本文選題：內(nèi)皮祖細(xì)胞 + 胸腺素β4��； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：目前,缺血性心血管疾病的發(fā)病率和致死率逐年上升,嚴(yán)重危害人類(lèi)健康。其發(fā)病制十分復(fù)雜,其中內(nèi)皮細(xì)胞功能障礙是導(dǎo)致缺血性心血管病的重要環(huán)節(jié)。雖然成熟內(nèi)皮細(xì)胞能夠修復(fù)損傷內(nèi)皮,但是其再生能力有限。內(nèi)皮祖細(xì)胞(endothelial progenitor cells, EPCs)是一類(lèi)血管內(nèi)皮細(xì)胞的前體細(xì)胞,具有向內(nèi)皮細(xì)胞分化的潛能。大量研究表明,EPCs在血管修復(fù)和新生中發(fā)揮多種作用。但是,目前EPCs移植在臨床應(yīng)用仍面臨諸多問(wèn)題,多種心血管疾病或心血管疾病危險(xiǎn)因素諸如老齡、高血壓、高膽固醇血癥、糖尿病等會(huì)減少循環(huán)EPCs數(shù)目,并損害EPCs的功能,這很大程度上限制了EPCs在缺血性心血管疾病中的應(yīng)用。因此,改善EPCs功能將是未來(lái)EPCs移植治療的重要策略。胸腺素P4 (thymosin β4, Tβ4),是一種由43個(gè)氨基酸殘基組成的小分子量蛋白,它介導(dǎo)了多種生物學(xué)反應(yīng),如血管新生、創(chuàng)傷愈合、炎癥控制等。我們此前的研究發(fā)現(xiàn)Tβ4能夠增強(qiáng)人外周血EPCs的增殖、遷移,并抑制人外周血EPCs的凋亡和衰老,但是Tβ4對(duì)于循環(huán)EPCs的成血管功能、旁分泌功能以及Tβ4預(yù)處理EPCs后進(jìn)行移植是否能進(jìn)一步提高EPCs移植治療的療效目前尚不明確�；谏鲜隹紤],我們首先觀察了體外實(shí)驗(yàn)中Tβ4對(duì)EPCs成血管功能的影響及相關(guān)機(jī)制；其次我們研究了Tβ4對(duì)EPCs旁分泌的作用；最后我們進(jìn)行了動(dòng)物實(shí)驗(yàn)探究了Tβ4預(yù)處理EPCs后進(jìn)行移植是否能進(jìn)一步提高EPCs移植治療的療效。以下分三部分對(duì)本研究的方法、結(jié)果及結(jié)論分別簡(jiǎn)述。1胸腺素β4對(duì)循環(huán)內(nèi)皮祖細(xì)胞成血管功能的影響目的：觀察Tβ4體外干預(yù)對(duì)外周血內(nèi)皮祖細(xì)胞成血管功能的影響及相關(guān)機(jī)制方法：采用密度梯度離心法從人外周血獲取單個(gè)核細(xì)胞,接種于人纖維連接蛋白(HFN)包被的培養(yǎng)板,培養(yǎng)7d后,收集貼壁細(xì)胞,采用FITC-UEA-I和DiI-acLDL染色,在激光共聚焦顯微鏡下觀察,雙染陽(yáng)性細(xì)胞鑒定為正在分化的EPCs。加入不同濃度的Tβ4 (10ng/mL, 100ng/mL和1000ng/mL)干預(yù),采用Matrigel膠體外成血管能力測(cè)定實(shí)驗(yàn)觀察EPCs的成血管能力。Western blot檢測(cè)EPCs Akt Ser473和eNOS Ser1177的磷酸化水平。隨后,在分別加入Akt siRNA 及 eNOS siRNA干擾后,再予1000ng/mL Tβ4干預(yù),再次采用Matrigel膠體外成血管能力測(cè)定實(shí)驗(yàn)觀察不同實(shí)驗(yàn)組EPCs的成血管能力。結(jié)果：Tβ4能明顯增加EPCs的成血管能力,并呈一定的量效關(guān)系,在1000ng/mL達(dá)到最大效應(yīng)(與對(duì)照組比較,33.33±1.86 vs 18.34±2.02,P0.05)。Western blot檢測(cè)顯示Tβ4能促進(jìn)Akt Ser473及eNOS Ser1177的磷酸化,且呈一定的量效關(guān)系。Akt siRNA及eNOS siRNA均顯著抑制了Tβ4促進(jìn)EPCs成血管的作用。結(jié)論：Tβ4可增強(qiáng)EPCs的體外成血管功能,且呈一定的量效關(guān)系。Akt/eNOS信號(hào)轉(zhuǎn)導(dǎo)途徑參與對(duì)Tβ4促進(jìn)EPCs體外成血管能力的調(diào)控。2胸腺素β4對(duì)循環(huán)內(nèi)皮祖細(xì)胞旁分泌作用的研究目的：觀察Tβ4影響外周血內(nèi)皮祖細(xì)胞旁分泌功能的影響及相關(guān)機(jī)制方法：分離和獲取外周血內(nèi)皮祖細(xì)胞(EPCs)方法同前。加入不同濃度的Tβ4(10ng/mL, 100ng/mL和1000ng/mL)干預(yù),采用熒光定量PCR檢測(cè)EPCs內(nèi)VEGF和IL-8的表達(dá),采用ELISA試劑盒檢測(cè)EPCs條件培養(yǎng)基(EPCs-derived conditioned medium,EPCs-CM)中VEGF和IL-8的表達(dá)。采用Transwell細(xì)胞遷移檢測(cè)和Matrigel膠毛細(xì)血管樣結(jié)構(gòu)形成分別檢測(cè)EPCs-CM、Tβ4-EPCs-CM及Tβ4-EPCs-CM加入中和性抗體(VEGF和IL-8中和性抗體)干預(yù)之后人臍靜脈內(nèi)皮細(xì)胞(Human umbilical vein endothelial cells, HUVECs)體外遷移和成血管能力。在分別加入Akt siRNA及eNOS siRNA干擾后,再予1000ng/mL Tβ4干預(yù),采用ELISA試劑盒檢測(cè)EPCs-CM中VEGF和IL-8的表達(dá)。結(jié)果： Tβ4干預(yù)能明顯增加EPCs內(nèi)和EPCs-CM中VEGF和IL-8的表達(dá),EPCs-CM能夠顯著增加內(nèi)皮細(xì)胞遷移和成血管作用,Tβ4干預(yù)能進(jìn)一步增強(qiáng)EPCs-CM對(duì)內(nèi)皮功能的影響,而VEGF和IL-8的中和性抗體可以分別拮抗T04對(duì)EPCs-CM促內(nèi)皮遷移和成血管的增強(qiáng)效應(yīng)。Akt siRNA可以顯著降低Tβ4-EPCs-CM中VEGF和IL-8的表達(dá)；eNOS siRNA可以顯著降低Tβ4-EPCs-CM中、VEGF而非IL-8的表達(dá)。結(jié)論：Tβ4能夠促進(jìn)EPCs的旁分泌作用,增加1EPCs內(nèi)和EPCs-CM中VEGF和IL-8的表達(dá),且這一作用與Akt/eNOS信號(hào)轉(zhuǎn)導(dǎo)途徑有關(guān)。3胸腺素β4對(duì)循環(huán)內(nèi)皮祖細(xì)胞移植作用的影響目的：觀察Tβ4對(duì)外周血內(nèi)皮祖細(xì)胞移植作用的影響方法：分離和獲取外周血內(nèi)皮祖細(xì)胞(EPCs)方法同前。采用前降支結(jié)扎法建立大鼠心肌梗死模型,按不同分組分別將150μL培養(yǎng)基、1×106個(gè)EPCs及相同數(shù)量的經(jīng)Tβ4預(yù)處理24小時(shí)后的EPCs采用心肌注射的方法移植于心臟缺血區(qū)。心梗4周后采用超聲心動(dòng)圖檢測(cè)大鼠的心臟功能,組織切片免疫熒光檢測(cè)心梗缺血邊緣區(qū)血管密度及移植EPCs駐留和參與血管修復(fù)情況；組織切片免疫組化及組織蛋白電泳檢測(cè)缺血區(qū)VEGF的表達(dá)及組織纖維化的程度。結(jié)果：Tβ4預(yù)處理能明顯提高EPCs在體駐留,并參與血管的修復(fù),較單純EPCs移植進(jìn)一步增加了缺血區(qū)血管密度,減低纖維化程度,最終改善大鼠心梗4周后的心臟功能。結(jié)論：Tβ4能明顯提高EPCs的移植效果,增加缺血區(qū)血管密度,降低纖維化程度,改善心功能。
[Abstract]:At present, the incidence and mortality rate of ischemic cardiovascular disease are increasing year by year, seriously endangering human health. Its pathogenesis is very complex, and endothelial cell dysfunction is an important part of ischemic cardiovascular disease. Although mature endothelial cells can repair damaged endothelium, but its regeneration ability is limited. Endothelial progenitor cells (endothelial Progenitor cells, EPCs, a kind of precursor cells of vascular endothelial cells, has the potential to differentiate into endothelial cells. A large number of studies have shown that EPCs plays a variety of roles in vascular repair and regeneration. However, there are still many problems in the clinical application of EPCs transplantation. Many cardiovascular and cardiovascular diseases such as aging and high risk factors are high. Blood pressure, hypercholesterolemia, diabetes and so on can reduce the number of circulating EPCs and damage the function of EPCs, which largely limits the use of EPCs in ischemic cardiovascular disease. Therefore, the improvement of EPCs function will be an important strategy for the future EPCs transplantation treatment. Thymosin P4 (thymosin beta 4, T beta 4) is a form of 43 amino acid residues. Small molecular weight protein, which mediates a variety of biological reactions, such as angiogenesis, wound healing, and inflammatory control. Our previous study found that T beta 4 could enhance the proliferation, migration, and apoptosis of human peripheral blood EPCs, and inhibit the apoptosis and senescence of human peripheral blood EPCs, but T beta 4 for the vascular function of circulating EPCs, paracrine function, and T beta 4 preconditioning EP It is not clear whether transplanting after Cs can further improve the therapeutic effect of EPCs transplantation. Based on the above considerations, we first observed the effect of T beta 4 on the vascular function of EPCs in vitro and related mechanisms; secondly, we studied the effect of T beta 4 on the paracrine secretion of EPCs; finally, we conducted an animal experiment to explore the preposition of T beta 4. Whether the transplantation of EPCs can further improve the therapeutic effect of EPCs transplantation. The following three parts give a brief account of the effects of.1 thymosin beta 4 on the vascular function of circulating endothelial progenitor cells: the effect of T beta 4 on the vascular function of peripheral blood progenitor cells in vitro and the related mechanism Methods: a density gradient centrifugation method was used to obtain mononuclear cells from human peripheral blood, inoculated with human fibronectin (HFN) coated culture plate, and cultured 7d to collect adherent cells. FITC-UEA-I and DiI-acLDL staining were used to observe with laser confocal microscopy. Double staining positive cells were identified as being differentiated EPCs. added to different concentrations of T. The intervention of beta 4 (10ng/mL, 100ng/mL and 1000ng/mL) was used to observe the vascular ability of Matrigel colloid to observe the vascular ability of EPCs..Western blot was used to detect the phosphorylation level of EPCs Akt Ser473 and eNOS Ser1177. The angiogenic ability of EPCs in different experimental groups was observed. Results: T beta 4 could significantly increase the vascular ability of EPCs, and showed a certain dose effect relationship. The maximum effect was achieved in 1000ng/mL (compared with the control group, 33.33 + 1.86 vs 18.34 + 2.02, P0.05).Western blot detection showed that T beta 4 could promote Akt Ser473 and eNOS eNOS. Phosphorylation and a certain dose effect relationship between.Akt siRNA and eNOS siRNA significantly inhibit the role of T beta 4 in promoting EPCs angiogenesis. Conclusion: T beta 4 can enhance the vascular function of EPCs in vitro, and is involved in a certain amount of.Akt/eNOS signal transduction pathway involved in the regulation of.2 thymosin beta 4 in the circulation of T beta 4. The objective of paracrine paracrine effect: To observe the effect of T beta 4 on the paracrine function of peripheral blood endothelial progenitor cells and the related mechanism methods: separation and acquisition of peripheral blood endothelial progenitor cells (EPCs) method in the same front. Adding different concentrations of T beta 4 (10ng/mL, 100ng/mL and 1000ng/mL) intervention, using fluorescence quantitative PCR to detect VEGF and IL-8 in EPCs The expression of VEGF and IL-8 in the EPCs conditioned medium (EPCs-derived conditioned medium, EPCs-CM) was detected by ELISA kit. Transwell cell migration detection and Matrigel gel capillary like structure were used to detect EPCs-CM. The human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVECs) were migrated and vasculogenic in vitro. After the interference of Akt siRNA and eNOS siRNA respectively, the intervention of 1000ng/mL T beta 4 was given. The expression of IL-8, EPCs-CM can significantly increase endothelial cell migration and angiogenesis, T beta 4 intervention can further enhance the effect of EPCs-CM on endothelial function, while VEGF and IL-8 neutralizing antibodies can antagonize T04 to EPCs-CM promoting endothelial migration and angiogenesis..Akt siRNA can significantly reduce T beta 4-EPCs-CM. ENOS siRNA can significantly reduce the expression of VEGF but not IL-8 in T beta 4-EPCs-CM. Conclusion: T beta 4 can promote the paracrine effect of EPCs, increase the expression of VEGF and IL-8 in 1EPCs and EPCs-CM, and this effect is related to the effect of thymosin beta 4 on the transplantation of endogenous progenitor cells. The effect of beta 4 on the transplantation of peripheral endothelial progenitor cells: isolation and acquisition of peripheral blood endothelial progenitor cells (EPCs) method. The model of rat myocardial infarction was established by anterior descending ligature. The 150 L medium, 1 x 106 EPCs and the same number of EPCs after 24 hours of T beta 4 were injected into the myocardium. Methods the cardiac function of rats was detected by echocardiography after 4 weeks of myocardial infarction. The density of blood vessels in the marginal zone of myocardial infarction, EPCs residing and vascular repair were detected by tissue section immunofluorescence. The expression of VEGF in ischemic area and tissue fibrosis were detected by immunohistochemistry and tissue protein electrophoresis. Results: T beta 4 preconditioning can significantly improve the retention of EPCs in body, and participate in the repair of blood vessels. Compared with simple EPCs transplantation, the density of blood vessels in the ischemic area, the degree of fibrosis and the cardiac function after 4 weeks of myocardial infarction can be improved. Conclusion: T beta 4 can obviously improve the effect of EPCs transplantation, increase the density of blood vessels in ischemic area, and reduce fiber. The degree of maintenance and improvement of heart function.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R543

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