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目的研究1例单克隆人胰腺干细胞系的细胞形态和表达特性。方法10例4～5月龄流产胎儿胰腺组织,胶原酶消化。原代细胞DMEM培养,胰蛋白酶消化传代。在传代中逐渐纯化胰腺干细胞。克隆环筛选单克隆人胰腺干细胞,扩增培养。活力较好的细胞液氮冷冻保存。采用HE和Giemsa染色,观察单克隆人胰腺干细胞的形态特征;通过透射电镜分析其超微结构。用免疫组织化学反应和RT-PCR方法,检测其蛋白水平和mRNA水平的表达特征。结果1例来源于4月龄男性胎儿的单克隆人胰腺干细胞建系,传50代。液氮保存细胞1×109个以上。单克隆人胰腺干细胞完全贴璧时呈多角形上皮样,单核,圆形或椭圆形,单、双或多核仁。细胞核质比大,线粒体、内质网等细胞器均不发达,胞质内无分泌颗粒,属于发育早期的胰腺上皮样细胞。共表达胰肠同源域因子1(pdx1)、胰高血糖素、巢蛋白及角蛋白19(CK19),不表达胰岛素、周期蛋白34(CD34)、CD44及CD45。转录表达pdx1、胰高血糖素及巢蛋白的mRNA,不转录表达胰岛素。结论首次证实1例共表达pdx1、胰高血糖素、巢蛋白及CK19蛋白的单克隆人胰腺干细胞系。
Objective To study the cell morphology and expression characteristics of 1 human pancreatic stem cell line. Methods Totally 10 miscarried fetal pancreases of 4-5 months old were digested with collagenase. Primary cells were cultured in DMEM, passaged by trypsinization. Pancreatic stem cells are gradually purified during passage. Cloning of monoclonal human pancreatic stem cells screening, expansion and culture. Vital cells of liquid nitrogen cryopreservation. Morphological characteristics of human pancreatic stem cells were observed by HE staining and Giemsa staining. The ultrastructure of pancreatic stem cells was analyzed by transmission electron microscopy. Immunohistochemical and RT-PCR methods were used to detect the protein expression and mRNA expression. Results One case of human pancreatic stem cells derived from 4-month-old male fetus was passaged for 50 passages. Liquid nitrogen to save more than 1 × 109 cells. Monoclonal human pancreatic stem cells completely polygamy when the polygon epithelium-like, mononuclear, round or oval, single, double or multiple nucleolus. Large nuclear and cytoplasmic ratio, mitochondria, endoplasmic reticulum and other organelles are not developed, no secreted particles within the cytoplasm, belonging to the early development of pancreatic epithelial cells. Co-expressing pancreatic homologous domain factor 1 (pdx1), glucagon, nestin and keratin 19 (CK19), not expressing insulin, cyclin 34 (CD34), CD44 and CD45. Transcriptional expression of pdx1, glucagon and nestin mRNA, does not transcription express insulin. Conclusions A monoclonal human pancreatic stem cell line co-expressing pdx1, glucagon, nestin and CK19 protein was confirmed for the first time.