番石榴叶总黄酮促进糖尿病模型小鼠胰岛再生机制的实验研究

发布时间：2018-06-29 08:44

本文选题：糖尿病 + 番石榴叶总黄酮　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:近年来,糖尿病(Diabetes Mellitus,DM)的患病率在世界范围内逐年攀升,尤其是其并发症严重威胁着人类的健康。DM病理变化均存在胰岛β细胞功能的受损或丧失,导致胰岛素分泌不足。目前DM的最常用治疗方法主要包括各类降糖药物治疗或胰岛素替代治疗,但这些治疗方法只能暂时降低血糖以及延缓并发症的发生,长期使用还易发生低血糖、胃肠道反应等诸多副作用,不能从根本上治疗糖尿病。近年来,胰岛或胰腺移植技术兴起,取得了显著进展,但由于其存在供体不足、排斥反应、技术不成熟等诸多瓶颈,临床难以广泛推广。而中医中药基于其副作用小、安全性高等优势,在治疗DM方面成效显著。因此,运用中医中药促进DM患者胰岛β细胞再生、维持其胰岛素分泌功能有可能从根本上治疗DM。本文将通过研究番石榴叶总黄酮对链脲佐菌素(Streptozocin,STZ)诱导的DM模型小鼠胰腺组织中胰岛发育相关转录因子胰腺十二指肠同源盒因子-1(Pancreas-duodenumhomeobox-1,PDX-1)、神经元素3(Neurogenin 3,Ngn3)、NK6转录因子相关基因座1(NK6 Transcri Ption Factor Related Locus 1,Nkx6.1)、SRY相关促HMG盒9(SRY-related HMG-box9,SOX9)、配对盒4(Paired box4,PAX4)表达的影响,探讨番石榴叶总黄酮促进DM胰岛β细胞再生机制,为治疗DM提供新的思路及方法。方法:将体重约19g的SPF级健康雄性BALB/c小鼠禁食(未禁水)过夜,次日测其空腹血糖水平及体重情况,然后按体重60 mg·kg~(-1)剂量经腹腔连续注射STZ 5天,2周后开始经小鼠尾静脉采血检测空腹血糖水平,以后每隔1周检测1次,待小鼠空腹血糖达到11.1mmol·L~(-1)以后,再每隔3天检测1次,连续2次空腹血糖浓度≥11.1 mmol·L~(-1)为DM小鼠模型成功,三次血糖平均值作为治疗前DM模型小鼠血糖,将注射后8周仍未达标(即空腹血糖浓度11.1mmol·L~(-1))小鼠丢弃。再将STZ诱导成功的DM模型小鼠随机分为模型组(10只)、番石榴叶总黄酮低剂量组(10只)、番石榴叶总黄酮高剂量组(10只)及二甲双胍组(10只),另设10只正常小鼠为对照。然后将番石榴叶总黄酮提取物溶于蒸馏水中行灌胃治疗,高、低剂量组按小鼠体重给予每天的提取物灌胃量分别为2g·kg~(-1)、1g·kg~(-1),即番石榴叶总黄酮的剂量为0.396g·kg~(-1)·d~(-1)、0.198g·kg~(-1)·d~(-1),二甲双胍灌胃量为0.0875g·kg~(-1)·d~(-1),正常组和DM模型组以等体积蒸馏水灌胃。灌胃2周后测其空腹血糖水平及体重情况,苏木素伊红(HE)染色评价胰岛形态学改变,逆转录聚合酶链反应(RT-PCR)法、蛋白质印迹法(Western Blot)及免疫组化(IHC)检测胰腺组织内与胰岛β细胞发育相关的转录因子:PDX-1、Ngn3、Nkx6.1、SOX9、PAX4m RNA及蛋白的表达情况。结果:本实验显示STZ诱导的DM模型小鼠的空腹血糖较正常组小鼠显著升高(P0.01),体重较正常组小鼠明显下降(P0.05);与STZ诱导的DM模型小鼠相比,番石榴叶总黄酮治疗组及二甲双胍组空腹血糖均明显降低(P0.05),体重增加(P0.05);HE染色显示模型组小鼠胰岛破坏严重,呈散在零星分布;番石榴叶总黄酮治疗组DM小鼠胰岛较模型组破坏减少,胰岛数量增加、体积增大,胰岛呈片状或沿胰腺导管分布。IHC染色显示,与正常组比较,DM模型组小鼠胰腺组织中PDX-1的表达量显著减少;番石榴叶总黄酮治疗组与DM模型组相比,小鼠胰腺组织中PDX-1的表达量均升高。RT-PCR及Western Blot检测显示,DM模型组小鼠胰腺组织中PDX-1、Ngn3、Nkx6.1、SOX9、PAX4m RNA及蛋白表达量较正常组显著下降(P0.01);而番石榴叶总黄酮治疗组与DM模型组比,胰腺组织中与胰岛再生相关的转录因子PDX-1、Ngn3、Nkx6.1、SOX9、PAX4m RNA及蛋白表达量也呈升高趋势,且与番石榴叶总黄酮的剂量呈相关性。结论:1.DM模型小鼠的胰岛细胞破坏严重,空腹血糖水平显著升高,胰腺组织中与胰岛再生有关的相关转录因子表达水平明显下降。2.番石榴叶总黄酮能显著降低DM小鼠的血糖水平,减轻胰岛细胞破坏,且能上调DM小鼠胰腺组织中与胰岛发育有关的相关转录因子表达水平。3.番石榴叶总黄酮对DM小鼠受损的胰岛具有促进再生作用,且其机制可能与上调调控胰岛β细胞再生相关转录因子的表达有关。
[Abstract]:Objective: in recent years, the prevalence of Diabetes Mellitus (DM) has been increasing worldwide, especially its complications are serious threat to human health,.DM pathological changes all have impaired or loss of islet beta cell function, resulting in insufficient insulin secretion. At present, the most commonly used treatment methods of DM include the treatment of various kinds of hypoglycemic drugs. Treatment or insulin replacement therapy, but these treatments can only temporarily reduce blood sugar and delay the occurrence of complications, long-term use of hypoglycemia, gastrointestinal reaction and many other side effects, can not be fundamentally treated with diabetes. In recent years, the rise of pancreatic islet or pancreas transplantation has made significant progress, but because of its presence in the donor Many bottlenecks such as foot, rejection and technology are not mature, and it is difficult to spread widely in clinic. Traditional Chinese medicine has the advantages of small side effect and high safety, so it is effective in the treatment of DM. Therefore, the use of traditional Chinese medicine to promote the regeneration of pancreatic islet beta cells in DM patients and to maintain its insulin secretion may be fundamentally treated by DM.. The pancreatic islet development related transcription factors, pancreatic duodenal homeobox factor -1 (Pancreas-duodenumhomeobox-1, PDX-1), nerve element 3 (Neurogenin 3, Ngn3), and NK6 transcriptional factor related loci 1 (NK6 Transcri Ption) were studied in the pancreatic tissue of DM model mice induced by Streptozocin (STZ). D Locus 1, Nkx6.1), the effect of SRY related HMG box 9 (SRY-related HMG-box9, SOX9), paired box 4 (Paired Box4, PAX4) expression, to explore the mechanism of beta cell regeneration by the total flavonoids of guava leaf to promote the regeneration of beta cell beta cells. The fasting blood glucose level and body weight condition were then injected into the abdominal cavity for 5 days at the dose of 60 mg. Kg~ (-1). After 2 weeks, the fasting blood glucose level was detected in the tail vein of mice. After 1 weeks, 1 times were detected every 1 weeks. After the fasting blood glucose of the mice reached 11.1mmol. L~ (-1), then 1 times every 3 days, 2 fasting blood glucose concentrations were more than 11.. 1 mmol / L~ (-1) was a successful DM mouse model, and the average of three blood glucose was used as the DM model mice before the treatment. The mice were discarded at 8 weeks after the injection (i.e., 11.1mmol L~ (-1) in the fasting plasma glucose concentration). Then the DM model mice were randomly divided into the model group (10 rats), the low dose group (10) of the guava leaf total flavonoids (10), and the guava leaf total. Huang Tonggao dose group (10) and metformin group (10 rats) and 10 normal mice were compared. Then the total flavonoids extract of guava leaf was dissolved in distilled water for gavage treatment. The dosage of the group was 2G. Kg~ (-1), 1g. Kg~ (-1), and the dosage of total flavonoids of guava leaf was 0.396g. K G~ (-1) / d~ (-1), 0.198g, kg~ (-1) and d~ (-1), and the gastric perfusion of metformin was 0.0875g. Kg~ (-1). The normal group and the model group were gavage with equal volume distilled water. After 2 weeks of gastric perfusion, the fasting blood glucose level and body weight were measured. The morphological changes of islets were evaluated by hematoxylin staining, reverse transcription polymerase chain reaction (reverse transcription) method, Western blot The expression of PDX-1, Ngn3, Nkx6.1, SOX9, PAX4m RNA and protein in the pancreatic tissue was detected by Western Blot and immunohistochemistry (IHC). Results: this experiment showed that the fasting blood glucose of DM model mice induced by STZ was significantly higher than that of normal mice (P0.01), and the weight of the mice was significantly lower than that of the normal mice. 0.05): compared with the DM model mice induced by STZ, the fasting blood glucose of the guava leaf total flavonoids treatment group and the metformin group decreased significantly (P0.05) and the body weight increased (P0.05); HE staining showed that the islet damage was serious and scattered in the model group, and the damage of the islets of the guava Ye Zonghuang ketone group was less than that in the model group and the number of islets increased. In addition, the volume increased, the islets were flaky or along the pancreatic duct distribution.IHC staining. Compared with the normal group, the expression of PDX-1 in the pancreas tissue of the DM model group was significantly reduced. The expression of PDX-1 in the pancreas tissue of the guava leaf total flavonoids was higher than that in the DM model group. The.RT-PCR and Western Blot detection showed in the pancreas tissue of the mice, and the DM model group was found. The expression of PDX-1, Ngn3, Nkx6.1, SOX9, PAX4m RNA and protein in the pancreatic tissue of mice was significantly lower than that in the normal group (P0.01). Compared with the DM model group, the total flavonoids in the guava leaf and the DM model group were also higher in the expression of the transcription factors, Ngn3, Nkx6.1, SOX9, and protein expression in the pancreatic tissue. Conclusion: the dose of ketone is related. Conclusion: the islet cells of the 1.DM model mice are seriously damaged, the level of fasting blood glucose is significantly increased, the expression level of the related transcription factors related to the pancreatic islet regeneration in the pancreatic tissue is significantly lower than that of the.2. guava leaf, which can significantly reduce the level of blood glucose level in DM mice, reduce the destruction of islet cells, and up regulate the DM mouse pancreas. The expression level of the related transcription factors related to islet development in the adeno tissue.3. can promote regeneration of the damaged islets of DM mice, and the mechanism may be related to the up regulation of the expression of the transcriptional factors related to the regulation of the regeneration of islet beta cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R-332

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