本文選題：內(nèi)皮祖細(xì)胞 + 骨髓��； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：糖尿病微血管病變已成為威脅人類健康的重要殺手。糖尿病病人拔牙創(chuàng)難以愈合是拔牙術(shù)后的主要并發(fā)癥。血管化困難是糖尿病難愈性創(chuàng)面發(fā)生的重要原因。大量研究結(jié)果顯示內(nèi)皮祖細(xì)胞(endothelial progenitor cells, EPCs)在血管化中起著重要作用。EPCs是內(nèi)皮細(xì)胞的前體細(xì)胞,不僅能直接分化為血管內(nèi)皮細(xì)胞參與血管發(fā)生和修復(fù),還可通過分泌細(xì)胞因子改善內(nèi)皮功能,促進(jìn)血管新生及創(chuàng)面愈合。堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor, bFGF)在創(chuàng)傷愈合及組織再生過程中起著重要作用,多用于口腔潰瘍等創(chuàng)面的治療。以往實驗研究顯示,bFGF可顯著改善EPCs的體外增殖、遷移等功能。而bFGF是否對糖尿病損害的內(nèi)皮祖細(xì)胞具有同樣的作用,尚未見相關(guān)研究報道。因此本實驗通過對糖尿病大鼠的內(nèi)皮祖細(xì)胞體外分離培養(yǎng),觀察不同濃度的bFGF對糖尿病大鼠內(nèi)皮祖細(xì)胞的作用,為利用bFGF和EPCs治療糖尿病難愈創(chuàng)面提供理論及實驗依據(jù)。本實驗分為以下兩個部分：第一部分大鼠骨髓和脾臟來源的內(nèi)皮祖細(xì)胞體外分離、培養(yǎng)鑒定及功能檢測目的：分別提取大鼠骨髓和脾臟來源的EPCs,比較其生物學(xué)特性,為下一步實驗EPCs的組織來源選擇提供指導(dǎo)。從大鼠骨髓和脾臟內(nèi)皮祖細(xì)胞提取,比較其生物學(xué)特性,為下一步的來源EPCs提供指導(dǎo)。方法：采用密度梯度離心法,從健康4周齡SD大鼠,獲取骨髓和脾臟的單個核細(xì)胞,培養(yǎng)7d后,采用細(xì)胞膜紅色熒光標(biāo)記的乙�；兔芏戎鞍�(DiI-ac-LDL)和綠色熒光標(biāo)記的荊豆凝集素(FITC-UEA-1)染色貼壁細(xì)胞鑒定EPCs,對比分析兩種EPCs的遷移、黏附和一氧化氮(nitric oxide, NO)分泌能力。結(jié)果：體外分離培養(yǎng)得到的骨髓和脾臟來源的EPCs細(xì)胞形態(tài)基本相似。絕大部分細(xì)胞吞噬DiI-ac-LDL和FITC-UEA-1雙染為黃色。骨髓來源的EPCs在細(xì)胞增殖、遷移、黏附和NO分泌能力方面均顯著高于脾臟來源的EPCs。結(jié)論：骨髓相對于脾臟,更適宜作為體外分離培養(yǎng)EPCs的組織來源。第二部分堿性成纖維細(xì)胞生長因子對糖尿病大鼠內(nèi)皮祖細(xì)胞增殖、遷移及成血管作用研究目的：觀察不同濃度bFGF對糖尿病大鼠骨髓EPCs體外增殖、遷移及成血管功能的影響,為利用bFGF和EPCs治療糖尿病難愈創(chuàng)面提供理論及實驗依據(jù)。方法：使用高糖飲食加鏈脲佐菌素(Streptozocin,STZ)注射的方法誘導(dǎo)形成Ⅱ型糖尿病大鼠模型。采用密度梯度離心法從糖尿病大鼠骨髓獲得單個核細(xì)胞,培養(yǎng)7d后收集貼壁細(xì)胞,DiI-ac-LDL和FITC-UEA-1雙吞實驗鑒定EPCs。取第1代EPCs,加入不同濃度bFGF (12.5.25.50、100、200ng/mL)培養(yǎng),MTT法和Matrigel基質(zhì)膠,Transwell小室檢測糖尿病內(nèi)皮祖細(xì)胞的增殖,遷移和體外血管生成能力的變化。結(jié)果：Ⅱ型糖尿病大鼠骨髓來源的單個核細(xì)胞絕大部分吞噬DiI-ac-LDL和FITC-UEA-1雙染為黃色,鑒定其為EPCs。糖尿病大鼠骨髓EPCs相對于正常大鼠EPCs,其增殖、遷移及體外成血管功能均明顯下降。12.5、25、50、100、200ng/mLbFGF均能提高糖尿病大鼠EPCs的增殖和遷移能力,且促進(jìn)糖尿病大鼠EPCs增殖的最佳濃度為25ng/mL;促進(jìn)糖尿病大鼠EPCs遷移的最適濃度為50ng/mL。體外Matrigel基質(zhì)膠上EPCs未形成明顯管狀結(jié)構(gòu)。結(jié)論：Ⅱ型糖尿病大鼠EPCs的增殖、遷移及成血管功能較正常大鼠EPCs明顯下降。糖尿病大鼠EPCs增殖和遷移能力在一定濃度的bFGF作用下能夠提升,但對體外血管形成能力無明顯作用。
[Abstract]:Diabetic microvascular lesions have become an important killer of human health. The difficult healing of diabetes patients is the main complication after extraction. The difficulty of vascularization is an important cause of diabetes refractory wound. A large number of research results show that endothelial progenitor cells (EPCs) plays a role in vascularization. The important role of.EPCs is the precursor cells of endothelial cells, which can not only directly differentiate into vascular endothelial cells to participate in angiogenesis and repair, but also improve endothelial function by secreting cytokines, promote angiogenesis and wound healing. Basic fibroblast growth factor (basic fibroblast growth factor, bFGF) reacts in wound healing and tissue re In the process of birth, it plays an important role in the treatment of wounds such as oral ulcers. Previous experimental studies have shown that bFGF can significantly improve the proliferation and migration of EPCs in vitro, and whether bFGF has the same effect on endothelial progenitor cells damaged by diabetes and has not yet been found in the related research. The progenitor cells were isolated and cultured in vitro to observe the effect of different concentrations of bFGF on the inner skin progenitor cells of diabetic rats and provide theoretical and experimental basis for the treatment of diabetic refractory wound surface using bFGF and EPCs. The experiment was divided into two parts: Part 1: isolation, culture identification and function of endothelial progenitor cells from bone marrow and spleen derived from the first part of rats Objective: to extract EPCs from the bone marrow and spleen of rats respectively, to compare their biological characteristics, and to provide guidance for the selection of the tissue source of EPCs in the next step. From the bone marrow and spleen endothelial progenitor cells of rats, the biological characteristics were compared, and the guidance for the next source of EPCs was provided. Method: density gradient centrifugation was used, from health 4. The mononuclear cells of bone marrow and spleen were obtained from SD rats of week age, and after 7d were cultured, EPCs was identified by acetylated low density lipoprotein (DiI-ac-LDL) and green fluorescent tagged agglutinin (FITC-UEA-1) stained adherent cells labeled with green fluorescence. The migration, adhesion and nitric oxide (nitric oxide, NO) secretion of two kinds of EPCs were compared and analyzed. Results: the form of EPCs cells derived from bone marrow and spleen in vitro was basically similar. Most of the cells phagocytic DiI-ac-LDL and FITC-UEA-1 double dye were yellow. The bone marrow derived EPCs was significantly higher in cell proliferation, migration, adhesion and NO secretion than that of the spleen from the EPCs. conclusion: bone marrow is more than the spleen. Suitable as a tissue source for in vitro isolation and culture of EPCs. Second basic fibroblast growth factor (BBF) on the proliferation, migration and angiogenesis of inner skin progenitor cells in diabetic rats: the effects of different concentrations of bFGF on the proliferation, migration and vascular function of bone marrow EPCs in diabetic rats, for the treatment of bFGF and EPCs The theoretical and experimental basis of diabetic wound healing was provided. Methods: the model of type II diabetic rats was induced by high glucose diet plus Streptozocin (STZ) injection. The density gradient centrifugation was used to obtain mononuclear cells from the bone marrow of diabetic rats, and 7d was collected to collect parietal cells, DiI-ac-LDL and FITC-UEA-1 double swallowing. EPCs. was tested for first generation of EPCs, adding different concentrations of bFGF (12.5.25.50100200ng/mL), MTT and Matrigel matrix, Transwell chamber to detect the proliferation, migration and angiogenesis of diabetic endothelial progenitor cells. Results: most of the mononuclear cells derived from bone marrow of type II diabetic rats were phagocytic DiI-ac-LD The double staining of L and FITC-UEA-1 was yellow. It was identified that the bone marrow EPCs of EPCs. diabetic rats was EPCs, its proliferation, migration and in vitro vascular function decreased obviously.12.5,25,50100200ng/mLbFGF can increase the proliferation and migration ability of EPCs in diabetic rats, and the optimum concentration of EPCs in diabetic rats was 25ng/mL. The optimum concentration of EPCs migration in diabetic rats was that EPCs did not form an obvious tubular structure on the 50ng/mL. Matrigel matrix gel. Conclusion: the proliferation, migration and vascular function of EPCs in type II diabetic rats were significantly lower than that of normal rats. The proliferation and migration energy of EPCs in diabetic rats could be enhanced under the action of bFGF in a certain concentration. However, there was no significant effect on the ability of angiogenesis in vitro.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.1

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本文編號：2010702