【作者】 张斌；

【导师】 郭刚；

【作者基本信息】 天津医科大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 目的:糖尿病是由于胰岛素分泌缺陷或其生物作用受损,或两者兼有引起的一组以高血糖为特征的代谢性疾病。通过之前的研究发现小肠能够分泌胰岛素,在糖尿病发病过程中起到一定的作用。为进一步研究大鼠小肠中的胰岛素是否为小肠上皮细胞分泌,我们通过本次研究提取大鼠小肠上皮细胞进行细胞体外培养,测定大鼠小肠上皮细胞胰岛素基因表达水平,从蛋白水平测定大鼠小肠上皮细胞是否分泌了胰岛素原,以及胰岛素原和C肽的水平。确定大鼠小肠上皮细胞胰岛素分泌的功能。方法:本实验通过脱颈处死Wistar孕鼠,剖宫取出胎鼠小肠,将胎鼠小肠上皮细胞以及胰岛细胞进行原代培养,用胰岛细胞作为对照,将两种细胞置于葡萄糖浓度分别为100mg/d L,200mg/d L,300mg/d L,400mg/d L的培养基中传代培养。经过传代培养后,分别取各细胞总RNA,反转录成c DNA,普通PCR检测出大鼠小肠上皮细胞胰岛素m RNA的存在,然后用实时荧光定量PCR检测大鼠小肠上皮细胞和胰岛细胞胰岛素m RNA的相对表达量,用统计学方法,相同浓度下两种细胞的胰岛素m RNA表达量作比较。用免疫组化的方法检测细胞中的胰岛素以及胰岛素原的表达情况。用蛋白层析分离蛋白,Western Blot鉴定胰岛素原的活性。结果:经过普通PCR检测出大鼠小肠上皮细胞确实有胰岛素基因的表达。实时荧光定量PCR数据显示大鼠小肠上皮细胞在正常情况下胰岛素m RNA表达量远低于大鼠胰岛细胞,葡糖糖浓度较高的情况下胰岛素m RNA表达水平有一定的提高,在葡萄糖浓度为200mg/d L的时候达到最高,接近大鼠胰岛细胞的表达水平。经过测定,可以确定大鼠小肠上皮细胞能够分泌胰岛素原,但是却没有胰岛素和C肽的生成。结论:本实验证明了大鼠小肠细胞能表达出胰岛素基因,并且在葡萄糖浓度为200mg/d L的时候大鼠小肠上皮细胞和胰岛细胞的胰岛素m RNA相对表达量没有显著差异(P>0.05)。说明小肠上皮细胞具有一定的分泌胰岛素原的功能,并且跟葡萄糖浓度有密切的关系,可能与糖尿病的发病机制有关。更多还原

【Abstract】 Objective: Diabetes mellitus is caused by defects in insulin secretion or its biological function is impaired,or both lead to a group characterized by hyperglycemia and metabolic diseases.Previous studies have found that the small intestine is capable of secreting insulin,which plays a role in the pathogenesis of diabetes.To further study the secretion of insulin in the small intestine of the rat,we extracted the cells of rat small intestine epithelial in vitro by this study.then determine the insulin gene in rat intestinal epithelial cell,and whether the rat intestinal epithelial cell can secrete proinsulin insulin and C peptide.To define the function of the insulin secretion of rat small intestine epithelial cells.Methods: The wistar pregnant rats were killed by cervical dislocation,split the uterus and get the small intestine of fetal rat,the epithelial cells of fetal rat intestine and islet cells were cultured in glucose concentrations were 100mg/dL,200mg/dL,300mg/dL,400mg/dL,islet cells group for comparison.After passage,we obtained the total RNA from the cells,reverse transcription into cDNA,PCR to detect the insulin of rat intestinal epithelial cells in primary mRNA,and then use Quantitative Real-time PCR to detect the relative expression of mRNA in rat intestinal epithelial cells and pancreatic cells.And use statistical methods to compare expression of insulin mRNA in two cells at the same concentration.We use the method of immunohistochemistry to detect insulin and proinsul in cells.Use the method fo protein chromatography to separate and purificat protein,then use Western Blot to identify the activity of proinsulin.Results: After PCR to detect the rat small intestinal epithelial cell have the expression of insulin gene.Data displayed from Quantitative Real-time PCR show that in the normal circumstances the rat intestinal epithelial cell insulin mRNA expression was lower than pancreatic cells,but under the condition of high concentration of glucose insulin mRNA expression level was improved,in the glucose concentration when 200mg/dL reached the highest,nearly the pancreatic cells in rats.Through determination,can determine the rat intestinal epithelial cells can secrete proinsulin,but without insulin and C peptide.Conclusion: The experiment proved that the small intestine of rats can express the gene of insulin.And there was no significant difference in the expression of insulin mRNA between the small intestine epithelial and the islet cells at a glucose concentration of 200mg/dL(P>0.05).This experiment shows that rat intestinal epithelial cells have the function to secrete proinsulin.And has a close relationship with the concentration of glucose,may be related to the pathogenesis of diabetes.更多还原