牛磺酸对胰腺干细胞增殖及分化为胰岛β细胞的调控作用及机制研究

The increase of pancreatic β cells is attributed to self replication and neogenesis, during which neurogenesis participated by pancreatic cells is the most important. Our previous studies proved that cysteine sulfinate decarboxylase (CSD) which is the limited enzyme of taurine synthesis was expressed in islet. Further studies certificated that taurine could improve the expression of stem cell marker in the islet of rats with diabetes, increase the number of beta cells of islet, indicating that taurine may play important role in the proliferation and differentiation of pancreatic stem cells. This study aims to combine the in vivo and in vitro experiment based on the previous studies, detecting the effect of taurine on the proliferation rate of islet stem cells, investigating the effect of taurine on the gene and protein expressions of cell proliferation nuclear antigen by real-time PCR and western-blot to inspect the effect of taurine on proliferation of pancreatic stem cells; inspecting the effect of taurine on cell differentiation by detecting the expressions of pancreatic stem cells and islet cell marker using Immunohistochemistry, Western-blot and RT-PCR. The effect and mechanism of taurine on the proliferation and differention of islet stem cells and the signal transduction pathway were revealed by analyzing the whole genome microarray hybridization, gene and protein expression of key factors in Wnt signal transduction pathway to determine its effect on cell proliferation, differentiation and signal transduction pathway in pancreatic stem cells, in order to provide theoretical basis for prevention and treatment of human and animal diabetes.

胰岛β细胞数量的增加有自我复制和新生两条途径，其中干细胞参与的新生过程至关重要。项目组前期研究发现，牛磺酸合成限速酶在胰腺组织内表达，同时发现牛磺酸能够促进链脲佐菌素（特异性破坏胰岛β细胞）诱导糖尿病大鼠胰腺干细胞标志物表达，胰岛β细胞数量增多，推测牛磺酸对胰腺干细胞增殖、分化为胰岛β细胞具有促进作用。本项目拟在此基础上结合体内外试验，通过检测糖尿病大鼠胰腺干细胞增殖率、细胞核增殖抗原基因及蛋白表达的影响，确定牛磺酸对胰腺干细胞增殖的作用；分析牛磺酸诱导后，胰腺干细胞标志物、成熟β胰岛细胞标志物基因及蛋白表达的动态变化，确定牛磺酸对胰腺干细胞分化为胰岛β细胞的作用；并通过全基因组芯片杂交及对Wnt信号通路关键因子基因及蛋白表达分析，确定其对胰腺干细胞增殖、分化及信号通路的影响，进而揭示牛磺酸对胰腺干细胞增殖、分化为胰岛β细胞的调控作用及机制，为应用牛磺酸防治人类及动物糖尿病提供理论依据。

作为全球范围内的代谢性常见疾病—糖尿病极大地影响着患者的生命质量。常规的药物和胰岛素注射并不足以根治糖尿病。近几年来，逐渐发展起来的干细胞的相关研究，有望从根本上治愈糖尿病及其并发症，因而成为治疗糖尿病的热点研究。本课题组前期研究表明牛磺酸能够抑制糖尿病模型大鼠血糖的升高，保护胰岛β细胞，促进体外培养大鼠胰腺干细胞的增殖，推测其可能通过促进胰腺干细胞的增殖及分化，促进胰岛β细胞数量增加。本研究拟通过体内、体外实验考察牛磺酸对胰腺干细胞分化为胰岛β细胞的作用及机制。. 体内试验：选取100只SPF级SD雄性大鼠，取50只大鼠腹腔注射50 mg/kg 链脲佐菌素（streptozocin， STZ），制备STZ诱导的糖尿病模型，分别于三天和一周后检测注射STZ大鼠的血糖水平，以随机血糖值均高于16.7 mmol/L判定为模型制备成功。随后将试验动物分为四组，分别为空白对照组（C）、牛磺酸对照组（T）、糖尿病模型组（M）、牛磺酸干预组（T+M），试验期为12周。试验结果显示，牛磺酸可上调STZ诱导的糖尿病模型大鼠胰腺干细胞标志物表达，促进胰岛β的增殖、分化，改善由STZ诱导的糖尿病模型大鼠的血糖升高，体重降低等糖尿病症状。. 体外试验：采用单次腹腔注射链脲佐菌素（streptozotocin, STZ）的方法建立大鼠糖尿病模型，从模型大鼠胰腺导管分离、提取、纯化胰腺干细胞，并进行体外传代培养；通过免疫细胞化学法鉴定胰腺干细胞表面标志物（Nestin、CK-19和PDX-1）的表达。将糖尿病大鼠胰腺干细胞分为对照组（基础培养基）和牛磺酸组（基础培养基中加入10mmol/L牛磺酸）。牛磺酸显著增加了糖尿病大鼠胰腺干细胞活力、细胞周期中DNA合成期的比例和增殖指数，显著上调了细胞增殖相关抗原mRNA和蛋白的表达；牛磺酸通过NF-κB和Ras/ERK信号通路促进了糖尿病大鼠胰腺干细胞的增殖；并能促进干细胞分化为活性良好的类胰岛细胞团。. 研究结果提示牛磺酸有望应用于人类或动物糖尿病治疗。