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供体不足已成为移植胰岛治疗Ⅰ型和部分Ⅱ型糖尿病的主要障碍,分离克隆胰腺干细胞作为种子细胞并诱导其分化为功能性胰岛可提供丰富的移植资源.本研究从人流产胎儿胰腺组织分离获得1例单克隆胰腺干细胞系.无菌取流产胎儿胰腺组织,0.1%Ⅳ型胶原酶消化分离为单个细胞和细胞团.低糖DMEM+10%FBS培养,单个细胞和细胞团贴壁,原代上皮样胰腺干细胞克隆性生长.0.25%胰蛋白酶+0.04%已二胺四乙酸(EDTA)消化传代,成纤维样细胞和其他细胞逐渐被消除,上皮样胰腺干细胞逐渐被纯化.克隆环筛选,获得单克隆人胰腺干细胞.在培养液中添加10ng/mL表皮生长因子(EGF),单克隆人胰腺干细胞快速生长至单层,呈铺路石样.继续传代培养,1例来源于4月龄男性流产胎儿胰腺干细胞已传50代.液氮冷冻保存细胞1×109个以上.染色体核型分析,该干细胞系为正常的二倍体细胞.免疫组织化学反应,共表达pdx1,glucagon,nestin及CK19蛋白,不表达insulin,CD34,CD44及CD45.RT-PCR检测,转录pdx1,glucagon,nestin及CK19的mRNA,不转录insulin.β-巯基乙醇诱导,分化为神经细胞,免疫组织化学反应表达NF蛋白.烟酰胺诱导,分化为DTZ染色阳性,转录表达insulin,分泌insulin和C肽的功能性类胰岛.将单克隆人胰腺干细胞体外诱导胰岛移植在STZ制备的糖尿病大鼠肾囊内,能降低糖尿病大鼠血糖水平,延长寿命.
Inadequate donor has become a major obstacle for the treatment of type I and partial type II diabetes mellitus after transplantation of pancreatic islets, isolation of cloned pancreatic stem cells as seed cells and induction of differentiation into functional islets provide abundant transplantation resources.In this study, we isolated human pancreatic tissue from human abortion One case of monoclonal pancreatic stem cell line was obtained. Aseptic abortion fetus pancreatic tissues were digested with 0.1% type Ⅳ collagenase and separated into single cells and cell clusters. Cells were cultured in low glucose DMEM + 10% FBS, Epithelial-like pancreatic stem cells clonally grow, digestive passage with 0.25% trypsin + 0.04% diaminetetraacetic acid (EDTA), fibroblast-like cells and other cells are gradually eliminated and epithelial-like pancreatic stem cells are gradually purified. Monoclonal human pancreatic stem cells were cultured in vitro, 10ng / mL epidermal growth factor (EGF) was added to the culture medium, and the human pancreatic stem cells rapidly grew to monolayer and became paving stones. Fetal pancreatic stem cells have been passed for 50 generations.Novirulent cryopreservation of cells more than 1 × 109. Chromosome karyotype analysis, the stem cell line is normal diploid cells.Immunohistochemistry The co-expression of pdx1, glucagon, nestin and CK19 protein, but not insulin, CD34, CD44 and CD45.RT-PCR detection, transcription pdx1, glucagon, nestin and CK19 mRNA, not transcribed insulin.β- mercaptoethanol induced differentiation NF-kappaB, NF-kappaB, NF-kappaB, NF-kappaB, NF-kappaB, nicotinamide induced and differentiated into DTZ-positive, insulin- Of diabetic rats kidney capsule, can reduce blood glucose levels in diabetic rats, prolong life.