本文選題：內(nèi)皮祖細(xì)胞 + 胸腺素β4��； 參考：《浙江大學(xué)》2009年博士論文

【摘要】： 內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs)是一類血管內(nèi)皮細(xì)胞的前體細(xì)胞,它可向血管內(nèi)皮細(xì)胞分化,但尚未完全表達(dá)成熟血管內(nèi)皮細(xì)胞表型。EPCs兼具造血干細(xì)胞及內(nèi)皮細(xì)胞的部分表面標(biāo)志,如CD34、CD133、VEGFR-2等。大量研究表明,EPCs在缺血組織的血管新生、損傷血管的再內(nèi)皮化、內(nèi)皮功能障礙的逆轉(zhuǎn)等方面扮演重要角色,因而在心血管系統(tǒng)的修復(fù)中發(fā)揮重要作用。然而,多種冠心病危險(xiǎn)因素諸如老齡、吸煙、高血壓、高膽固醇血癥、糖尿病等會(huì)使循環(huán)EPCs的功能受損,從而限制了基于EPCs的細(xì)胞治療手段在臨床中的應(yīng)用。因此,探求有效途徑增強(qiáng)EPCs功能至關(guān)重要。 胸腺素(thymosins)是一類多肽分子,最初從小牛胸腺中提取得到,根據(jù)其等電點(diǎn)的不同而分為三個(gè)亞家族(α,β和γ亞家族)。胸腺素β4(thymosinβ4,Tβ4)是胸腺素β家族中的一員,由43個(gè)氨基酸殘基組成,廣泛分布于人體內(nèi)多種組織與細(xì)胞中。Tβ4介導(dǎo)了多種生物學(xué)反應(yīng),如血管新生、創(chuàng)傷愈合等。此前研究表明,Tβ4能增強(qiáng)多種干/祖細(xì)胞的增殖、遷移、分化等功能,進(jìn)而促進(jìn)血管新生及心肌修復(fù)。但是,目前尚未明確Tβ4是否可通過(guò)增強(qiáng)循環(huán)EPCs的功能而發(fā)揮心血管保護(hù)作用。 基于上述理論基礎(chǔ),我們提出假設(shè):Tβ4能增強(qiáng)EPCs的功能,促進(jìn)其修復(fù)血管內(nèi)皮,維持內(nèi)皮的完整性,從而發(fā)揮心血管保護(hù)作用。由于目前對(duì)Tβ4影響EPCs的功能及其機(jī)制方面知之甚少,我們首先觀察了Tβ4對(duì)EPCs增殖、遷移、粘附等功能的影響,并探討了可能參與其中的信號(hào)轉(zhuǎn)導(dǎo)途徑,然后觀察了Tβ4對(duì)EPCs凋亡及衰老的作用。以下分三部分對(duì)本研究的方法、結(jié)果及結(jié)論作一簡(jiǎn)述。 1胸腺素β4對(duì)外周血內(nèi)皮祖細(xì)胞功能的影響 目的:觀察Tβ4體外干預(yù)對(duì)外周血EPCs功能的影響 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,將其接種于人纖維連接蛋白包被的培養(yǎng)板,培養(yǎng)7d后,收集貼壁細(xì)胞,予FITC-UEA-I及DiI-acLDL染色,激光共聚焦顯微鏡下觀察,雙染色陽(yáng)性細(xì)胞鑒定為正在分化的EPCs,流式細(xì)胞儀檢測(cè)其表面標(biāo)志(VEGFR-2,CD34,CD133),進(jìn)一步鑒定EPCs。加入不同濃度Tβ4(1ng/mL,10ng/mL,100ng/mL和1000ng/mL)干預(yù),并進(jìn)行細(xì)胞功能學(xué)實(shí)驗(yàn)。分別采用MTT比色法、集落形成實(shí)驗(yàn)、Transwell遷移實(shí)驗(yàn)和粘附能力測(cè)定實(shí)驗(yàn)觀察EPCs的增殖能力、集落形成能力、遷移能力和粘附能力。 結(jié)果:Tβ4能明顯增加EPCs的增殖、集落形成、遷移和粘附能力,并呈一定的量效關(guān)系,在1000ng/mL組達(dá)到最大效應(yīng)(與對(duì)照組比較,增殖能力0.409±0.020 vs0.257±0.015,P0.05;集落形成能力16.7±2.6 vs 6.0±1.0,P0.05;遷移能力79.6±5.6 vs 31.5±3.4,P0.05;粘附能力45.3±4.7 vs 18.8±3.0,P0.05)。 結(jié)論:Tβ4可增強(qiáng)EPCs的功能,且Tβ4對(duì)EPCs功能的影響呈一定的量效關(guān)系。 2胸腺素β4影響外周血內(nèi)皮祖細(xì)胞功能的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制探討 目的:觀察Tβ4影響外周血內(nèi)皮祖細(xì)胞功能的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制 方法:采用密度梯度離心法從外周血獲取單個(gè)核細(xì)胞,將其接種在人纖維連接蛋白包被的培養(yǎng)板,培養(yǎng)7d后,收集貼壁細(xì)胞,予FITC-UEA-I及DiI-acLDL染色,激光共聚焦顯微鏡下觀察,雙染色陽(yáng)性細(xì)胞鑒定為正在分化的EPCs,流式細(xì)胞儀檢測(cè)其表面標(biāo)志(VEGFR-2,CD34,CD133),進(jìn)一步鑒定EPCs。加入Tβ4干預(yù),western blot檢測(cè)EPCs Akt Ser~(473)、eNOS Ser~(1177)和ERK1/2的磷酸化水平。隨后,在分別加入磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)抑制劑、內(nèi)皮型-氧化氮合酶(endothelial nitric oxide synthase,eNOS)抑制劑、細(xì)胞外信號(hào)調(diào)節(jié)激酶(extracellular signal-regulated protein kinase,ERK)1/2抑制劑預(yù)處理EPCs30min后,再予1000ng/mLTβ4干預(yù),采用MTT比色法、Transwell遷移實(shí)驗(yàn)和粘附能力測(cè)定實(shí)驗(yàn)分別觀察EPCs的增殖能力、遷移能力和粘附能力。 結(jié)果:Westernblot檢測(cè)顯示Tβ4能促進(jìn)Akt ser~(473)、eNOS Ser~(1177)和ERK1/2的磷酸化,且均呈一定的時(shí)效和量效關(guān)系。PI3K抑制劑(LY294002,Wortmannin)及eNOS抑制劑(L-NAME)均顯著抑制了Tβ4促進(jìn)EPCs增殖、遷移和粘附的作用,而ERK1/2抑制劑(PD98059)則均無(wú)明顯影響。 結(jié)論:PI3K/Akt/eNOS而非ERK1/2信號(hào)轉(zhuǎn)導(dǎo)途徑參與對(duì)Tβ4促進(jìn)EPCs增殖、遷移、粘附的調(diào)控。 3胸腺素β4對(duì)外周血內(nèi)皮祖細(xì)胞凋亡和衰老的影響 目的:觀察Tβ4對(duì)外周血內(nèi)皮祖細(xì)胞凋亡和衰老的影響 方法:采用密度梯度離心法從外周血獲取單個(gè)核細(xì)胞,將其接種在人纖維連接蛋白包被的培養(yǎng)板,培養(yǎng)7d后,收集貼壁細(xì)胞,予FITC-UEA-I及DiI-acLDL染色,激光共聚焦顯微鏡下觀察,雙染色陽(yáng)性細(xì)胞鑒定為正在分化的EPCs,流式細(xì)胞儀檢測(cè)其表面標(biāo)志(VEGFR-2,CD34,CD133),進(jìn)一步鑒定EPCs。貼壁細(xì)胞培養(yǎng)7d后,采用去血清培養(yǎng)以誘導(dǎo)EPCs凋亡。在去血清培養(yǎng)EPCs 24h后加入不同濃度Tβ4(1ng/mL,10ng/mL,100ng/mL和1000ng/mL)干預(yù),繼續(xù)培養(yǎng)24h,Alexa Fluor~((?))488-Annexin V/PI流式細(xì)胞儀檢測(cè)細(xì)胞凋亡。貼壁細(xì)胞培養(yǎng)4d后,加入不同濃度Tβ4(1ng/mL,10ng/mL,100ng/mL,1000ng/mL)干預(yù),繼續(xù)培養(yǎng)7d后,采用SA-β-半乳糖苷酶染色試劑盒檢測(cè)細(xì)胞衰老。 結(jié)果:Tβ4能明顯抑制EPCs的凋亡和衰老,且均呈一定的量效關(guān)系,在1000ng/mL組達(dá)到最大效應(yīng)(與對(duì)照組比較,細(xì)胞凋亡率6.67±0.72% vs 16.58±1.56%,P0.05;SA-β-半乳糖苷酶陽(yáng)性細(xì)胞數(shù)量19.3±2.6% vs 42.7±4.3%,P0.05)。 結(jié)論:Tβ4能明顯抑制EPCs的凋亡和衰老,且Tβ4對(duì)EPCs凋亡和衰老的影響均呈一定的量效關(guān)系。
[Abstract]:Endothelial progenitor cells (EPCs) is a kind of precursor cells of a kind of vascular endothelial cells. It can differentiate into vascular endothelial cells, but it has not completely expressed the partial surface markers of the mature vascular endothelial cell phenotype.EPCs with both hematopoietic stem cells and endothelial cells, such as CD34, CD133, VEGFR-2 and so on. A large number of studies have shown that EPCs is in ischemia. It plays an important role in vascular regeneration, endothelialization of blood vessels, the reversal of endothelial dysfunction, and thus plays an important role in the repair of the cardiovascular system. However, multiple coronary risk factors such as aging, smoking, hypertension, hypercholesterolemia, diabetes, etc. can damage the function of circulatory EPCs, thus limiting the function of circulation. The application of cell therapy based on EPCs in clinical practice, therefore, it is very important to explore effective ways to enhance EPCs function.
Thymosin (thymosins) is a class of polypeptide molecules, originally extracted from the calf thymus and divided into three subfamilies (alpha, beta and gamma subfamilies) according to their isoelectric points. Thymosin beta 4 (thymosin beta 4, T beta 4) is a member of the thymosin beta family, consisting of 43 amino acid residues and widely distributed in a variety of tissues and cells in the human body and.T beta 4. A variety of biological reactions, such as angiogenesis and wound healing, have been introduced. Previous studies have shown that T beta 4 can enhance the proliferation, migration, differentiation and other functions of multiple stem / progenitor cells to promote angiogenesis and myocardial repair. However, it is not clear whether T beta 4 can play a role in cardiovascular protection by enhancing the function of cyclic EPCs.
Based on the above theoretical basis, we propose that T beta 4 can enhance the function of EPCs, promote the repair of vascular endothelium, maintain endothelium integrity, and play the role of cardiovascular protection. As we know little about the function and mechanism of T beta 4 affecting EPCs, we first observed the effects of T beta 4 on EPCs proliferation, migration, adhesion and other functions, The signal transduction pathway may be involved, and the effect of T beta 4 on EPCs apoptosis and senescence is observed. The following three parts give a brief account of the methods, results and conclusions of this study.
Effect of 1 thymosin beta 4 on peripheral blood endothelial progenitor cells
Objective: To observe the effect of T beta 4 intervention on peripheral blood EPCs function in vitro.
Methods: density gradient centrifugation was used to obtain mononuclear cells from human peripheral blood and inoculated into the culture plate of human fibronectin package. After culture 7d, the adherent cells were collected and stained with FITC-UEA-I and DiI-acLDL. The two staining positive fine cells were identified as the differentiated EPCs, and the flow cytometry was used to detect the cells. The surface markers (VEGFR-2, CD34, CD133), further identified EPCs. intervention with different concentrations of T beta 4 (1ng/mL, 10ng/mL, 100ng/mL and 1000ng/mL), and carried out cell functional experiments. The MTT colorimetric assay, colony formation experiment, Transwell Migration Experiment and adhesion ability test were used to observe the proliferation, colony forming ability and migration ability of EPCs. And adhesion ability.
Results: T beta 4 could significantly increase the proliferation of EPCs, colony formation, migration and adhesion, and the maximum effect in 1000ng/mL group. Compared with the control group, the proliferation ability was 0.409 + 0.020 vs0.257 + 0.015, P0.05; the colony forming ability was 16.7 + 2.6 vs 6 + 1, P0.05; migration ability was 79.6 + 5.6 vs 31.5 + 3.4, P0.05; adhesion energy The force was 45.3 + 4.7 vs 18.8 + 3, P0.05).
Conclusion: T beta 4 can enhance the function of EPCs, and T beta 4 has a dose-dependent relationship with EPCs function.
Effect of 2 thymosin beta 4 on signal transduction mechanism of endothelial progenitor cells in peripheral blood
Objective: To observe the signal transduction mechanism of T beta 4 on the function of endothelial progenitor cells in peripheral blood.
Methods: density gradient centrifugation was used to obtain mononuclear cells from peripheral blood and inoculated into the culture plate of human fibronectin package. After culture 7d, the adherent cells were collected and stained with FITC-UEA-I and DiI-acLDL. The two staining positive cells were identified as the differentiated EPCs, and the flow cytometry was used to detect the cells. VEGFR-2 (CD34, CD133), further identification of T beta 4 intervention by EPCs., Western blot to detect EPCs Akt Ser~ (473), eNOS Ser~ (1177) and phosphorylation level. OS) inhibitor, extracellular signal regulated kinase (extracellular signal-regulated protein kinase, ERK) 1/2 inhibitor pretreated EPCs30min, and then 1000ng/mLT beta 4 intervention. MTT colorimetric assay, Transwell Migration Experiment and adhesion assay test were used to observe the proliferation, migration and adhesion of EPCs.
Results: Westernblot test showed that T beta 4 could promote the phosphorylation of Akt ser~ (473), eNOS Ser~ (1177) and ERK1/2, and all of them showed a certain aging and dose effect relationship. Both.PI3K inhibitor (LY294002, Wortmannin) and eNOS inhibitor (L-NAME) all significantly inhibited the effect of beta 4 on promoting proliferation, migration and adhesion. Influence.
Conclusion: PI3K/Akt/eNOS, rather than ERK1/2 signal transduction pathway, is involved in the regulation of T beta 4 on EPCs proliferation, migration and adhesion.
Effects of 3 thymosin beta 4 on apoptosis and senescence of peripheral blood endothelial progenitor cells
Objective: To observe the effect of T beta 4 on apoptosis and aging of endothelial progenitor cells in peripheral blood.
Methods: density gradient centrifugation was used to obtain mononuclear cells from peripheral blood and inoculated into the culture plate of human fibronectin package. After culture 7d, the adherent cells were collected and stained with FITC-UEA-I and DiI-acLDL. The two staining positive cells were identified as the differentiated EPCs, and the flow cytometry was used to detect the cells. VEGFR-2, CD34, CD133), after further identification of EPCs. adherent cell culture, 7d was cultured to induce the apoptosis of EPCs. After the serum culture of EPCs 24h, EPCs 24h was cultured with different concentrations of T beta 4 (1ng/mL, 10ng/mL, 100ng/mL, etc.) to detect cell apoptosis. After the cells were cultured for 4D, the cell senescence was detected by SA- beta galactosidase staining kit after adding different concentrations of T beta 4 (1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL) and continuing to cultivate 7d.
Results: T beta 4 could significantly inhibit the apoptosis and senescence of EPCs, and all showed a certain dose effect relationship. The maximum effect was achieved in the 1000ng/mL group (compared with the control group, the apoptosis rate was 6.67 + 0.72% vs 16.58 + 1.56%, P0.05; the number of SA- beta galactosidase positive cells was 19.3 + 2.6% vs 42.7 + 4.3%, P0.05).
Conclusion: T beta 4 can significantly inhibit the apoptosis and senescence of EPCs, and the effect of T beta 4 on EPCs apoptosis and senescence is dose effect relationship.
【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】：2009
【分類號(hào)】：R329

【共引文獻(xiàn)】

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相關(guān)碩士學(xué)位論文 前10條

1 鄭莉萍;大鼠外周血內(nèi)皮祖細(xì)胞培養(yǎng)及鑒定的實(shí)驗(yàn)研究[D];鄭州大學(xué);2010年

2 那娜;強(qiáng)化控制血糖、血壓、血脂對(duì)2型糖尿病患者動(dòng)脈內(nèi)中膜厚度、血清AGEs及其可溶性受體水平的影響[D];第二軍醫(yī)大學(xué);2011年

3 李秀芩;鈣調(diào)神經(jīng)磷酸酶/活化T細(xì)胞核因子信號(hào)通路在大鼠主動(dòng)脈平滑肌細(xì)胞鈣化中的作用[D];河北醫(yī)科大學(xué);2011年

4 郭顯蓉;rHDL抑制CACs衰老的機(jī)制研究[D];北京協(xié)和醫(yī)學(xué)院;2011年

5 曹賽;胸腺肽β4（Tβ4）的生物合成及其相關(guān)研究[D];湖南農(nóng)業(yè)大學(xué);2011年

6 程鵬;Thymosinβ4及β-aescin早期應(yīng)用對(duì)脊髓損傷的保護(hù)作用[D];第四軍醫(yī)大學(xué);2011年

7 康玲;降糖藥物對(duì)冠心病合并2型糖尿病患者外周血EPCs的影響[D];泰山醫(yī)學(xué)院;2011年

8 吳青;黃芪多糖對(duì)2型糖尿病患者外周血內(nèi)皮祖細(xì)胞NO濃度和PI3K/AKT/eNOS信號(hào)通路的影響[D];貴陽(yáng)中醫(yī)學(xué)院;2011年

9 丁茹;血管緊張素轉(zhuǎn)換酶抑制劑對(duì)冠心病患者血管內(nèi)皮功能及循環(huán)內(nèi)皮祖細(xì)胞數(shù)量的影響[D];第二軍醫(yī)大學(xué);2006年

10 宗小娟;阿托伐他汀對(duì)ox-LDL引起的骨髓來(lái)源內(nèi)皮前體細(xì)胞增殖凋亡及功能等變化的影響[D];第四軍醫(yī)大學(xué);2006年

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