昆明小鼠胰腺干细胞分离培养及特异性标志物巢蛋白的鉴定

Isolation and culture of pancreatic stem cells from Kunming mouse and the identification of Nestin as its specific marker

探讨胰腺干细胞的分离、培养及其特异性标志物巢蛋白鉴定的基本技术方法。选取新生昆明种小鼠25只,分离胰腺组织,V型胶原酶消化,结合自然沉降技术形成不连续密度梯度离心。分别从磷酸盐缓冲液/1.068g/L Percoll液(第一界面)、1.068g/L Percoll液/1.096g/L Percoll液(第二界面)、1.096g/L Percoll液/1.118g/L Percoll液(第三界面)收集细胞。各界面细胞均加入含体积分数为0.1胎牛血清的DMEM低糖培养基,置于经多聚赖氨酸处理的培养瓶中常规培养。24h后将培养基更换为不含胎牛血清的DMEM低糖培养基,并加入10mg/L的角质细胞生长因子和20μg/L的碱性成纤维细胞生长因子,观察体外培养的各界面细胞的形态学特征。胰腺的内分泌部存在β细胞,双硫腙能与β细胞分泌颗粒中的锌螯合而呈现铁红色。将收集的各界面细胞进行双硫腙染色,检测细胞来源。免疫组织化学法检测各界面巢蛋白的表达。结果第一、二界面细胞65%～90%双硫腙染色呈阳性,来源于胰腺的内分泌部;第三界面细胞双硫腙染色呈阴性,来源于胰腺的外分泌部。从胰腺的内分泌部获得的细胞聚集呈胰岛样细胞团贴壁生长,48～72h后可见许多大、圆、单个核的细胞从胰岛样细胞团中向周围长出,以附壁生长的方式在局部形成细胞集落。从胰腺的外分泌部获得的细胞主要呈上皮样生长,并逐渐汇合成片,在上皮样细胞集落的周围可见大、圆、单个核的细胞生长并形成集落。各界面大、圆、单个核的细胞,巢蛋白均呈阳性表达。提示从胰腺内、外分泌部的组织中能成功获取胰腺干细胞特异性标志Nestin表达阳性的干样细胞。

To explore the basic technique to isolate and culture the pancreatic stem cells and identify its specific marker of Nestin, we selected 25 newbom Kunming mice. The pancreatic tissue was submitted to digestion by collegenase V, and separated by discontinuous density gradient centrifugation binding physio-subsidence. Cells were collected from the first interface of phosphate buffer/1.068 g/L Percoll, second interface of 1.068 g/L Percoll/1.096 g/L Percoll and third interface of 1.096 g/L Percoll/1.118 g/L Percoll, and cultivated in a culture flask treated with polylysine after adding low carbohydrates DMEM contained 0.1 volume fraction FBS. Twenty-four hours later, the medium was replaced by serum-free DMEM medium with the addition of 10 mg/L keratinocyte growth factors and 20 μg/L basic fibroblast growth factor to observe the morphological characters of cells at each interface. 13 cells were found in the intemal secretory portion of pancreas; dithizone chelated with zine secreted from βcells and presented siren red. All cells collected from each interface were given dithizone staining to detect their sources. The expression of protein Nestin, the specific marker of pancreatic stem cells, was detected by immunocytochemical assay. 65%-90% cells from the first and second interfaces showed positive in dithizone staining and derived from the intemal secretory portion of pancreas; the cells from the third interface were negative and came from the extemal secretory portion of pancreas. Cells aggregation from the portion of pancreas adhered as insulin-like cell group, and 48-72 hours later, many big, round and mononuclear cells appeared from those cell groups; these cells aggregated just like colonies. Cells from the extemal secretory portion of pancreas epithelium-likely grew and gradually fused to slice. Big, round and mononuclear cells appeared around those cell groups and aggregated just like colonies. Nestin of the big, round and mononuclear cells showed positive expression, indicating that the stem cells with immunoreactive positive Nestin as the pancreatic stem cells marker could be harvested successfully from the intemal and extemal secretory portion of pancreas tissue.