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研究生:洪千惠
研究生(外文):Chien Hui Hung
論文名稱:RANTES與其受體CCR5對胰臟胰島素分泌的影響
論文名稱(外文):The Influence of RANTES and Its Receptor (CCR5) on Pancreatic Insulin Secretion
指導教授:洪麗滿
指導教授(外文):L. M. Hung
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:69
中文關鍵詞:胰臟功能胰島素
外文關鍵詞:Pancreatic functioninsulinCCL5CCR5
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CC趨化因子(chemokine)是一種低分子量的蛋白質家族,參與促發炎反應,先前的研究指出C-C Chemokine ligand 5 (CCL5, RANTES)與C-C chemokine receptor type 5 (CCR5, CD195)結合後會參與發炎、癌症和糖尿病病程的發展及促進血管内皮生長因子(vascular endothelial growth factor, VEGF)生成而影響胰臟功能性;然而兩者對於胰島素敏感性與胰島素分泌的影響目前仍沒有清楚的結論。
本研究目的在探討CCL5及CCR5對胰島素敏感性與胰島分泌胰島素功能的影響。實驗利用28週齡野生型(Wild-Type, WT)、CCL5 knockout (L5KO)、CCR5 knockout (R5KO)及CCL5/CCR5 double knockout (LR5KO)四組小鼠,利用口服葡萄糖耐受性試驗(Oral glucose tolerance test, OGTT)與腹腔注射胰島素耐受性試驗(Intraperitoneal insulin tolerance test, IPITT)來檢測葡萄糖耐受性及胰島素敏感性。此外,實驗小鼠於第28週齡犧牲,取胰臟組織切片,並利用H&E染色來比較各實驗動物胰臟型態上的差異。實驗結果發現L5KO小鼠的體重與WT相比沒有差異,然而R5KO會使體重顯著上升;而LR5KO小鼠的卻會使體重顯著下降。在三組基因剔除小鼠的空腹血糖皆有上升,但僅R5KO小鼠之空腹胰島素有顯著上升;合併葡萄糖耐受性測試的結果,R5KO小鼠的葡萄糖耐受性變差,因此推測R5KO小鼠有葡萄糖不耐的情形,但特別的是R5KO小鼠在給予葡萄糖刺激後胰島素分泌(Glucose-stimulated insulin secretion, GSIS)與WT比較無任何差異。除此之外胰臟組織H&E與IHC染色以及RT-qPCR結果發現,R5KO小鼠的胰島有增大而且胰島內胰島素含量有增加的趨勢。Western blot實驗結果也發現R5KO小鼠胰臟中ATP-sensitive K channel subunit Kir6.2蛋白質表現量之稍微增加可能參與R5KO小鼠胰島素上升之機制。
經由本實驗結果推測R5KO小鼠的胰島素釋放上升可能與Kir6.2蛋白質表現量增加有關,而L5KO小鼠和LR5KO小鼠小鼠並不會影響胰島素分泌能力。
CC chemokine, a superfamily of low molecular weight protein that can promote inflammation. Previous studies have demonstrated that the interaction of C-C chemokine ligand 5 (CCL5, RANTES) and C-C chemokine receptor type 5 (CCR5, CD195), participates in inflammation, up-regulation of vascular endothelial growth factor (VEGF), progression of diabetes, and subsequently affect pancreatic function. However, the impacts of CCL5 and CCR5 in insulin sensitivity and insulin secretion have not yet been elucidated.
Therefore, we aimed to investigate the effect of CCL5 and CCR5 in insulin sensitivity and pancreatic islet insulin secretion. Twenty-eight weeks old C57BL6 mice were separated into 4 groups as wild-type (WT), CCL5-knockout (L5KO), CCR5-knockout (R5KO), and CCL5/CCR5 double knockout (LR5KO). Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT) were used to examine the glucose tolerance and insulin sensitivity. In addition, mice were sacrificed at the age of 28 weeks, and morphology of pancreatic islet was analyzed by H&E staining. The body weight of L5KO mice has no change compared with WT mice;whereas R5KO mice exhibited a significantly increasing of body weight, while the body weight of LR5KO mice was decreased. The fasting blood glucose level was increased in all three knockout mice. However, hyperinsulinemia was only observed in R5KO mice. Combination with the higher glucose level under OGTT in R5KO mice, we suggest a glucose intolerance in R5KO mice. Interestingly, the glucose-stimulated insulin secretion (GSIS) in R5KO mice had no difference compared with WT. In addition, the enlargement of pancreatic islet and high level of insulin production were observed by H&E, IHC and RT-qPCR. Moreover, the results of western blot show an increasing trend of ATP-sensitive K channel subunit Kir6.2 in pancreas, suggesting a contributor to hyperinsulinemia in R5KO mice.
These results suggest that the up-regulated Kir6.2 protein expression in CCR5 knockout mice may contribute to enhance insulin secretion; whereas L5KO and LR5KO have no effect on pancreatic insulin secretion.
指導教授推薦書
口試委員會審定書
致謝 iii
中文摘要(Abstract) v
Abstract vii
目錄 x
圖目錄 xiv
表目錄 xv
1.前言(Introduction) - 1 -
1.1胰臟的功能及其重要性 - 1 -
1.1.1胰島素 - 2 -
1.1.2胰島素分泌機制 - 2 -
1.1.3胰島素訊號傳遞路徑 - 4 -
1.1.4胰島素阻抗 - 5 -
1.1.5胰臟與第二型糖尿病 - 6 -
1.2趨化因子 - 7 -
1.2.1 CCL5 - 9 -
1.2.2 CCR5 - 9 -
1.3胰臟和趨化因子 - 10 -
1.4 CCL5和CCR5對血糖的調控 - 11 -
2.研究目的(Research Aims) - 13 -
3.材料與方法(Materials andMethods) - 14 -
3.1實驗流程 - 14 -
3.2實驗動物與飼料 - 14 -
3.3血液生化值 - 15 -
3.3.1胰島素之測定(Insulin) - 15 -
3.3.2血糖值之測定(Glucose) - 16 -
3.3.3血漿膽固醇之測定(Cholesterol) - 17 -
3.3.4血清三酸甘油脂之測定(Triglycerides) - 18 -
3.3.5血清非酯化游離脂肪酸 - 19 -
3.4腹腔胰島素耐受性測試(Intraperitoneal insulin tolerance test,IPITT) - 20 -
3.5口服葡萄糖耐受性試驗(Oral Glucose Tolerance Test,OGTT) - 20 -
3.6組織採集 - 21 -
3.7胰臟之蛋白質萃取 - 21 -
3.8蛋白質定量及分裝 - 22 -
3.9蛋白質電泳及西方點墨法 - 22 -
3.10胰臟之RNA萃取 - 23 -
3.11 RNA轉成cDNA - 24 -
3.12即時聚合酶鏈鎖反應(Real-time polymerase chain reaction,Real-time PCR) - 25 -
3.13組織染色切片 - 27 -
3.13.1蘇木精-伊紅染色(Hematoxylin and Eosin stain,H&E Stain) - 27 -
3.13.2免疫組織化學染色(Immunohistochemistry,IHC) - 28 -
3.14胰島定量方法 - 29 -
3.15統計分析 - 30 -
4結果(Results) - 31 -
4.1比較28週齡WT、L5KO、R5KO及LR5KO小鼠的體重、血糖、胰島素及血液生化值 - 31 -
4.2比較WT、L5KO、R5KO及LR5KO小鼠的口服葡萄糖耐受性測試(OGTT)血糖及胰島素分泌量 - 33 -
4.3比較WT、L5KO、R5KO及LR5KO小鼠的腹腔注射胰島素耐受性測試(IPITT) - 34 -
4.4比較WT、L5KO、R5KO及LR5KO小鼠之胰島外觀及大小 - 34 -
4.5比較28週齡WT、L5KO、R5KO及LR5KO小鼠胰島內的胰島素含量 - 35 -
4.6比較WT、L5KO、R5KO及LR5KO小鼠胰島素mRNA表現量 - 36 -
4.7比較WT、L5KO、R5KO及LR5KO小鼠胰臟Kir6.2 蛋白質表現量 - 36 -
5.討論(Discussion) - 37 -
6.參考文獻(References) - 40 -
7.圖表(Figures and Tables) - 46 -

圖目錄
Figure 1. 利用口服葡萄糖耐受性測試比較WT、L5KO、R5KO及LR5KO小鼠之葡萄糖的耐受性。 - 47 -
Figure 2. 利用口服葡萄糖耐受性測試及血液胰島素變化比較WT、L5KO、R5KO及LR5KO小鼠之口服葡萄糖期間血液胰島素分泌變化。 - 48 -
Figure 3. 利用腹腔注射胰島素耐受性試驗比較WT、L5KO、R5KO及LR5KO小鼠對胰島素的敏感性。 - 49 -
Figure 4. 利用H&E染色比較16週齡和28週齡WT、L5KO、R5KO及LR5KO小鼠胰島的型態外觀與面積。 - 50 -
Figure 5. 量化H&E染色結果比較16週齡(A)和28週齡(B)WT、L5KO、R5KO及LR5KO小鼠胰島的平均大小。 - 51 -
Figure 6. 利用IHC染色比較28週齡WT、L5KO、R5KO及LR5KO小鼠胰島β細胞胰島素之表現。 - 52 -
Figure 7. 利用RT-qPCR比較WT、L5KO、R5KO及LR5KO小鼠胰島素mRNA表現量。 - 53 -
Figure 8. 比較WT、L5KO、R5KO及LR5KO小鼠胰臟Kir6.2蛋白質表現量。 - 54 -

表目錄
Table 1.比較28週齡WT、L5KO、R5KO及LR5KO小鼠之體重、血液中葡萄糖、胰島素及生化值 - 46 -
6.參考文獻(References)
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