本文選題：糖尿病 + 番石榴葉總黃酮　； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:近年來(lái),糖尿病(Diabetes Mellitus,DM)的患病率在世界范圍內(nèi)逐年攀升,尤其是其并發(fā)癥嚴(yán)重威脅著人類(lèi)的健康。DM病理變化均存在胰島β細(xì)胞功能的受損或喪失,導(dǎo)致胰島素分泌不足。目前DM的最常用治療方法主要包括各類(lèi)降糖藥物治療或胰島素替代治療,但這些治療方法只能暫時(shí)降低血糖以及延緩并發(fā)癥的發(fā)生,長(zhǎng)期使用還易發(fā)生低血糖、胃腸道反應(yīng)等諸多副作用,不能從根本上治療糖尿病。近年來(lái),胰島或胰腺移植技術(shù)興起,取得了顯著進(jìn)展,但由于其存在供體不足、排斥反應(yīng)、技術(shù)不成熟等諸多瓶頸,臨床難以廣泛推廣。而中醫(yī)中藥基于其副作用小、安全性高等優(yōu)勢(shì),在治療DM方面成效顯著。因此,運(yùn)用中醫(yī)中藥促進(jìn)DM患者胰島β細(xì)胞再生、維持其胰島素分泌功能有可能從根本上治療DM。本文將通過(guò)研究番石榴葉總黃酮對(duì)鏈脲佐菌素(Streptozocin,STZ)誘導(dǎo)的DM模型小鼠胰腺組織中胰島發(fā)育相關(guān)轉(zhuǎn)錄因子胰腺十二指腸同源盒因子-1(Pancreas-duodenumhomeobox-1,PDX-1)、神經(jīng)元素3(Neurogenin 3,Ngn3)、NK6轉(zhuǎn)錄因子相關(guān)基因座1(NK6 Transcri Ption Factor Related Locus 1,Nkx6.1)、SRY相關(guān)促HMG盒9(SRY-related HMG-box9,SOX9)、配對(duì)盒4(Paired box4,PAX4)表達(dá)的影響,探討番石榴葉總黃酮促進(jìn)DM胰島β細(xì)胞再生機(jī)制,為治療DM提供新的思路及方法。方法:將體重約19g的SPF級(jí)健康雄性BALB/c小鼠禁食(未禁水)過(guò)夜,次日測(cè)其空腹血糖水平及體重情況,然后按體重60 mg·kg~(-1)劑量經(jīng)腹腔連續(xù)注射STZ 5天,2周后開(kāi)始經(jīng)小鼠尾靜脈采血檢測(cè)空腹血糖水平,以后每隔1周檢測(cè)1次,待小鼠空腹血糖達(dá)到11.1mmol·L~(-1)以后,再每隔3天檢測(cè)1次,連續(xù)2次空腹血糖濃度≥11.1 mmol·L~(-1)為DM小鼠模型成功,三次血糖平均值作為治療前DM模型小鼠血糖,將注射后8周仍未達(dá)標(biāo)(即空腹血糖濃度11.1mmol·L~(-1))小鼠丟棄。再將STZ誘導(dǎo)成功的DM模型小鼠隨機(jī)分為模型組(10只)、番石榴葉總黃酮低劑量組(10只)、番石榴葉總黃酮高劑量組(10只)及二甲雙胍組(10只),另設(shè)10只正常小鼠為對(duì)照。然后將番石榴葉總黃酮提取物溶于蒸餾水中行灌胃治療,高、低劑量組按小鼠體重給予每天的提取物灌胃量分別為2g·kg~(-1)、1g·kg~(-1),即番石榴葉總黃酮的劑量為0.396g·kg~(-1)·d~(-1)、0.198g·kg~(-1)·d~(-1),二甲雙胍灌胃量為0.0875g·kg~(-1)·d~(-1),正常組和DM模型組以等體積蒸餾水灌胃。灌胃2周后測(cè)其空腹血糖水平及體重情況,蘇木素伊紅(HE)染色評(píng)價(jià)胰島形態(tài)學(xué)改變,逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)法、蛋白質(zhì)印跡法(Western Blot)及免疫組化(IHC)檢測(cè)胰腺組織內(nèi)與胰島β細(xì)胞發(fā)育相關(guān)的轉(zhuǎn)錄因子:PDX-1、Ngn3、Nkx6.1、SOX9、PAX4m RNA及蛋白的表達(dá)情況。結(jié)果:本實(shí)驗(yàn)顯示STZ誘導(dǎo)的DM模型小鼠的空腹血糖較正常組小鼠顯著升高(P0.01),體重較正常組小鼠明顯下降(P0.05);與STZ誘導(dǎo)的DM模型小鼠相比,番石榴葉總黃酮治療組及二甲雙胍組空腹血糖均明顯降低(P0.05),體重增加(P0.05);HE染色顯示模型組小鼠胰島破壞嚴(yán)重,呈散在零星分布;番石榴葉總黃酮治療組DM小鼠胰島較模型組破壞減少,胰島數(shù)量增加、體積增大,胰島呈片狀或沿胰腺導(dǎo)管分布。IHC染色顯示,與正常組比較,DM模型組小鼠胰腺組織中PDX-1的表達(dá)量顯著減少;番石榴葉總黃酮治療組與DM模型組相比,小鼠胰腺組織中PDX-1的表達(dá)量均升高。RT-PCR及Western Blot檢測(cè)顯示,DM模型組小鼠胰腺組織中PDX-1、Ngn3、Nkx6.1、SOX9、PAX4m RNA及蛋白表達(dá)量較正常組顯著下降(P0.01);而番石榴葉總黃酮治療組與DM模型組比,胰腺組織中與胰島再生相關(guān)的轉(zhuǎn)錄因子PDX-1、Ngn3、Nkx6.1、SOX9、PAX4m RNA及蛋白表達(dá)量也呈升高趨勢(shì),且與番石榴葉總黃酮的劑量呈相關(guān)性。結(jié)論:1.DM模型小鼠的胰島細(xì)胞破壞嚴(yán)重,空腹血糖水平顯著升高,胰腺組織中與胰島再生有關(guān)的相關(guān)轉(zhuǎn)錄因子表達(dá)水平明顯下降。2.番石榴葉總黃酮能顯著降低DM小鼠的血糖水平,減輕胰島細(xì)胞破壞,且能上調(diào)DM小鼠胰腺組織中與胰島發(fā)育有關(guān)的相關(guān)轉(zhuǎn)錄因子表達(dá)水平。3.番石榴葉總黃酮對(duì)DM小鼠受損的胰島具有促進(jìn)再生作用,且其機(jī)制可能與上調(diào)調(diào)控胰島β細(xì)胞再生相關(guān)轉(zhuǎn)錄因子的表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R587.1;R-332

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