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为探讨用单纯生物学制剂诱导人脐带间充质干细胞(mesenchymal stem cells derived from human umbilical cord,hUC-MSCs)向胰岛素分泌细胞分化的可行性,本研究用胶原酶Ⅱ、胰蛋白酶次序消化及两步离心法从人胎儿完整脐带中分离、纯化出hUC-MSCs;用表皮生长因子、碱性成纤维生长因子、银杏提取液和高糖培养基IMDM诱导hUC-MSCs向胰岛素分泌细胞分化。在hUC-MSCs诱导前后,用倒置显微镜观察其形态变化,RT-PCR检测其胰岛相关基因的表达;双硫腙染色鉴定胰岛样细胞团(islet-like clusters,ILCs);细胞免疫荧光染色检测ILCs中PDX-1和免疫活性胰岛素(immunoreactive insulin,IRI)的表达;化学发光法检测ILCs的IRI分泌量;Western blot鉴定IRI的性质。结果显示:纯化的hUC-MSCs呈间充质干细胞特有的形态特征:长梭形,平行或螺旋形排列;在上述单纯生物学制剂的诱导下,hUC-MSCs逐渐变圆并聚集成团;在25cm2培养瓶的细胞生长面可见上百个ILCs;ILCs表达胰岛特异性基因pdx-1、insulin;ILCs呈PDX-1和IRI免疫染色阳性反应,双硫腙染色呈阳性;ILCs可分泌IRI,但多为胰岛素原(proinsulin,PI)。以上结果提示,用表皮生长因子、碱性成纤维生长因子、银杏提取液和高糖培养基IMDM可诱导hUC-MSCs快速分化为胰岛素分泌细胞,但ILCs功能不够成熟,难以产生足量真胰岛素。
In order to explore the feasibility of inducing human umbilical cord stem cells derived from human umbilical cord (hUC-MSCs) to differentiate into insulin-secreting cells with simple biological agents, collagenase Ⅱ, trypsin digestion and two The hUC-MSCs were isolated from intact human umbilical cord by centrifugation step by step. The hUC-MSCs were induced to differentiate into insulin-secreting cells by using epidermal growth factor, basic fibroblast growth factor, ginkgo biloba extract and high glucose medium IMDM. The morphology of islet-like clusters (ILCs) was detected by dithizone staining before and after induction of hUC-MSCs by inverted microscopy. The expression of islet-like clusters was detected by RT-PCR. ILCs were detected by immunofluorescent staining The expression of PDX-1 and immunoreactive insulin (IRI) was detected by chemiluminescence method. The IRI secretion of ILCs was detected by chemiluminescence assay. The properties of IRI were identified by Western blot. The results showed that the purified hUC-MSCs were characterized by mesenchymal stem cells: long fusiform, parallel or spiral arrangement; hUC-MSCs gradually rounded and clustered into clusters under the above simple biological preparation; ILCs expressed islet-specific gene pdx-1, insulin; ILCs showed positive immunostaining for PDX-1 and IRI, and dithizone staining was positive; ILCs secreted IRI, but ILCs secreted IRI, Mostly proinsulin (proinsulin, PI). These results suggest that epidermal growth factor, basic fibroblast growth factor, ginkgo extract and high glucose medium IMDM can induce rapid differentiation of hUC-MSCs into insulin-secreting cells, but ILCs are not mature enough to produce enough true insulin.