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背景:已有报道,二甲基亚砜可使骨髓基质细胞分化为神经元,那么可否用二甲基亚砜诱导骨髓基质细胞向nestin阳性细胞分化,继而向胰岛分化。目的:探讨二甲基亚砜对成年大鼠骨髓基质细胞定向诱导分化为胰岛样细胞团的作用。设计:以细胞为观察对象,随机对照,体外实验。单位:河北北方学院医学院组织学与胚胎学教研室。材料:实验于2006-05/2007-07在河北北方学院组胚教研室细胞室完成。选取6~8周龄Wistar大鼠10只,性别不拘,由解放军军事医学科学院实验动物中心提供。实验用分析纯二甲基亚砜购自北京市化学试剂厂,反转录聚合酶链反应系统为Promega公司产品(A3500),Taq酶和DNAmarkerDL2000购自北京天为时代生物技术有限公司,鼠抗人胰岛素单克隆抗体(ZM20155)、兔抗人胰高血糖素多克隆抗体(ZA20119)购自北京中杉金桥生物技术有限公司,兔抗人nestin多克隆抗体(与大鼠组织有良好的交叉反应,可用于检测人、大鼠等哺乳动物组织)、SABC试剂盒购自武汉博士德公司,大鼠胰岛素放射免疫试剂盒,购自美国Linco公司。方法:骨髓基质细胞以含体积分数0.1胎牛血清的H-DMEM培养60min,收集未贴壁细胞以含10g/L二甲基亚砜的无血清H-DMEM作为诱导液,以109/cm2密度接种于6孔板,诱导3d,继之用体积分数0.1胎牛血清的H-DMEM培养7d。主要观察指标:用DTZ染色观察诱导细胞团的形态,免疫细胞化学染色检测巢蛋白、胰岛素、胰高血糖素、生长抑素的定位;反转录聚合酶链反应检测诱导细胞团的内分泌基因表达;放免分析检测细胞团的胰岛素分泌量。结果:①骨髓基质细胞分化的细胞团形态:骨髓基质细胞为典型的成纤维细胞样或葡萄样贴壁细胞,经二甲基亚砜诱导3d后,出现胰岛样细胞团,经DTZ染色显示了和胰岛同样的特性。②免疫细胞化学染色显示:二甲基亚砜诱导后1d骨髓基质细胞表达nestin蛋白,诱导10d表达胰岛素和胰高血糖素;未经二甲基亚砜诱导的骨髓基质细胞则不表达胰岛素和胰高血糖素。③反转录聚合酶链反应结果显示:骨髓基质细胞经二甲基亚砜诱导后胰岛样细胞团表达insulin1,insulin2,glucagon和somatostain基因。④细胞团的胰岛素分泌量:胰岛样细胞团对葡萄糖刺激敏感,低糖(3.3mmol/L)和高糖条件下(16.7mmol/L)胰岛素分泌量分别为5.56和24.5μU/mL。结论:体外经二甲基亚砜诱导的大鼠骨髓基质细胞可定向分化为胰岛样细胞。
BACKGROUND: It has been reported that dimethyl sulfoxide can differentiate bone marrow stromal cells into neurons. Then whether dimethylsulfoxide can induce bone marrow stromal cells to differentiate into nestin positive cells and then differentiate into islets. Objective: To investigate the effect of dimethyl sulfoxide (DMSO) on the induction of bone marrow stromal cells into islet-like cells in adult rats. Design: The cells were observed, randomized controlled, in vitro experiments. Unit: Department of Histology and Embryology, Hebei North University School of Medicine. MATERIALS: The experiment was performed in the cell room of the embryo department of Hebei North University from May 2006 to July 2007. 10 Wistar rats aged 6 to 8 weeks were selected, sexually and without exception, provided by the Experimental Animal Center of PLA Academy of Military Medical Sciences. Experimental analytical grade dimethylsulfoxide was purchased from the Beijing Chemical Reagent Factory, reverse transcription polymerase chain reaction system Promega company products (A3500), Taq enzyme and DNAmarkerDL2000 were purchased from Beijing Tianyi Times Biotechnology Co., Ltd., mouse anti The human insulin monoclonal antibody (ZM20155) and rabbit anti-human glucagon polyclonal antibody (ZA20119) were purchased from Beijing Zhongshang Jinqiao Biotechnology Co., Ltd. The rabbit anti-human nestin polyclonal antibody (good cross-reaction with rat tissue, Can be used to detect human, rat and other mammalian tissue), SABC kit purchased from Wuhan Boster Company, rat insulin radioimmunoassay kit, purchased from the United States Linco company. METHODS: BMSCs were cultured in H-DMEM containing 0.1% fetal bovine serum for 60 min and the non-adherent cells were collected as serum-free H-DMEM containing 10 g / L dimethylsulfoxide (DMSO) Inoculated in 6-well plates, induced 3d, followed by the volume fraction of 0.1 fetal bovine serum H-DMEM cultured 7d. MAIN OUTCOME MEASURES: Cell morphology was observed by DTZ staining, nestin, insulin, glucagon and somatostatin were detected by immunocytochemical staining; endocrine gene expression was detected by reverse transcriptase-polymerase chain reaction ; Radioimmunoassay detection of cell mass of insulin secretion. Results: ①Cell morphology of bone marrow stromal cells: The bone marrow stromal cells were typical fibroblast-like or grape-like adherent cells. After induced by dimethyl sulfoxide for 3 days, the islet-like cell mass appeared, and DTZ staining showed And islet the same characteristics. ② Immunocytochemical staining showed that nestin protein was expressed in bone marrow stromal cells on the 1st day after dimethyl sulfoxide induction, and induced insulin and glucagon expression on the 10th day after DMSO induction; while bone marrow stromal cells induced by dimethyl sulfoxide did not express insulin and pancreatic Glucagon. ③ Reverse transcriptase-polymerase chain reaction showed that insulin-1, insulin2, glucagon and somatostain genes were expressed in islet-like cell mass of bone marrow stromal cells induced by dimethyl sulfoxide. ④ Cell mass insulin secretion: Islet-like cell mass was sensitive to glucose stimulation, insulin secretion was 5.56 and 24.5μU / mL under low glucose (3.3mmol / L) and high glucose (16.7mmol / L) respectively. CONCLUSION: Rat bone marrow stromal cells induced by dimethyl sulfoxide can be differentiated into islet-like cells in vitro.