Hu Ying, Wang Qiu-ying, Ma Li, Ma Guan-jie, Jiang Xue-ying, Zhao Chun-hua
State Key Lab of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, CAMS, PUMC, Tianjin 300020, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2002 Feb;24(1):45-9.
To isolate and identify the phenotype and biological characteristics of pancreas derived mesenchymal stem cell.
Fresh pancreas of 4-5 months old aborted fetus was dissected free from connective tissue, and was cut into small pieces. The adherent cells were harvested and subcultured, after the third subculture, the cells were used for examination. Cell cycle was analyzed by measuring DNA content by FACScan flow cytometer. Phenotype of MSCs was analyzed by immunohistochemical SA technique and differentiated cells were identified by relevant specific staining.
Fetal pancreas derived cells gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast like morphology. By transmission electron microcopy, MSC had few endoplasmic reticulums and mitochondrias. During the log phase of growth, MSC proliferated with a two fold population at 30 h. MSC can be ex vivo expanded by successive cycles of trypsinization, seeding, and culture. Under these conditions, MSC had capability of passaging up to 30 times without displaying significant changes in morphology, with 2-fold increase in cell number after each passage. This indicates the high ex vivo expansion potential of MSC. The results showed that the yield of CFU-Fs was above 200 clones even after the 6th passage. Cell cycle analysis by flow cytometry revealed that more than 83% of cells were in the G0/G1 phases, while a small population of cells were actively engaged in proliferation (S + G2 + M = 17%). We also showed that more than 86% of cells were positive stained by FITC labeled CD44, CD29, CD13, and only about 1% of cells were positive for CD34, HLA-DR. Expression of collagen I, III was positive while vWF was negative. In the differentiation study, we found culture-expanded pancreas MSCs could be directed into the osteogenic lineage as detected by osteoblastic morphology, expression of alkaline phosphatase, modulation of osteocalcin mRNA production and the formation of a mineralized extracellular matrix. We also found that MSCs could give rise to the adipogenic and chondrogenic lineage as evidenced by accumulation of lipid-rich vacuoles within cells and the expression of lipoprotein lipase mRNA or the expression of collagen II and the deposition of proteoglycans.
Mesenchymal stem cells existing in human pancreas can be isolated by their adherent ability and should be essential to sustain a steady supply of primitive cells in tissue remodeling.
分离并鉴定胰腺来源间充质干细胞的表型及生物学特性。
取4 - 5月龄流产胎儿的新鲜胰腺，剔除结缔组织后切成小块。收集贴壁细胞并传代培养，第3代后用于检测。采用FACScan流式细胞仪检测DNA含量分析细胞周期。用免疫组织化学SA技术分析间充质干细胞的表型，用相关特异性染色鉴定分化细胞。
胎儿胰腺来源的细胞产生了一群贴壁细胞，其主要细胞类型具有典型的成纤维细胞样形态。透射电镜观察显示，间充质干细胞内质网和线粒体较少。在生长对数期，间充质干细胞在30小时内数量翻倍增殖。间充质干细胞可通过胰蛋白酶消化、接种和培养的连续循环进行体外扩增。在此条件下，间充质干细胞能够传代达30次，形态无明显变化，每次传代后细胞数量增加2倍。这表明间充质干细胞具有较高的体外扩增潜力。结果显示，即使在第6代后，集落形成单位 - 成纤维细胞（CFU - Fs）的产量仍高于200个克隆。流式细胞仪分析细胞周期显示，超过83%的细胞处于G0/G1期，而一小部分细胞处于活跃增殖状态（S + G2 + M = 17%）。我们还发现，超过86%的细胞对异硫氰酸荧光素（FITC）标记的CD44、CD29、CD13呈阳性染色，而只有约1%的细胞对CD34、HLA - DR呈阳性。I型、III型胶原表达阳性，而血管性血友病因子（vWF）表达阴性。在分化研究中，我们发现培养扩增的胰腺间充质干细胞可定向分化为成骨谱系，表现为成骨细胞形态、碱性磷酸酶表达、骨钙素mRNA产量的调节以及矿化细胞外基质的形成。我们还发现，间充质干细胞可分化为脂肪生成和软骨生成谱系，表现为细胞内富含脂质的空泡积累、脂蛋白脂肪酶mRNA表达或II型胶原表达以及蛋白聚糖沉积。
人胰腺中存在的间充质干细胞可通过其贴壁能力分离，对于在组织重塑中维持原始细胞的稳定供应至关重要。
