本文選題：內(nèi)皮祖細(xì)胞 + 分離�。� 參考：《蘇州大學(xué)》2016年博士論文

【摘要】：下肢深靜脈血栓形成(Deep Venous Thrombosis,DVT)是常見的外周血管疾病,可引起血栓后綜合征(Post-thrombotic syndrome,PTS)和致死性肺動脈栓塞(Pulmonary embolism,PE)。目前深靜脈血栓治療方法包括藥物抗凝溶栓、手術(shù)取栓和導(dǎo)管溶栓,但都未能消除血栓后綜合征發(fā)生、遠(yuǎn)期通暢率不高、易于復(fù)發(fā)等缺點(diǎn),因此需要一種更加安全有效的方法治療深靜脈血栓。在缺血性疾病中,干細(xì)胞研究取得了積極進(jìn)展,其中內(nèi)皮祖細(xì)胞(Endothelial progenitor cells,EPCs)近年來研究較多,內(nèi)皮祖細(xì)胞是來源于骨髓中的血管內(nèi)皮前體細(xì)胞,在新生血管形成過程中起著重要作用。我們前期研究結(jié)果發(fā)現(xiàn),在大鼠靜脈血栓模型中,移植的大鼠EPCs可歸巢到靜脈血栓中,改善血栓微環(huán)境,促進(jìn)急性血栓的溶解和慢性血栓的機(jī)化再通。但EPCs應(yīng)用面臨很多問題,移植的EPCs僅少量可分化為血管內(nèi)皮細(xì)胞,如何改善EPCs功能,成為缺血性疾病重要的研究方向。MicroRNAs(miRNAs,miR)是一類長度約22nt的非編碼RNA,miRNAs通過完全匹配或者不完全匹配的方式識別靶基因3’-UTR區(qū)域,抑制蛋白翻譯或者影響mRNA穩(wěn)定性,在轉(zhuǎn)錄后水平調(diào)控蛋白表達(dá),發(fā)揮重要的生物學(xué)功能。近年研究證實(shí)miRNAs參與EPCs功能調(diào)節(jié),在血管新生和血管生成中扮演重要角色。但miRNAs在深靜脈血栓患者和正�；颊咄庵苎狤PCs中表達(dá)有無差異,這些差異性的miRNAs能否調(diào)控EPCs功能和影響靜脈血栓溶解再通,帶著這些問題,設(shè)計了本課題。我們采集DVT患者和健康人外周血樣本,通過密度梯度離心法分離外周血單個核細(xì)胞,體外誘導(dǎo)培養(yǎng)EPCs,利用基因芯片篩選DVT患者和健康人EPCs中miRNAs的表達(dá)譜差異,用實(shí)時熒光定量PCR(qRT-PCR)驗(yàn)證芯片結(jié)果,對和芯片結(jié)果一致的miRNAs,通過文獻(xiàn)檢索和生物信息學(xué)分析挑選認(rèn)為值得深入研究的miRNAs做細(xì)胞功能實(shí)驗(yàn)(我們挑選了6個miRNAs),觀察這些miRNAs對EPCs的遷移、成血管和凋亡等細(xì)胞功能是否有影響,細(xì)胞功能結(jié)果顯示miR-483-3p對EPCs功能有影響,我們對miR-483-3p做更深入研究,通過生物信息學(xué)預(yù)測miR-483-3p可能的靶基因,通過熒光素酶報告基因?qū)嶒?yàn)、上調(diào)和下調(diào)miR-483-3p、共轉(zhuǎn)染和基因沉默后用Western blot檢測蛋白變化進(jìn)一步確認(rèn)靶基因,以及共轉(zhuǎn)染和基因沉默后對EPCs功能的影響。體內(nèi)實(shí)驗(yàn)通過建立大鼠血栓模型,共聚焦熒光顯微鏡、HE染色和DSA觀察mi R-483-3p調(diào)控EPCs后對EPCs歸巢和血栓溶解再通的影響。結(jié)果發(fā)現(xiàn):miR-483-3p在DVT患者EPCs中高表達(dá);下調(diào)EPCs中mi R-483-3p表達(dá)會促進(jìn)EPCs遷移、成血管能力,抑制EPCs凋亡,促進(jìn)EPCs歸巢和EPCs對血栓的溶解再通。我們的研究為干細(xì)胞治療靜脈血栓探索新的思路。本實(shí)驗(yàn)研究,將分為5部分,主要研究方法及結(jié)果如下。第一部分人外周血內(nèi)皮祖細(xì)胞和大鼠骨髓源性內(nèi)皮祖細(xì)胞的培養(yǎng)和鑒定目的:建立人外周血內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs)和大鼠骨髓源性內(nèi)皮祖細(xì)胞的分離、培養(yǎng)及鑒定方法,為后續(xù)體外和體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。方法:采用密度梯度離心法分離人外周血和大鼠骨髓單個核細(xì)胞,將單個核細(xì)胞懸于含20%胎牛血清(FBS)EGM-2培養(yǎng)基中,培養(yǎng)2~3周,顯微鏡下觀察細(xì)胞形態(tài)特征,流式細(xì)胞儀檢測細(xì)胞表面標(biāo)記物CD34、CD133、VEGFR-2表達(dá)量,雙熒光染色檢測細(xì)胞攝取DiI-ac-LDL和FITC-UEA-1能力。結(jié)果:剛分離的人外周血單個核細(xì)胞(peripheral blood mononuclear cells,PBMC)和骨髓單個核細(xì)胞(Bone marrow-derived mononuclear cells,BMMNC)圓形體積小,2天后可見少數(shù)細(xì)胞貼壁,3天后細(xì)胞體積逐漸變大,貼壁細(xì)胞增多,5天后部分細(xì)胞呈紡錘形生長,出現(xiàn)細(xì)胞集落,四周細(xì)胞呈放射樣排列,第10至14天,細(xì)胞呈現(xiàn)鋪路石或鵝卵石樣改變。流式細(xì)胞儀顯示細(xì)胞表面主要表達(dá)內(nèi)皮標(biāo)記物VEGFR-2,CD34、CD133表達(dá)低。雙熒光染色顯示細(xì)胞能夠吞噬DiI-ac-LDL和FITC-UEA-1。結(jié)論:在EGM-2培養(yǎng)基誘導(dǎo)下,成功的從人外周血單個核細(xì)胞和大鼠骨髓單個核細(xì)胞中培養(yǎng)出EPCs,2~3周培養(yǎng)后呈現(xiàn)晚期EPCs特征。第二部分下肢深靜脈血栓患者EPCs中mi RNAs差異表達(dá)譜篩選及驗(yàn)證目的:利用基因芯片篩選DVT患者和健康人外周血EPCs中mi RNAs的表達(dá)譜差異,并用q RT-PCR驗(yàn)證芯片結(jié)果的可靠性。方法:采集DVT患者和健康人外周血樣本,利用密度梯度離心法分離外周血單個核細(xì)胞進(jìn)行EPCs培養(yǎng),通過mi RNA基因芯片篩選DVT患者和健康人EPCs的mi RNAs表達(dá)譜差異,并用q RT-PCR驗(yàn)證芯片結(jié)果。結(jié)果:Mi RNA基因芯片顯示mi R-483-3p等多個mi RNAs在DVT患者和健康人外周血EPCs中表達(dá)存在差異,q RT-PCR結(jié)果和芯片一致。結(jié)論:Mi R-483-3p等多個mi RNAs在DVT患者和健康人外周血EPCs中表達(dá)有差異。第三部分Mi R-483-3p對內(nèi)皮祖細(xì)胞功能的影響及靶基因預(yù)測和驗(yàn)證目的:探討mi R-483-3p對人外周血EPCs遷移、成血管能力和凋亡的影響,預(yù)測其靶基因并驗(yàn)證。方法:用Lipofectamine 3000將mi R-483-3p模擬物(agomir)、抑制物(antagomir)和陰性對照物轉(zhuǎn)染到EPCs中,用transwell實(shí)驗(yàn)檢測mi R-483-3p對EPCs遷移的影響,用matrigel管腔形成實(shí)驗(yàn)檢測mi R-483-3p對EPCs成血管能力的影響,用流式細(xì)胞儀檢測mi R-483-3p對EPCs凋亡的影響。利用生物信息學(xué)預(yù)測mi R-483-3p可能的靶基因,利用熒光素酶報告基因?qū)嶒?yàn)、q RT-PCR和western blots等實(shí)驗(yàn)確認(rèn)靶基因。結(jié)果:上調(diào)EPCs中mi R-483-3p表達(dá)會抑制EPCs遷移和成血管能力,促進(jìn)EPCs凋亡,下調(diào)EPCs中mi R-483-3p表達(dá)結(jié)果和上調(diào)相反,生物信息學(xué)預(yù)測血清反應(yīng)因子(serum response factor,SRF)可能是靶基因,q RT-PCR和western blots等實(shí)驗(yàn)驗(yàn)證SRF就是mi R-483-3p的靶基因。結(jié)論:上調(diào)EPCs中mi R-483-3p表達(dá)會抑制EPCs遷移和成血管能力,促進(jìn)EPCs凋亡;SRF是mi R-483-3p靶基因。第四部分Mi R-483-3p慢病毒載體的構(gòu)建和表達(dá)目的:構(gòu)建mi R-483-3p/mi R-483-3p sponge慢病毒表達(dá)載體,感染大鼠EPCs,并驗(yàn)證EPCs中mi R-483-3p表達(dá)情況,為后續(xù)體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。方法:將mi R-483-3p前體序列和慢病毒載體經(jīng)酶切連接產(chǎn)生p GLV3-H1-GFP-mi R-483-3p和p GLV3-H1-GFP-mi R-483-3p sponge,與輔助包裝載體一起轉(zhuǎn)染293T細(xì)胞,收集病毒上清感染EPCs,用熒光顯微鏡觀察轉(zhuǎn)染效率,用q RT-PCR檢測感染后EPCs內(nèi)mi R-483-3p表達(dá)情況。結(jié)果:慢病毒載體p GLV3-H1-GFP-mi R-483-3p/p GLV3-H1-GFP-mi R-483-3p sponge構(gòu)建成功,轉(zhuǎn)染EPCs后能有效上調(diào)和下調(diào)EPCs內(nèi)的mi R-483-3p表達(dá)。結(jié)論:成功構(gòu)建了攜帶目的基因mi R-483-3p的慢病毒載體p GLV3-H1-GFP-mi R-483-3p/p GLV3-H1-GFP-mi R-483-3p sponge。第五部分Mi RNA-483-3p調(diào)控內(nèi)皮祖細(xì)胞對靜脈血栓溶解再通的影響目的:研究mi R-483-3p調(diào)控大鼠EPCs后對EPCs歸巢和EPCs對靜脈血栓溶解再通的影響。方法:將慢病毒載體p GLV3-H1-GFP vector、p GLV3-H1-GFP-mi R-483-3p和p GLV3-H1-GFP-mi R-483-3p sponge轉(zhuǎn)染至EPCs中,采用結(jié)扎左腎靜脈下方的下腔靜脈構(gòu)建大鼠深靜脈血栓模型,再將轉(zhuǎn)染的EPCs通過大鼠尾靜脈移植到血栓模型中。分四組:A組(10只),空白對照組,經(jīng)尾靜脈注入1 ml PBS;B組(10只),EPCs/p GLV3-H1-GFP vector(EPCs/vector組),經(jīng)尾靜脈注入1 ml含有1.0×106EPCs/vector的PBS細(xì)胞懸液;C組(10只),EPCs/p GLV3-H1-GFP-mi R-483-3p(EPCs/mi R-483-3p組),經(jīng)尾靜脈注入1 ml含有1.0×106 EPCs/mi R-483-3p的PBS細(xì)胞懸液;D組(10只),EPCs/p GLV3-H1-GFP-mi R-483-3p sponge(EPCs/mi R-483-3p sponge組),經(jīng)尾靜脈注入1 ml含有1.0×106EPCs/mi R-483-3p sponge的PBS細(xì)胞懸液。術(shù)后7天收集標(biāo)本,經(jīng)熒光顯微鏡觀察EPCs在血栓中的歸巢,經(jīng)HE染色、數(shù)字減影血管造影(digital subtract angiography,DSA)觀察靜脈血栓溶解再通情況。結(jié)果:經(jīng)GFP熒光標(biāo)記的EPCs出現(xiàn)在靜脈血栓中,不同實(shí)驗(yàn)組的陽性細(xì)胞數(shù)比較:EPCs/mi R-483-3p sponge組EPCs/vector組EPCs/mi R-483-3p組,提示mi R-483-3p抑制EPCs歸巢至靜脈血栓中。各實(shí)驗(yàn)組血栓重量比較:blank control組EPCs/mi R-483-3p組EPCs/vector組EPCs/mi R-483-3p sponge組,提示mi R-483-3p抑制EPCs的溶栓能力。HE染色觀察各實(shí)驗(yàn)組血栓溶解再通情況比較:EPCs/mi R-483-3p sponge組EPCs/vector組EPCs/mi R-483-3p組blank control組;DSA觀察各實(shí)驗(yàn)組血栓溶解再通情況比較:EPCs/mi R-483-3p sponge組EPCs/vector組EPCs/mi R-483-3p組blank control組;提示過表達(dá)mi R-483-3p會抑制EPCs對靜脈血栓的溶解再通,下調(diào)mi R-483-3p能促進(jìn)EPCs的溶栓能力。結(jié)論:移植轉(zhuǎn)染后的EPCs能夠歸巢至靜脈血栓中,上調(diào)mi R-483-3p抑制了EPCs歸巢能力和EPCs對靜脈血栓的溶解再通,下調(diào)mi R-483-3p能促進(jìn)EPCs歸巢和溶栓能力。
[Abstract]:Deep Venous Thrombosis (DVT) is a common peripheral vascular disease, which can cause Post-thrombotic syndrome (PTS) and fatal pulmonary embolism (Pulmonary embolism, PE). At present, the methods of deep venous thrombosis include anticoagulant thrombolysis, surgical thrombolysis and catheter thrombolysis, but they have not been eliminated. After thrombus syndrome, the long-term patency rate is not high and it is easy to relapse. Therefore, it needs a more safe and effective method for the treatment of deep vein thrombosis. In the ischemic disease, the stem cell research has made positive progress, in which the Endothelial progenitor cells (EPCs) has been studied more in recent years, the endothelial progenitor cell is the source. The vascular endothelial progenitor cells in the bone marrow play an important role in the formation of neovascularization. In our previous study, we found that in the rat venous thrombosis model, the transplanted rat EPCs could return to the venous thrombosis, improve the microenvironment of thrombus, promote the dissolution of acute thrombus and the pathogenesis of chronic thrombus. But the application of EPCs is facing Many problems, only a small number of transplanted EPCs can differentiate into vascular endothelial cells, how to improve the function of EPCs, and become an important research direction of ischemic disease.MicroRNAs (miRNAs, miR) is a class of non coded RNA with a length of about 22nt, miRNAs can identify the target gene 3 '-UTR region by fully matched or incomplete matching, and inhibit the translation of protein or protein translation. Influence the stability of mRNA and regulate protein expression at post transcriptional level and play an important biological function. In recent years, studies have shown that miRNAs plays an important role in the regulation of EPCs function and plays an important role in angiogenesis and angiogenesis. However, there is no difference in the expression of miRNAs in the peripheral blood of patients with deep venous thrombosis and normal patients, and these differences of miRNAs can be found. The EPCs function and the effect of venous thrombosis and recanalization were not regulated. With these problems, we designed this topic. We collected peripheral blood samples from DVT patients and healthy people, isolated peripheral blood mononuclear cells by density gradient centrifugation, induced EPCs in vitro, and screened the difference of miRNAs expression profiles in DVT patients and healthy people EPCs by gene chip. Using real time fluorescence quantitative PCR (qRT-PCR) to verify the results of the chip, the miRNAs, which is consistent with the results of the chip, is selected by literature search and bioinformatics analysis to select the miRNAs to do cell function experiments (we select 6 miRNAs), observe the migration of these miRNAs to EPCs, the function of cell formation and apoptosis and so on. The results of cell function show that miR-483-3p has an impact on the function of EPCs. We do a more thorough study of miR-483-3p, predict the possible target gene of miR-483-3p through bioinformatics, up and down miR-483-3p through the luciferase reporter gene experiment, CO transfection and gene silencing, and further confirm the target gene with the change of Western blot detection protein. And the effect of CO transfection and gene silencing on the function of EPCs. In vivo, the effect of rat thrombus model, confocal fluorescence microscopy, HE staining and DSA observation on the effect of MI R-483-3p on EPCs homing and thrombolytic recanalization after EPCs were observed in vivo. The results showed that miR-483-3p was highly expressed in EPCs of DVT suffering from EPCs; Promoting EPCs migration, vascular capacity, inhibiting EPCs apoptosis, promoting EPCs homing and dissolution of thrombus by EPCs. Our study will explore new ideas for the treatment of venous thrombosis by stem cells. This experimental study will be divided into 5 parts. The main research methods and results are as follows. Cell culture and identification Objective: to establish the isolation, culture and identification of human peripheral blood endothelial progenitor cells (endothelial progenitor cells (EPCs) and rat bone marrow derived endothelial progenitor cells. Methods: the separation of human peripheral blood and rat bone marrow mononuclear cells by density gradient centrifugation method will be established by the method of density gradient centrifugation. The nuclear cells were suspended in the EGM-2 medium containing 20% fetal bovine serum (FBS), cultured for 2~3 weeks and observed the morphological characteristics of the cells under microscope. Flow cytometry was used to detect the cell surface markers CD34, CD133, VEGFR-2 expression, and double fluorescence staining was used to detect the uptake of DiI-ac-LDL and FITC-UEA-1 in cells. Results: isolated human peripheral blood mononuclear cells (peripheral) Blood mononuclear cells, PBMC) and bone marrow mononuclear cells (Bone marrow-derived mononuclear cells, BMMNC) have small round volume. After 2 days, a few cells are adhered to the wall. After 3 days, the cell volume becomes larger and the adherent cells increase. After 5 days, the cells are spindle shaped, cell colonies appear, and the surrounding cells are arranged radiated from tenth to 14 days. The cells showed the paving stone or cobblestone like changes. Flow cytometry showed that the cell surface mainly expressed the endothelial markers VEGFR-2, CD34 and CD133. The double fluorescence staining showed that the cells were able to swallow DiI-ac-LDL and FITC-UEA-1. conclusion: under the inducement of EGM-2 medium, the cells were successfully derived from human peripheral blood mononuclear cells and rat bone marrow mononuclear cells. EPCs and 2~3 weeks were cultured for advanced EPCs. The screening and verification of MI RNAs differential expression profiles in EPCs in second patients with deep vein thrombosis in the lower extremities were screened and verified by using gene chip to screen the difference of MI RNAs expression in the peripheral blood EPCs of DVT patients and healthy people, and the reliability of the result of Q RT-PCR test chip. Using the density gradient centrifugation method, the peripheral blood mononuclear cells were isolated from the peripheral blood samples from the healthy people for EPCs culture. The MI RNA gene chip was used to screen the MI RNAs expression profiles of the DVT patients and the healthy people EPCs, and the Q RT-PCR was used to verify the results of the chip. The expression of EPCs in peripheral blood was different, and the results of Q RT-PCR were consistent with the chip. Conclusion: the expression of multiple mi RNAs, such as Mi R-483-3p, is different in the peripheral blood EPCs of DVT patients and healthy people. Third part Mi R-483-3p on the function of endothelial progenitor cells and target gene prediction and verification purpose: To explore the migration of human peripheral blood and angiogenesis. The effect of ability and apoptosis was predicted and the target gene was predicted. Methods: Mi R-483-3p mimics (agomir), inhibitor (antagomir) and negative control were transfected into EPCs with Lipofectamine 3000. The effect of MI R-483-3p on EPCs migration was detected by Transwell test, and Matrigel lumen formation was used to detect the capacity of MI. The effect of MI R-483-3p on EPCs apoptosis was detected by flow cytometry. Using bioinformatics to predict the possible target genes of MI R-483-3p, the target genes were confirmed by luciferase reporter gene experiment, Q RT-PCR and Western blots. The result: up regulation mi R-483-3p expression in EPCs inhibits migration and angiogenesis, and promotes apoptosis. The result and up regulation of MI R-483-3p expression in EPCs were down, and bioinformatics predicted that the serum reaction factor (serum response factor, SRF) might be the target gene, and Q RT-PCR and Western blots showed that SRF was the target gene. SRF is the target gene of MI R-483-3p. The construction and expression of the fourth part of the Mi R-483-3p lentivirus vector: construct mi R-483-3p/mi R-483-3p sponge Lentivirus Expression Vector, infect rat EPCs, and verify the MI expression in EPCs. P GLV3-H1-GFP-mi R-483-3p and P GLV3-H1-GFP-mi R-483-3p sponge were connected by the enzyme, and 293T cells were transfected with the auxiliary package carrier, and the infection EPCs in the virus supernatant was collected. The transfection efficiency was observed by the fluorescence microscope. The expression of the infection was detected by Q RT-PCR. 3-H1-GFP-mi R-483-3p sponge was successfully constructed. After transfection of EPCs, the expression of MI R-483-3p in EPCs was up and down effectively. Conclusion: a lentiviral vector carrying the target gene mi R-483-3p P GLV3-H1-GFP-mi R-483-3p/p, which regulates the thrombolysis of venous thrombosis, is regulated by the fifth part. Objective: To study the effect of MI R-483-3p on EPCs homing and EPCs on the dissolving and recanalization of venous thrombosis after EPCs in rats. Methods: transfection of the lentivirus carrier P GLV3-H1-GFP vector, P GLV3-H1-GFP-mi R-483-3p and P GLV3-H1-GFP-mi into the inferior vena cava under the left renal vein to construct the rat Deep vein thrombosis model, and then transfected EPCs through rat tail vein to thrombus model, divided into four groups: A group (10), blank control group, 1 ml PBS via tail vein, B group (10 rats), EPCs/p GLV3-H1-GFP vector (EPCs/vector group), 1 ml containing 1 x 106EPCs/vector PBS cell suspension through tail vein injection; 10 group (10) GFP-mi R-483-3p (group EPCs/mi R-483-3p) was injected into the tail vein for 1 ml PBS cell suspension containing 1 x 106 EPCs/mi R-483-3p; D group (10), EPCs/p GLV3-H1-GFP-mi R-483-3p, and injected into the tail vein for 1 * 7 cells suspension. The specimens were collected on 7 days after the operation. A microscope was used to observe the homing of EPCs in thrombus, HE staining, and digital subtract angiography (DSA) to observe the dissolving and recanalization of venous thrombosis. Results: the GFP labeled EPCs appeared in the venous thrombosis, and the number of positive cells in the different experimental groups was compared with the EPCs/mi R-483-3p sponge group EPCs/vector group Group 3P, suggesting that MI R-483-3p inhibited EPCs homing to venous thrombosis. Compared with the experimental group, the thrombus weight of the experimental group was compared with the EPCs/vector group EPCs/mi R-483-3p sponge group in group blank control EPCs/mi R-483-3p, and the thrombolytic ability of the experimental groups was compared. Tor group EPCs/mi R-483-3p group blank control group; DSA observation of thrombolytic recanalization in each experimental group: EPCs/mi R-483-3p sponge group EPCs/vector group EPCs/mi R-483-3p group. The transfected EPCs can be returned to the venous thrombosis. Up regulation of MI R-483-3p inhibits the EPCs homing ability and the dissolution and repassage of EPCs to venous thrombosis, and the downregulation of MI R-483-3p can promote the homing and thrombolytic ability of EPCs.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R543.6
，

本文編號：1915938