胰岛和共培养组诱导形成的胰岛样细胞团的胰岛素释放量差异无统计学意义。培养14 d后，共培养组胰岛样细胞团内出现大量棕黄色颗粒，超微结构中出现较多分泌颗粒和粗面内质网，呈现较活跃的蛋白质分泌功能。 结论:BMSCs与胰岛细胞的共培养体系能诱导BMSCs分化为胰岛样细胞团，该细胞团表达胰岛素蛋白，具有较成熟的胰岛素分泌作用。

abstractsObjective:To examine the possibility of the differentiation into islet-like cell clusters from the co-culture system of bone marrow mesenchymal stem cells (BMSCs) and islet cells.Methods:Rat BMSCs from the femur and tibia of Wistar rats were isolated and purified taken under aseptic conditions; the surface markers CD 44 and CD 90 expressions of BMSCs were detected by flow cytometry; and alizarin red staining and oil red O staining were used to identify the cells induced in the osteogenic direction and adipogenic direction, respectively. Rat islet cells from the pancreas of Wistar rats were isolated and purified; and dithiazone staining was performed for validation. The basal insulin level of the culture was detected by ELISA method. 5.6mmol/L (low glucose) and 25.0 mmol/L (high glucosa) glucose were added to the culture, respectively, and insulin release was detected by ELISA. 5-generation BMSCs and islet cells were collected and divided randomly into stem cell culture alone group (stem cell group), stem cell-islet co-culture group (co-culture group), and islet culture alone group (islet group). The morphological changes of BMSCs during co-culture were observed using an inverted phase contrast microscope; basal insulin secretion and insulin secretion stimulated by low and high glucose were tested by ELISA. Insulin protein expression in induced islet-like cell masses in co-culture group were detected by immunocytochemical staining. The ultrastructure of islet-like cells was observed by using transmission electron microscopy. Results:The positive rates of CD 44 and CD 90 were 99.48% and 99.50%, respectively; BMSCs were induced the formation of multiple calcium nodules outside the differentiation cells in the osteogenic direction, and many lipid droplets in the cytoplasm of differentiated cells in the adipogenic direction. Dithiazone staining showed that β cells in pancreatic islet were brown red and about 450 islets could be obtained per pancreas with a mean purity up to 80%. The insulin release in the low sugar group and the high sugar group were (7.105±1.551) mIU/ml and (20.231±1.592) mIU/ml, respectively, with a statistically significant difference ( P<0.05). It can be seen that local stem cells began to gather and grow upward into small clumps in the budding manner until finally forming a spherical islet-like cell cluster structure after 7 days of culture in the co-culture group. The basal insulin secretion in the stem cell group was <0.5 mIU/L. In the islet group, insulin secretion peaked on the 5th day and then gradually decreased to about 20% of the highest value on the 13th day. The insulin level of the co-culture group peaked on the 5th day, and the 13th day remained at about 40% of the peak level. There were statistically significant differences on basal insulin secretion on the 8th, 10th and 13th day between islet group and co-culture group (all P value >0.05). There was no statistically significant difference between the insulin release by islet in islet group under the stimulation of low and high sugar and that by islet-like cell cluster in co-culture group. There were a large number of brownish-yellow granules in the islet-like cell clusters after the co-culture for 14 days; and there were more secretory granules and coarse endoplasmic reticulum in the ultrastructure, showing more active protein secretion functions. Conclusions:The co-culture system of BMSCs and islet cells could induce BMSCs into differentiating into islet-like cell clusters, which can express insulin protein and had relatively mature function of insulin secretion.