本文選題：二氮嗪 + 糖尿病�。� 參考：《第二軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：研究目的： 糖尿病(Diabetes mellitus，DM)是一種全身系統(tǒng)性代謝紊亂疾病，分為胰島素依賴型(Insulin-dependent)與非胰島素依賴型(Insulin-independent)。大血管及微血管病變是糖尿病患者主要的致殘致死原因，而內(nèi)皮功能的損傷(Endothelial dysfunction)則是引起傷口愈合缺陷和血管病變的始發(fā)因子。內(nèi)皮細(xì)胞(Endothelial cells，ECs)的活性是維持內(nèi)皮功能的基礎(chǔ)，如果內(nèi)皮細(xì)胞的凋亡率超過正常水平，它就會(huì)影響血管的完整性。內(nèi)皮祖細(xì)胞(Endothelial progenitor cells, EPCs)來源于成體骨髓，并可分化為內(nèi)皮細(xì)胞，進(jìn)而參與血管新生和內(nèi)皮損傷后的修復(fù)過程。已有研究表明，骨髓來源的內(nèi)皮祖細(xì)胞(Bone marrow-derived endothelial progenitor cells, BM-EPCs)可以定向結(jié)合到血管再生部位并參與受損內(nèi)皮的修復(fù)。循環(huán)中的EPCs的數(shù)量與發(fā)生血管疾病的概率呈反比。因此EPCs與內(nèi)皮功能有著至關(guān)重要的聯(lián)系。凝血酶敏感蛋白-1(Thrombospondin-1,TSP-1)是一種有效的內(nèi)源性的血管生成抑制劑，糖尿病會(huì)引起EPCs的TSP-1mRNA的表達(dá)量增加, EPCs功能障礙也會(huì)導(dǎo)致TSP-1的表達(dá)增加。腫瘤抑制基因p53可以激活由THBS1基因編碼的TSP-1的啟動(dòng)子，有文獻(xiàn)指出：p53可促進(jìn)TSP-1的產(chǎn)生，使成纖維細(xì)胞的血管新生受損。二氮嗪(Diazoxide)是線粒體KATP通道開放劑，可通過使細(xì)胞膜超極化，K+外流增多，Ca2+內(nèi)流減少，具有抑制葡萄糖刺激的胰島素分泌，從而為胰島B細(xì)胞細(xì)胞提供休息，發(fā)揮延緩和阻礙糖尿病及其并發(fā)癥的發(fā)展的作用。本課題研究二氮嗪能否加速糖尿病小鼠傷口愈合，改善BM-EPCs功能，并探索其可能機(jī)制。 研究?jī)?nèi)容及結(jié)果： 動(dòng)物模型：使用鏈脲佐菌素(Streptozotocin, STZ)誘導(dǎo)的1型糖尿病小鼠模型；細(xì)胞模型：以葡萄糖33mM誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞(Human umbilical veinendothelial cells, HUVECs)和BM-EPCs高糖損傷的細(xì)胞模型。在動(dòng)物水平，研究了二氮嗪對(duì)糖尿病小鼠內(nèi)皮功能的影響，從EPCs角度圍繞TSP-1和p53兩個(gè)作用靶點(diǎn)探討了其機(jī)制；在細(xì)胞水平，通過細(xì)胞功能實(shí)驗(yàn)(遷移功能、粘附功能和小管形成功能)來觀察二氮嗪對(duì)血管生成的影響；并通過測(cè)定TSP-1和p53的表達(dá)來明確二氮嗪影響血管生成的機(jī)制。 1.二氮嗪促進(jìn)1型糖尿病小鼠傷口愈合 選取5周齡的C57BL/6小鼠，18-20g左右，隨機(jī)分為正常組(Control)、1型糖尿病模型組(STZ)和二氮嗪治療組(30mg/kg/d灌胃4周)。小鼠麻醉狀態(tài)下，在其背部切取一個(gè)半徑6mm的圓形傷口，連續(xù)12天每2天觀察其傷口愈合的情況。第3、6、9天分別取傷口周圍6mm內(nèi)的皮膚作為標(biāo)本，CD31免疫組化檢測(cè)血管密度情況。取2-3mm小鼠的主動(dòng)脈，通過血管張力系統(tǒng)來檢測(cè)其血管舒張功能。結(jié)果顯示：1型糖尿病小鼠的傷口愈合速度明顯降低，毛細(xì)血管密度顯著下降，血管依賴性內(nèi)皮舒張功能明顯下降；二氮嗪治療后傷口愈合能力增強(qiáng)，血管密度增加，血管依賴性內(nèi)皮舒張功能增強(qiáng)。 2.二氮嗪改善1型糖尿病小鼠BM-EPCs功能 采用Dil-acLDL和FITC-UEA-1雙陽性鑒定BM-EPCs。通過CD34和Flk-1雙陽性的百分比檢測(cè)小鼠循環(huán)中的EPCs數(shù)量。通過Boyden小室和成管實(shí)驗(yàn)檢測(cè)遷移功能和小管形成能力。結(jié)果顯示：1型糖尿病小鼠循環(huán)中EPCs的數(shù)目明顯降低，二氮嗪治療后可以顯著增加循環(huán)中EPCs的數(shù)量；1型糖尿病小鼠BM-EPCs的小管形成能力、遷移功能和粘附功能明顯下降，二氮嗪治療后可以顯著改善BM-EPCs的小管形成能力、遷移功能和粘附功能。 3.二氮嗪改善1型糖尿病小鼠BM-EPCs功能的可能機(jī)制 通過免疫印跡法檢測(cè)1型糖尿病小鼠BM-EPCs中p53和TSP-1表達(dá)。結(jié)果表明：1型糖尿病小鼠BM-EPCs的p53表達(dá)明顯升高，二氮嗪治療后可以顯著降低BM-EPCs中p53的水平；1型糖尿病小鼠BM-EPCs的TSP-1的表達(dá)明顯增加，二氮嗪治療后可以顯著降低TSP-1的表達(dá)。 4.二氮嗪改善高糖誘導(dǎo)的BM-EPCs功能障礙及機(jī)制 正常小鼠骨髓分離的BM-EPCs培養(yǎng)7天后，使用高糖(33mM)誘導(dǎo)細(xì)胞損傷模型，二氮嗪(30μM)處理24h后，檢測(cè)細(xì)胞功能和蛋白表達(dá)情況。結(jié)果顯示：高糖誘導(dǎo)的BM-EPCs小管形成能力、遷移功能和粘附功能明顯降低，二氮嗪(30μM)處理后可以顯著改善高糖誘導(dǎo)對(duì)BM-EPCs小管形成能力、遷移功能和粘附功能的損傷。高糖誘導(dǎo)的BM-EPCs中p53和TSP-1的表達(dá)明顯升高，，而二氮嗪處理后可以下調(diào)高糖誘導(dǎo)的BM-EPCs中p53和TSP-1的水平升高。說明高糖誘導(dǎo)引起血管生成抑制因子TSP-1的增加，進(jìn)而導(dǎo)致BM-EPCs的功能受損，二氮嗪處理后可以顯著改善因高糖誘導(dǎo)引起的BM-EPCs功能受損。 5.二氮嗪改善高糖誘導(dǎo)的HUVECs功能障礙 使用高糖(33mM)誘導(dǎo)的HUVECs損傷模型，二氮嗪(30μM)處理24h后，檢測(cè)其細(xì)胞功能的變化。結(jié)果顯示：高糖誘導(dǎo)后會(huì)明顯降低HUVECs的小管形成能力、遷移功能和粘附功能，二氮嗪(30μM)處理后HUVECs的小管形成能力、遷移和粘附功能顯著增強(qiáng)。 結(jié)論： ATP敏感的鉀離子通道開放劑二氮嗪能夠顯著加速鏈脲佐菌素誘導(dǎo)的1型糖尿病小鼠傷口愈合速度，促進(jìn)傷口組織血管新生；這一效應(yīng)可能與二氮嗪改善糖尿病BM-EPCs功能相關(guān)，機(jī)制涉及p53/TSP-1通路。
[Abstract]:The purpose of the study is:
Diabetes mellitus (DM) is a systemic metabolic disorder that is divided into insulin dependent (Insulin-dependent) and non insulin dependent (Insulin-independent). Major vascular and microvascular lesions are the main cause of death in diabetic patients, and endothelial dysfunction (Endothelial dysfunction) is caused by the injury of endothelial function (Endothelial dysfunction). The initiation factor of wound healing defects and vascular lesions. The activity of Endothelial cells (ECs) is the basis for maintaining endothelial function. If the apoptosis rate of endothelial cells exceeds the normal level, it will affect the integrity of blood vessels. Endothelial progenitor cells (Endothelial progenitor cells, EPCs) are derived from adult bone marrow and can be differentiated into internal cells. The number of Bone marrow-derived endothelial progenitor cells (BM-EPCs) derived from bone marrow can be directed to vascular regeneration sites and involved in the repair of damaged endothelium. The number of EPCs in the cycle and the incidence of vascular disease in the circulation have been studied. The rate is inversely proportional. Therefore, EPCs has a vital link with endothelial function. -1 (Thrombospondin-1, TSP-1) is an effective endogenous angiogenesis inhibitor. Diabetes causes an increase in the expression of TSP-1mRNA in EPCs, and EPCs dysfunction also leads to an increase in the expression of TSP-1. The tumor suppressor gene p53 can be stimulated. The promoter of TSP-1, encoded by the THBS1 gene, indicates that p53 can promote the production of TSP-1 and damage the angiogenesis of fibroblasts. Two azazazine (Diazoxide) is a mitochondrial KATP channel opening agent, which can induce the hyperpolarization of the cell membrane, the increase of K+ Exodus, the decrease of the flow in Ca2+, and the secretion of insulin to inhibit glucose stimulation. The islet B cell cells provide a rest and play a role in retarding and hindering the development of diabetes and its complications. This topic studies whether two azozine can accelerate wound healing in diabetic mice, improve the function of BM-EPCs, and explore its possible mechanism.
Research content and results:
Animal model: the model of type 1 diabetic mice induced by Streptozotocin (STZ); cell model: cell model induced by glucose 33mM (Human umbilical VeinEndothelial cells, HUVECs) and BM-EPCs high glucose injury with glucose 33mM. At animal level, the endothelium of diabetic mice was studied. The effect of function was discussed from the EPCs point of view around the two targets of TSP-1 and p53. At the cell level, the effects of two azazazine on angiogenesis were observed by cell function experiments (migration function, adhesion function and tubule formation function), and the mechanism of two azazazine to influence angiogenesis was determined by measuring the expression of TSP-1 and p53.
1. two azazazine promotes wound healing in type 1 diabetic mice
5 weeks old C57BL/6 mice, 18-20g, were randomly divided into normal group (Control), type 1 diabetes model group (STZ) and two azinazine treatment group (30mg/kg/d gavage 4 weeks). Under the anesthetic state of the mice, a circular wound with a radius of 6mm was cut on the back of the mice, and the wound healing was observed every 2 days for 12 days. 6mm on day 3,6,9 took the surrounding 6mm respectively. The blood vessel density was detected by CD31 immunohistochemistry. The vasodilatation function of the aorta of 2-3mm mice was measured by vascular tension system. The results showed that the wound healing speed of type 1 diabetic mice decreased significantly, the capillary density decreased significantly, and the vascular dependent endothelial diastolic function decreased significantly; two After nitrozine treatment, wound healing ability was enhanced, vascular density increased and vascular dependent endothelial function increased.
2. two azazazine improves BM-EPCs function in type 1 diabetic mice
The Dil-acLDL and FITC-UEA-1 double positive identification of BM-EPCs. was used to detect the number of EPCs in mice circulated by the percentage of CD34 and Flk-1 positive. The migration function and tubule formation ability were detected by Boyden compartment and tube test. The results showed that the number of EPCs in the cycle of type 1 diabetic mice decreased significantly, and two azazazine could be significant after the treatment of azazazine. The number of EPCs in the circulation was increased; the tubuloforming ability of BM-EPCs in type 1 diabetic mice, the migration function and adhesion function decreased significantly. After two azazine treatment, the tubuloforming ability, migration function and adhesion function of BM-EPCs could be significantly improved.
3. possible mechanism of two azazine improving BM-EPCs function in type 1 diabetic mice
The expression of p53 and TSP-1 in BM-EPCs of type 1 diabetic mice was detected by Western blot. The results showed that the p53 expression of BM-EPCs in type 1 diabetic mice increased significantly, and the level of p53 in BM-EPCs could be significantly reduced after two azazazine treatment. The TSP-1 expression of BM-EPCs in type 1 diabetic mice was significantly increased, and the TSP-1 of two azazazine could significantly reduce TSP-1. Expression.
4. two azazine improves BM-EPCs dysfunction induced by high glucose and its mechanism
7 days after BM-EPCs culture of normal mice bone marrow separation, the cell damage model was induced by high glucose (33mM), and two azazazine (30 mu M) was treated with 24h to detect the cell function and protein expression. The results showed that high sugar induced BM-EPCs tubule formation ability, migration function and adhesion function decreased significantly, and two azazazine (30 micron M) treatment could be significantly improved. High sugar induced BM-EPCs tubule formation, migration and adhesion damage. The expression of p53 and TSP-1 in BM-EPCs induced by high glucose was significantly increased, and two azazazine treatment could reduce the level of p53 and TSP-1 in high glucose induced BM-EPCs. It indicated that high glucose induced the increase of angiogenesis inhibitor TSP-1, and thus lead to BM-. The function of EPCs is impaired. Two azazine treatment can significantly improve BM-EPCs damage induced by high glucose.
5. two azazazine improves HUVECs dysfunction induced by high glucose
Using high glucose (33mM) induced HUVECs damage model, two azazazine (30 mu M) treated 24h, the changes in cell function were detected. The results showed that high glucose induced obviously lower tube formation ability, migration function and adhesion function, two azazazine (30 micron M) treatment, HUVECs tubule formation ability, migration and adhesion function significantly enhanced.
Conclusion:
ATP sensitive potassium channel opener, two azazazine, can significantly accelerate the healing speed of streptozotocin induced wound healing in type 1 diabetic mice and promote angiogenesis in wound tissue. This effect may be related to the improvement of diabetic BM-EPCs function by two azinazine, and the mechanism involves the p53/TSP-1 pathway.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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本文編號(hào)：1802213