二甲基亚砜诱导成年大鼠骨髓基质细胞转分化为胰岛样细胞

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200605／2007—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Prornega公司产品（A3500），Taq酶和DNA marker DL2000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／cm^2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。

In vitro trans-differentiation of bone marrow stromal cells into islet-like clusters by dimethyl sulphoxide

BACKGROUND: Recent findings suggest that bone marrow stromal cells (MSCs) have the capacity to differentiate into neurons induced by dimethyl sulphoxide (DMSO). So we think whether MSCs can trans-differentiate to nestin positive precurosor cells, then to endocrine cells of the pancreas.OBJECTIVE: To investigate the possibility of differentiating functional insulin-producing cells from MSCs induced by DMSO. DESIGN: Randomized and controlled trails for cells in vitro.SETTING: Department of Histology and Embryology, Medical College of Hebei North University. MATERIALS: This study was performed in the Cell Culture Room of Department of Histology and Embryology, Hebei North University from May 2006 to July 2007. Ten SPF Wistar rats of 6-8 weeks age without sex constraint weighing 180-230 g, were purchased from the Center of Experimental Animal Academy of Military Medical Sciences (license: SCXK-armed forces 2002-001). Fetal bovine serum was Gibco products, Trizol and RT-PCR (A3500) kit were purchased from Promega; DMSO from ChemicalReagent Beijing Co., Ltd.; Taq enzyme and DNA marker DL2000 from Tianwei Biotech Beijing Co., Ltd.; mice anti-human insulin monoclonal antibody, rabbit anti-human glucagon and nestin polyclonal antibody from Zhongshan Golden Bridge Biotechnology Co., Ltd.; The polyclonal antibodies had well cross-reaction with the rat's tissue. So they could be used to tissues of human, rats or other mammals. SABC kit was provided by Boster Biotechnology Co., Ltd.; radioimmunoassay kit by Linco Co., Ltd.METHODS: The bone marrow cells woe cultured in H-DMEM supplemented with 0.1 volume fraction of FBS. After 60-minute incubation, non-adherent cells were collected. The cells were re-plated in serum-free DMEM medium at a cell density of 109/cm2 in the presence of 10 mL/L DMSO. The cells were then cultured in DMEM supplemented with 10 mL/L DMSO for 3 days followed by high (25 mmol/L) glucose DMEM media supplemented with 0.1 volume fraction of FBS for 7 additional days. They were plated in plastic six well plates on slide.MAIN OUTCOME MEASURES: The morphology were observed and diphenyl thiocarbazone (DTZ) staining. Immuno-activity was detected using the cytochemical method for the protein expression, such as nestin, insulin, glucagons and somatostatin. The endocrine gene expressions of induced clusters were detected by RT-PCR. Measurement of Insulin content and secretion were detected by immunoassay.RESULTS: The MSC cells woe typically fibrocyte-like, grape-like coherent dnstered cells. They were observed under experiment group alter the 3rd day after DMSO induction, which were similar to pancreatic islet cells. DTZ staining of the cell aggregates were positive as the natural pancreatic islets, Immunocytochernistry also confirmed that these aggregates were positive for nestin on the 1st day after DMSO induction, and positive for insulin, glucagon on the 10th day after DMSO; while the control groups were negatively stained for the characteristics of endocrine. RT-PCR results showed the insulin 1, insulin 2, glucagon and somatostatin geae expressions were on the 7th day after DMSO induction. The aggregates have good reactions to glucose, the secretion of insulin content respectively 5.56 and 24.5 μ U/mL under low glucose (3.3 mmol/L) and high glucose (16.7 mmol/L) conditions.CONCLUSION: The MSCs can be induced to differentiate into pancreatic endocrine hormone-producing cells in serum-flee with DMSO.