诱导干细胞分化的胰岛细胞形成胰岛样结构的实验研究

目的建立一种体外诱导干细胞分化的胰岛细胞形成胰岛样结构的技术方法。方法体外诱导人胚胎胰腺干细胞分化为胰岛细胞,将胰岛细胞制备成浓度分别为1×10^4／ml、3×10^4／ml、1×10^5／ml、3×10^5／ml（1×10^4／ml、3×10^4／ml、1×10^5／ml、3×10^5／ml组）单细胞悬液,在含有细胞外基质（ECM）的培养基中悬浮培养24h以诱导形成胰岛样结构,在荧光体视显微镜下观察胰岛样结构的形态和大小;采用免疫荧光染色方法检测胰岛样结构中ECM成分及其细胞组成与定位。将胰岛样结构分别在含5．6和30．0mmol／L葡萄糖的培养基中体外培养2h,采用放射免疫法测定上清中胰岛素水平。建立糖尿病大鼠模型,经门静脉将胰岛样结构移植至大鼠肝脏,移植后连续3d经鼠尾静脉检测血糖水平。结果90％人胚胎胰腺干细胞分化的胰岛细胞形成了胰岛样结构,其包膜、大小均与天然人胰岛组织结构相似;以形成直径150μm大小胰岛样结构的比例为标准,比较各组细胞浓度与胰岛样结构形成率的关系,结果显示分别与1×10^4／ml、3×10^4／ml、3×10^5／ml组（25．0％±2．5％、28．0％±3．0％、21．5％±2．5％）相比,1×10^5／ml组胰岛样结构形成率最佳（36．5％±4．0％,均P〈0．01）。胰岛样结构包膜上含有与天然人胰岛组织类似的ECM成分,且其中含有胰岛素、胰高血糖素和C-肽阳性细胞,其细胞比例与分布均与天然人胰岛组织类似。30．0mmol／L高糖浓度刺激后胰岛样结构胰岛素释放水平[（110±12）μIU／ml]较5．6mmol／L基础糖浓度[（59．5±8．0）pIU／ml]明显增加（P〈0,01）,胰岛样结构移植后糖尿病大鼠血糖水平较移植前均明显降低（P〈0．01）。结论本研究建立的技术方法可以将干细胞分化的胰岛细胞在体外大量、快速、高效地诱导形成具有功能的胰岛样结构。

Experimental study of inducing pancreatic stem cells differentiating into islet-like structures

Objective To establish a method that induced islet cells differentiated from pancreatic stem cells to form the islet-like structures in vitro.Methods Inducing human fetal pancreatic stem cells differentiating into the islet cells in vitro, and the islet cells prepared into density of 1× 10^4/ml, 3× 10^4/ml, 1 × 10^5/ml, 3× 10^5/ml respectively （1 × 10^4/ml, 3 × 10^4/ml, 1 × 10^5/ml, 3 × 10^5/ml group respectively） were suspended in medium containing extra-cellular matrix （ECM） and cultured for 24 h to induce the formation of the islet-like structures. The morphology and size of the islet-like structures were detected under fluorescence stereomicroscope; the ECM components and cellular types and location in the islet-like strucatres were detected by immunofluorescence staining. The islet-like structures were cultured in medium containing 5.6 and 30.0 mmol/L glucose respectively for 2 h, and the insulin release levels in the supernatant were detected by radioimmunoassay. To establish the rat models of diabetes, and transplanting the islet-like structures into the liver of rats through the portal vein, detecting the glycemia levels for 3 d continuously through the tail vein of rats. Results 90% of islet cells differentiated from human fetal pancreatic were aggregated spontaneously into the islet-like structures, which displayed obvious integrity outer membrane and similar size with native human islets. Using the diameter of 150 μm as a standard, the relationship between cells concentration and the formation rates of the islet-like structures was analyzed, and the resultshowed that the formation rate of the islet-like structures in the 1 × 10^5/ml group （36.5% ±4.0%, P 〈 0.01） was best compared with 1× 10^4/ml, 3 × 10^4/ml, 3 × 10^5/ml group respectively （25.0% ±2.5%, 28.0% ±3.0%, 21.5% ±2.5%, respectively）. These islet-like structures contained similar ECM components with native human islets on outer membrane, and the proportion and distribution of insulin-positive cells, glucagons