發(fā)布時(shí)間：2018-01-17 22:02

  本文關(guān)鍵詞：血管緊張素II對(duì)大鼠骨髓源內(nèi)皮祖細(xì)胞生物學(xué)特性的影響　出處：《第四軍醫(yī)大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 內(nèi)皮祖細(xì)胞 血管緊張素 纈沙坦 增殖 凋亡 一氧化氮 粘附 血管內(nèi)皮生長(zhǎng)因子受體

【摘要】： 實(shí)驗(yàn)背景及目的 內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs),又稱成血管細(xì)胞,存在于骨髓(bone marrow,BM)、臍帶血(umbilical cord blood,UCB)及外周血(peripheral blood,PB)中,可以分化為成熟的內(nèi)皮細(xì)胞(endothelial cells,ECs),是血管ECs的前體細(xì)胞。研究表明,EPCs不僅參與胚胎時(shí)期血管生成,而且在缺血、血管損傷等刺激下,可動(dòng)員并遷移至病變局部參與出生后的血管發(fā)生及受損內(nèi)皮的修復(fù),在缺血組織的血運(yùn)重建方面發(fā)揮著重要作用,給缺血性疾病的治療帶來(lái)了新的希望。但對(duì)EPCs的生物學(xué)特性及其影響因素尚缺乏全面了解,而這是對(duì)于更好的應(yīng)用EPCs為人類造福所必需的。 最新的研究發(fā)現(xiàn),EPCs的數(shù)量、功能等生物學(xué)特性與心血管疾病的危險(xiǎn)因素如高脂血癥、糖尿病、C-反應(yīng)蛋白(C-reactive protein,CRP)等成負(fù)相關(guān)。血管緊張素II(AngII)是一種與多種心血管疾病的病理生理過(guò)程密切相關(guān)的致病因素,它的水平是否也會(huì)影響EPCs的生物學(xué)特性尚不清楚。另一方面,近些年不斷有研究報(bào)道AngII可以促進(jìn)新生血管的形成,提示它可能與EPCs存在一定的相互關(guān)系。 AngII可以促進(jìn)ECs的增殖,抑制凋亡,上調(diào)其粘附分子的表達(dá),增加其內(nèi)皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)mRNA的合成,從而增加一氧化氮(nitric oxide,NO)的分泌。因此,我們假設(shè)AngII對(duì)EPCs也具有類似作用。本實(shí)驗(yàn)在體外培養(yǎng)大鼠BM-EPCs的基礎(chǔ)上,觀察一定濃度的AngII對(duì)其增殖、凋亡、NO分泌及粘附能力等生物學(xué)特性的影響,并進(jìn)一步探討其可能機(jī)制,以期為臨床更好地利用EPCs治療缺血性疾病提供思路。 實(shí)驗(yàn)方法 第一部分:大鼠BM-EPCs的體外分離、誘導(dǎo)分化及鑒定。無(wú)菌分離大鼠股骨及脛骨,沖洗骨髓腔,收集細(xì)胞懸液,利用Ficoll密度梯度離心法分離BM單個(gè)核細(xì)胞(mononuclear cells,MNCs),接種在含有特定濃度血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)、堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor,bFGF)的培養(yǎng)基中進(jìn)行誘導(dǎo)分化。對(duì)原代細(xì)胞的表面標(biāo)志分子CD133和血管內(nèi)皮生長(zhǎng)因子2型受體(vascular endothelial growth factor type 2 receptor,VEGFR-2/Flk-1)進(jìn)行雙染,激光共聚焦鑒定,雙陽(yáng)性為EPCs。 第二部分:AngII對(duì)BM-EPCs增殖能力的影響及機(jī)制探討。取原代培養(yǎng)的EPCs,分成若干組,在其培養(yǎng)基中添加不同濃度的AngII,至培養(yǎng)終點(diǎn),四甲基偶氮唑鹽(methyl thiazolyl tetrazolium,MTT)比色法測(cè)定細(xì)胞增殖活性。利用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription-polymerase chain raction,RT-PCR)對(duì)不同實(shí)驗(yàn)組的Flk-1 mRNA進(jìn)行擴(kuò)增,半定量分析其表達(dá)量的差異。 第三部分:AngII對(duì)BM-EPCs凋亡、NO分泌及粘附能力的影響。取原代培養(yǎng)的EPCs,不同濃度的AngII干預(yù)后,Hoechst染色測(cè)定凋亡率,試劑盒測(cè)定NO分泌量,結(jié)晶紫染色測(cè)定粘附能力。 實(shí)驗(yàn)結(jié)果 1、體外成功培養(yǎng)出大鼠BM-EPCs,并經(jīng)鑒定證實(shí)。 2、與空白對(duì)照組比較,在VEGF存在的前提下,不同濃度的AngII可以使EPCs的增殖活性增強(qiáng)(P0.05),且呈濃度依賴性;AngII可以促進(jìn)EPCs Flk-1 mRNA的表達(dá)。血管緊張素II 1型受體(angiotensin II type 1 receptor,AT1R)拮抗劑纈沙坦、蛋白激酶C(protein kinase C,PKC)抑制劑均可以拮抗這種作用(P0.05)。 3、與空白對(duì)照組比較,不同濃度的AngII可以使無(wú)血清條件下EPCs的凋亡率下降(P0.05);不同濃度的AngII可以使EPCs NO的分泌量增加(P0.05);不同濃度的AngII可以使短期內(nèi)EPCs的貼壁細(xì)胞數(shù)量增加(P0.05)。作用均呈濃度依賴性,纈沙坦可拮抗AngII的上述作用(P0.05)。 結(jié)論 一定濃度的AngII可以改善BM-EPCs的生物學(xué)特性,表現(xiàn)為細(xì)胞增殖活性增強(qiáng)、凋亡率下降、NO分泌量增加、粘附能力增強(qiáng),而AngII的上述作用是通過(guò)AT1R介導(dǎo)的。結(jié)果提示AngII可能是通過(guò)對(duì)EPCs的作用產(chǎn)生促進(jìn)血管生成的效應(yīng)。
[Abstract]:Experimental background and purpose
Endothelial progenitor cells (endothelial progenitor cells, EPCs), also known as vascular cells in bone marrow (bone, marrow, BM), umbilical cord blood (umbilical cord blood, UCB) and peripheral blood (peripheral, blood, PB) can differentiate into mature endothelial cells (endothelial, cells, ECs), is a former somatic cell vascular ECs. The results show that EPCs is not only involved in embryonic angiogenesis, but also in ischemia, vascular damage and other stimuli, can mobilize and migrate to the lesion involved in postnatal neovascularization and repair of damaged endothelium, plays an important role in ischemic tissue revascularization, brings new hope for the treatment of ischemic diseases. But the factors of biological characteristics and its effect on EPCs is still lack of comprehensive understanding, which is better for the application of EPCs for the benefit of mankind is necessary.
The latest study found that the number of EPCs, such as hyperlipidemia and diabetes risk factors, biological function and cardiovascular disease, C- reactive protein (C-reactive protein, CRP) and a negative correlation. Angiotensin II (AngII) is closely related to a variety of pathogenic factors and cardiovascular disease pathophysiology the process, it will also affect the level of the biological characteristics of EPCs is not clear. On the other hand, in recent years it has been reported that AngII can promote the formation of new blood vessels, suggesting that it may have a certain correlation with EPCs.
AngII can inhibit apoptosis, promote the proliferation of ECs by up regulating the expression of adhesion molecules, increase the endothelial nitric oxide synthase (endothelial nitric oxide synthase, eNOS) mRNA synthesis, thereby increasing nitric oxide (nitric oxide, NO) secretion. Therefore, we suppose that AngII had a similar effect on EPCs in this experiment. In vitro cultured rat BM-EPCs based on the observation of a certain concentration of AngII on the proliferation, apoptosis, affect the biological characteristics of NO secretion and adhesion ability, and further explore its possible mechanism, in order to make better use of EPCs for the clinical treatment of ischemic diseases to provide ideas.
Experimental method
The first part: isolation of rat BM-EPCs in vitro, differentiation and identification. The separation of aseptic femoral and tibial bone marrow cavity of rats, flushing, cell suspension was collected, separated BM mononuclear cells by Ficoll density gradient centrifugation (mononuclear, cells, MNCs), growth factor in vascular endothelial specific inoculation concentration (vascular endothelial growth factor, VEGF), basic fibroblast growth factor (basic fibroblast, growth factor, bFGF) of the culture medium to induce the differentiation of primary cells. The surface markers of CD133 and vascular endothelial growth factor receptor 2 (vascular endothelial growth factor type 2 receptor, VEGFR-2/Flk-1) by double staining, laser scanning confocal microscope double positive for EPCs.
The second part: To investigate the effect of AngII on the proliferation of BM-EPCs and its mechanism. The primary cultured EPCs, divided into several groups, in the culture medium with different concentrations of AngII, to train end point, four methyl thiazolyl tetrazolium salt (methyl thiazolyl, tetrazolium, MTT) determination of cell proliferation activity by reverse transcriptase polymerase assay. Chain reaction (reverse transcription-polymerase chain raction, RT-PCR) of the different groups of Flk-1 mRNA were amplified, the expression difference and semi quantitative analysis.
The third part: the effect of AngII on the apoptosis, NO secretion and adhesion of BM-EPCs. After primary culture of EPCs and AngII with different concentrations, the apoptosis rate was determined by Hoechst staining, the NO secretion was determined by kit, and the adhesion ability was determined by crystal violet staining.
experimental result
1, rat BM-EPCs was successfully cultured in vitro and confirmed by identification.
2, compared with the control group, in the premise of the existence of VEGF under different concentrations of AngII can make the EPCs proliferation enhancement (P0.05), in a dose-dependent manner; AngII can promote the expression of EPCs Flk-1 mRNA. Angiotensin II type 1 receptor (angiotensin II type 1 receptor, AT1R) antagonist valsartan protein kinase C (protein, kinase, C, PKC) inhibitors were able to antagonize this effect (P0.05).
3, compared with the blank control group, different concentration of AngII can cause apoptosis under serum free EPCs decreased (P0.05); different concentrations of AngII can make the EPCs secretion of NO increased (P0.05); different concentrations of AngII can make the number of adherent cells in the short term the increase of EPCs (P0.05) are. In a concentration dependent manner, the effect of valsartan can inhibit AngII (P0.05).
conclusion
The biological characteristics of a certain concentration of AngII can improve the performance of BM-EPCs, in order to enhance the activity of cell proliferation, decreased apoptosis rate, NO secretion increased, enhanced adhesion ability, and the effect of AngII is mediated by AT1R. The results suggest that AngII may promote angiogenesis effect is produced by the effect on EPCs.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 杜瑞;FoxO4在血管緊張素Ⅱ誘導(dǎo)的內(nèi)皮祖細(xì)胞凋亡中的機(jī)制研究[D];山西醫(yī)科大學(xué);2010年

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本文編號(hào)：1438134