發(fā)布時(shí)間：2018-08-07 22:04

【摘要】： 1.背景與目的: 血管內(nèi)皮損傷是動脈粥樣硬化、高血壓、糖尿病、介入治療后再狹窄等多種血管性疾病共同的病理生理基礎(chǔ),盡早促進(jìn)受損血管再內(nèi)皮化、恢復(fù)內(nèi)皮功能為抑制血管損傷不良修復(fù)、預(yù)防或防治血管再狹窄及血栓形成的有效策略。因此,揭示內(nèi)皮再生機(jī)理,促進(jìn)內(nèi)皮細(xì)胞有益再生是損傷性血管疾病治療中亟待解決的問題。既往認(rèn)為血管損傷主要依靠損傷部位鄰近的內(nèi)皮細(xì)胞再生修復(fù),但病損情況下鄰近內(nèi)皮的增殖能力有限。近年研究顯示除了血管損傷局部附近內(nèi)皮細(xì)胞再生外,不同來源的血管前體細(xì)胞同樣是參與損傷內(nèi)皮修復(fù)的重要力量。業(yè)已證實(shí),內(nèi)皮前體細(xì)胞即所稱的內(nèi)皮祖細(xì)胞(Endothelial precusor cells, EPCs),能“歸巢”于血管損傷處定向分化為內(nèi)皮細(xì)胞并通過旁分泌機(jī)制促進(jìn)內(nèi)皮修復(fù)和血管新生。然而,目前對于調(diào)控EPCs增殖、遷移等生理功能的機(jī)制或某些關(guān)鍵因子在其中的作用尚不完全清楚。 分化抑制因子(Inhibitor of DNA binding/differentiation, Id)屬于螺旋-環(huán)-螺旋(Helix-loop-helix, HLH)轉(zhuǎn)錄因子家族,Id包括Id1-Id4四個(gè)亞型,各成員均包括高度保守的HLH結(jié)構(gòu)區(qū),但缺乏堿性DNA結(jié)合區(qū)。Id與其他堿性HLH蛋白(如E2A,E12,E47,c-Myc)形成異二聚體,從而抑制了這些堿性HLH蛋白與DNA及其他組織特異性堿性LHL轉(zhuǎn)錄因子結(jié)合,影響特異性蛋白的表達(dá),抑制細(xì)胞分化。目前發(fā)現(xiàn),Id參與了包括表皮、肌肉、神經(jīng)等多種類細(xì)胞增殖分化及及腫瘤形成,且Id1與骨髓EPCs動員等密切相關(guān),但I(xiàn)d是否也對EPCs功能有調(diào)控作用并參與血管損傷修復(fù)尚不清楚。研究結(jié)果發(fā)現(xiàn):①Id與血管系統(tǒng)發(fā)生密切相關(guān),胚胎發(fā)育中Id1-Id4表達(dá)呈復(fù)雜的時(shí)空模式,但I(xiàn)d1、Id3廣泛表達(dá)且重疊貫穿整個(gè)胚胎腦血管系統(tǒng),Id1/Id3雙基因敲除小鼠由于明顯的血管畸形、分叉障礙導(dǎo)致胚胎E13.5期死亡;②Id參與腫瘤血管新生:Id在內(nèi)皮細(xì)胞過表達(dá)導(dǎo)致血管新生,缺失導(dǎo)致促血管生成基因如FGFR-1的下調(diào),而野生型骨髓內(nèi)皮前體細(xì)胞高表達(dá)Id1、Id3,移植后摻合到腫瘤血管床參與血管新生,且Id1、Id3陽性表達(dá)與血管密度顯著正相關(guān);③體外細(xì)胞實(shí)驗(yàn)中,Id能促進(jìn)人臍靜脈內(nèi)皮細(xì)胞(HUVEC)的增殖、激活及管樣形成,促血管生長因子VEGF、TGF-β則誘導(dǎo)HUVEC及骨髓源EPCs表達(dá)Id1和Id3,提示Id1、Id3可能是VEGF等促血管生長作用的下游關(guān)鍵靶點(diǎn)。研究結(jié)果提示:Id1可能表達(dá)于EPCs,并有調(diào)控其增殖、血管新生的功效,推測Id1為決定EPCs增殖的一類開關(guān)基因,受上游特殊信號刺激Id1的表達(dá)左右著增殖重要基因的轉(zhuǎn)錄,在EPCs介導(dǎo)的血管損傷修復(fù)中發(fā)揮重要作用。 在本課題中,我們將分別從細(xì)胞及在體動物水平探討Id1在EPCs增殖、遷移以及血管損傷修復(fù)中的作用。為研究EPCs生理功能的調(diào)控機(jī)制及EPCs在血管損傷修復(fù)中的作用提供實(shí)驗(yàn)依據(jù),為深入探討促進(jìn)血管損傷后內(nèi)皮有益再生提供新的思路。 2.方法: 2.1重組腺病毒Ad-Id1的構(gòu)建 由大鼠組織中提取RNA,經(jīng)RT-PCR擴(kuò)增得到目的基因Id1片段,經(jīng)pMD19T-Simple和AdEasy細(xì)菌內(nèi)同源重組系統(tǒng)構(gòu)建Id1的病毒過表達(dá)載體,即Ad-Id1,通過測序、PCR和酶切鑒定重組病毒Ad-Id1的構(gòu)建。 2.2 Id1對EPCs增殖、遷移功能的影響 用密度梯度離心法及選擇性培養(yǎng)的方法,體外分離、培養(yǎng)小鼠脾源性EPCs,通過細(xì)胞形態(tài)學(xué)、表面分子標(biāo)志以及Dil-acLDL/FITC-UEA-I雙陽性等方法進(jìn)行鑒定;轉(zhuǎn)染Ad- Id1以及si-RNA-Id1,觀察過表達(dá)或沉默Id1基因?qū)PCs增殖、遷移的作用。 2.3觀察Id1在血管損傷后局部血管壁的表達(dá)及參與血管損傷修復(fù)過程的作用 用體重為20g-30g的雄性昆明小鼠復(fù)制頸動脈損傷模型,通過熒光定量RT-PCR以及Western blot分別觀察Id1在血管損傷修復(fù)過程中mRNA和蛋白表達(dá)水平的變化;將過表達(dá)Id1的EPCs注入頸動脈損傷小鼠尾靜脈,14d后觀察損傷血管局部再內(nèi)皮化及新生內(nèi)膜增殖程度。 3.結(jié)果; 3.1.重組腺病毒Ad-Id1的構(gòu)建 由大鼠組織提取RNA,進(jìn)行RT-PCR獲得含酶切位點(diǎn)的Id1基因全CDS區(qū),將目的基因連接到pMD19T-Simple載體中進(jìn)行擴(kuò)增,經(jīng)酶切、連接、轉(zhuǎn)化等步驟后得到pAdTrack-Id1,與骨架質(zhì)粒pAdEasy-1在BJ5183細(xì)菌內(nèi)進(jìn)行同源重組,通過篩選、293T細(xì)胞包裝后獲得重組病毒Ad-Id1,經(jīng)鑒定、擴(kuò)增獲得高滴度的Ad-Id1病毒顆粒以用于下一步的實(shí)驗(yàn)。Ad-Id1的病毒滴度約為1.2×1010 -2.8×1011 pfu/ml。 3.2 Id1對體外培養(yǎng)脾源性EPCs增殖、遷移功能的影響 3.2.1脾源性EPCs鑒定 分離培養(yǎng)的脾源性EPCs經(jīng)過誘導(dǎo)分化后向內(nèi)皮細(xì)胞表型轉(zhuǎn)變,流式細(xì)胞檢測Scal-1、VEGFR-2在培養(yǎng)細(xì)胞中的陽性率分別為83.5%、57.6%,Dil-acLDL/FITC- UEA-I雙陽性細(xì)胞約占90%,說明所培養(yǎng)脾源性細(xì)胞是EPCs。 3.2.2 Id1在脾源性EPCs的表達(dá)情況 Id1在脾源性EPCs呈低水平表達(dá),而當(dāng)受到血清或VEGF刺激后,Id1的基因及蛋白水平均明顯增高;靜止?fàn)顟B(tài)下Id1定位表達(dá)于EPCs細(xì)胞漿內(nèi)。 3.2.3基因轉(zhuǎn)染脾源性EPCs 轉(zhuǎn)染后細(xì)胞狀態(tài)良好,貼壁生長,無變圓、縮小或脫落等病理跡象,經(jīng)過熒光倒置顯微鏡、RT-PCR以及Western blot觀察發(fā)現(xiàn)Ad- Id1的轉(zhuǎn)染效率在第24小時(shí)約60%,48-72小時(shí)達(dá)高峰,約80%。si-RNA-Id1轉(zhuǎn)染后4天,Id1在EPCs的表達(dá)被顯著抑制,轉(zhuǎn)染率約50%。 3.2.4 Ad- Id1對EPCs增殖的作用 采用MTT法分析發(fā)現(xiàn),Ad- Id1對EPCs增殖顯著影響。無論與未轉(zhuǎn)染對照組還是Ad-GFP對照組比較,Ad- Id1對EPCs的增殖有統(tǒng)計(jì)學(xué)差異(均* P0.05),提示Ad- Id1對EPCs有促進(jìn)增殖的作用。 3.2.5 Ad- Id1對EPCs遷移的作用 外源性Id1過表達(dá)顯著促進(jìn)EPCs遷移。在Ad- Id1作用誘導(dǎo)下,平均每個(gè)視野中遷移EPCs的數(shù)目從7.1±1.8增加到26.1±2.8,增加了近3倍(* P0.01)。加入Id1封閉性抗體Id1-Ab后,Ad- Id1促遷移作用明顯減弱(# P0.05),提示EPCs遷移能力的增強(qiáng)是過表達(dá)Id1導(dǎo)致。 3.2.6利用小片段RNA(si-RNA)干擾沉默Ad- Id1對EPCs增殖、遷移的影響 結(jié)果表明si-RNA- Id1介導(dǎo)的Id1基因的沉默明顯抑制了EPCs的增殖、遷移功能,與未干預(yù)組及陰性對照組比較均有顯著差異(* p0.05)。 3.3 Id1在血管損傷修復(fù)中的表達(dá)及作用 3.3.1小鼠頸動脈損傷模型的建立 經(jīng)損傷血管組織切片HE染色證實(shí),本研究成功復(fù)制了小鼠頸動脈損傷模型,鏡下觀察到血管損傷后7天局部內(nèi)膜有增生,14天時(shí)新生內(nèi)膜增生明顯,到28天新生的內(nèi)膜幾乎堵塞整個(gè)血管腔。內(nèi)膜/中膜比值(IA/MA)14d組為1.30±0.15,28d組為4.10±0.20較損傷7d組的0.28±0.02顯著增加(分別為P0.01,P=0.000)。 3.3.2 Id1在血管損傷局部血管壁的表達(dá) 免疫組化實(shí)驗(yàn)顯示Id1在損傷血管的新生內(nèi)膜、中膜以及外膜組織均有表達(dá)。Id1mRNA在正常血管組織低表達(dá),血管損傷后表達(dá)迅速上升,114d即達(dá)到高峰,之后逐漸下降維持到血管損傷后第28天仍較對照組高。Western blot檢測到Id1蛋白表達(dá)在血管損傷后第7d開始上調(diào),14d時(shí)達(dá)到高峰,然后逐漸回落,在損傷后第28天仍有表達(dá)。 3.3.3移植過表達(dá)Id1的EPCs對損傷血管再內(nèi)皮化的影響 損傷14d時(shí)Ad-Id1-EPCs轉(zhuǎn)染組再內(nèi)皮化率為68.36±4.51%,而Ad-GFP-EPCs轉(zhuǎn)染組及未轉(zhuǎn)染組再內(nèi)皮化率分別為43.1±6.59%、40.5±7.82%,兩者之間無統(tǒng)計(jì)學(xué)差異,但與Ad-Id1-EPCs轉(zhuǎn)染組比較均存在明顯差異(P0.05),說明轉(zhuǎn)染Id1過表達(dá)的EPCs可促進(jìn)第14d時(shí)損傷血管再內(nèi)皮化。 3.3.4移植過表達(dá)Id1的EPCs對損傷血管局部新生內(nèi)膜增殖的影響 血管損傷后第14d,Ad-Id1-EPCs轉(zhuǎn)染組小鼠損傷頸動脈內(nèi)、中膜比值為1.08±0.15,結(jié)果與Ad-GFP-EPCs組(1.16±0.14)及未轉(zhuǎn)染組(1.15±0.17)比較三組之間比較無明顯差異(P0.05),而三組中膜面積均無明顯差異(P0.05),提示轉(zhuǎn)移植表達(dá)Id1的EPCs未減輕血管損傷后新生內(nèi)膜的增殖程度。 4.結(jié)論: 4.1 Id1在靜止?fàn)顟B(tài)的脾源性EPCs呈低表達(dá),定位于細(xì)胞漿內(nèi); 4.2 Id1影響EPCs增殖、遷移功能:過表達(dá)Id1可促進(jìn)EPCs的增殖、遷移,干擾Id1則抑制EPCs的增殖、遷移; 4.3 Id1在損傷血管局部呈動態(tài)表達(dá),14d為其表達(dá)高峰期; 4.4移植過表達(dá)Id1的EPCs到頸動脈損傷動物模型,可促進(jìn)14d后損傷血管再內(nèi)皮化,但未明顯抑制局部新生內(nèi)膜的增殖。
[Abstract]:1. background and purpose:
Vascular endothelial injury is the common pathophysiological basis of various vascular diseases such as atherosclerosis, hypertension, diabetes, and restenosis after interventional therapy. It is an effective strategy to promote endothelialization of damaged vessels as soon as possible, restore endothelial function as an effective strategy to prevent or prevent vascular restenosis and thrombosis. The mechanism of endothelial regeneration and promoting the beneficial regeneration of endothelial cells are an urgent problem in the treatment of damaged vascular diseases. It was believed that vascular injury mainly relied on the regeneration of endothelial cells adjacent to the injured part of the endothelial cells, but the proliferation ability of adjacent endothelial cells was limited under the condition of lesion. In addition, vascular precursor cells from different sources are also important forces involved in the repair of damaged endothelium. It has been proved that endothelial progenitor cells (Endothelial precusor cells, EPCs) can be "homing" in vascular injury to differentiate into endothelial cells and promote endothelial repair and vascular neovascularization through paracrine mechanism. However, the mechanism of regulating the proliferation and migration of EPCs or the role of some key factors in them are not fully understood.
The differentiation inhibitor (Inhibitor of DNA binding/differentiation, Id) belongs to the helix loop helix (Helix-loop-helix, HLH) transcription factor family, and Id includes four Id1-Id4 subtypes. Each member includes a highly conserved HLH structural region. It inhibits the combination of these alkaline HLH proteins with DNA and other tissue specific basic LHL transcription factors, which affects the expression of specific proteins and inhibits cell differentiation. At present, Id has been found to be involved in the proliferation and differentiation of many kinds of cells, including epidermis, muscle and nerve, and the formation of tumor, and Id1 is closely related to the mobilization of bone marrow EPCs, but is Id also the same It is not clear to regulate the function of EPCs and to repair the repair of vascular injury. The results are as follows: (1) Id is closely related to the vascular system, and the expression of Id1-Id4 in the embryonic development is complex spatio-temporal pattern, but Id1, Id3 is widely expressed and overlaps throughout the whole embryonic cerebral vascular system, and the Id1/Id3 double gene knockout mice are due to obvious vascular malformation. The bifurcation obstacle leads to the death of the E13.5 phase of the embryo; (2) Id participates in neovascularization of tumor: the overexpression of Id in the endothelial cells leads to angiogenesis, and the deletion leads to the downregulation of the angiogenesis gene, such as FGFR-1, while the wild type bone marrow endothelial progenitor cells express Id1, Id3, and participate in the angiogenesis after transplantation, and the positive expression of Id1, Id3 Id can promote the proliferation, activation and tube formation of human umbilical vein endothelial cells (HUVEC) in vitro, and promote vascular growth factor VEGF, TGF- beta induce HUVEC and bone marrow EPCs to express Id1 and Id3, suggesting Id1, Id3 may be the downstream key target of VEGF equal vascular growth. The results suggest: Id1 It may be expressed in EPCs, and it regulates its proliferation and angiogenesis. It is presumed that Id1 is a class of switch genes that determine the proliferation of EPCs, which is stimulated by the special signal of the upstream signal to stimulate the transcription of the important gene of the proliferation of Id1, and plays an important role in the repair of vascular injury mediated by EPCs.
In this subject, we will explore the role of Id1 in the proliferation, migration and vascular damage repair of EPCs from the level of cell and body animal, and provide experimental basis for the study of the regulatory mechanism of the physiological function of EPCs and the role of EPCs in the repair of vascular injury, and provide a new way of thinking for the further exploration of the beneficial regeneration of endothelium after vascular injury.
The 2. method:
Construction of 2.1 recombinant adenovirus Ad-Id1
RNA was extracted from rat tissues and Id1 fragments of the target gene were amplified by RT-PCR. The viral overexpression vector of Id1, Ad-Id1, was constructed by the homologous recombination system of pMD19T-Simple and AdEasy bacteria. The recombinant virus Ad-Id1 was constructed by sequencing, PCR and enzyme digestion.
The effect of 2.2 Id1 on the proliferation and migration of EPCs
With the method of density gradient centrifugation and selective culture, the spleen derived EPCs of mice was isolated and identified by cell morphology, surface molecular markers and Dil-acLDL/FITC-UEA-I double positive methods, and Ad- Id1 and si-RNA-Id1 were transfected to observe the effect of Id1 gene on EPCs proliferation and migration.
2.3 to observe the expression of Id1 in local vessel wall after vascular injury and its role in vascular injury repair process.
The carotid artery injury model was replicated in male Kunming mice with weight of 20g-30g, and the changes of mRNA and protein expression in the process of vascular injury repair were observed by fluorescence quantitative RT-PCR and Western blot. The EPCs in the carotid artery was injected into the tail vein of the carotid artery injury in mice, and the local re endothelialization of the injured blood vessels was observed and the new endothelialization of the injured vessels was observed after 14d. The degree of proliferation of endocardium.
3. results;
Construction of recombinant adenovirus Ad-Id1 of 3.1.
RNA was extracted from the rat tissue, and the whole CDS region of the Id1 gene containing the enzyme cut site was obtained by RT-PCR. The target gene was connected to the pMD19T-Simple vector for amplification. After the enzyme digestion, connection, transformation and other steps, pAdTrack-Id1 was obtained, and the skeleton plasmid pAdEasy-1 was reorganized in the BJ5183 bacteria. Through screening, the 293T cell was packaged and reorganized. The virus Ad-Id1 was identified and amplified to obtain high titer of Ad-Id1 virus particles for further experiments. The virus titer of Ad-Id1 was about 1.2 *1010-2.8 *1011 pfu/ml.
Effect of 3.2 Id1 on the proliferation and migration of splenic derived EPCs in vitro
Identification of 3.2.1 spleen derived EPCs
The splenic EPCs was transformed into the endothelial cell phenotype after differentiation, and the flow cytometry was used to detect Scal-1. The positive rate of VEGFR-2 in the cultured cells was 83.5%, 57.6%, and Dil-acLDL/FITC- UEA-I double positive cells accounted for 90%, indicating that the cultured spleen derived cells were EPCs..
Expression of 3.2.2 Id1 in spleen derived EPCs
Id1 was expressed at a low level in the spleen derived EPCs, but when stimulated by serum or VEGF, the gene and protein levels of Id1 were significantly increased, and Id1 was expressed in the EPCs cytoplasm.
Transfection of 3.2.3 gene to spleen derived EPCs
After transfection, the cells were in good condition, adherent growth, no circle, reduction or shedding. After fluorescence inversion microscope, RT-PCR and Western blot observation found that the transfection efficiency of Ad- Id1 was about 60%, 48-72 hours reached the peak, and the expression of Id1 in EPCs was significantly inhibited and the transfection rate was about 50%. about 50%. after 80%.si-RNA-Id1 transfection.
The effect of 3.2.4 Ad- Id1 on the proliferation of EPCs
The results of MTT analysis showed that Ad- Id1 had a significant effect on the proliferation of EPCs. The proliferation of Ad- Id1 to EPCs was statistically significant (all * P0.05), no matter compared with the untransfected control group or the Ad-GFP control group, suggesting that Ad- Id1 could promote the proliferation of EPCs.
The effect of 3.2.5 Ad- Id1 on EPCs migration
The overexpression of exogenous Id1 significantly promoted the migration of EPCs. The number of EPCs migrated from 7.1 + 1.8 to 26.1 + 2.8, increased by nearly 3 times (* P0.01) in each field of visual field under the induction of Ad- Id1. The migration of Ad- Id1 decreased significantly (P0.05) after adding the Id1 blocking antibody Id1-Ab, suggesting that the enhancement of the EPCs migration ability was caused by overexpression.
3.2.6 interferes with the proliferation and migration of EPCs by silencing Ad- Id1 with small fragment RNA (si-RNA).
The results showed that the silence of the Id1 gene mediated by si-RNA- Id1 significantly inhibited the proliferation of EPCs, and the migration function was significantly different from that of the untreated group and the negative control group (* P0.05).
Expression and role of 3.3 Id1 in repair of vascular injury
Establishment of carotid artery injury model in 3.3.1 mice
The damaged vascular tissue section HE staining proved that the mouse carotid artery injury model was successfully replicated in this study. Under the microscope, the local intima hyperplasia was observed 7 days after vascular injury, and the neointimal hyperplasia was obvious at 14 days, and the neointima of the new endometrium almost blocked the whole vascular cavity in 28 days. The ratio of intima / middle membrane (IA/MA) 14d group was 4.10 + 0.20 in the 1.30 + 0.15,28d group. Compared with the injury group 7d, 0.28 + 0.02 increased significantly (P0.01, P=0.000 respectively).
Expression of 3.3.2 Id1 in local vascular wall of vascular injury
The immunohistochemical experiment showed that Id1 expressed.Id1mRNA in the neointima, middle membrane and outer membrane tissues of the injured blood vessels, and expressed the low expression in the normal vascular tissue. After the vascular injury, the expression of 114d rose rapidly, and the peak was reached. After the gradual descent to the vascular injury, the expression of Id1 protein in the blood was still higher than that of the control group at the twenty-eighth day. The expression of Id1 protein in the blood was still detected in the blood. The 7d began to increase after injury, reached its peak at 14d, then gradually decreased, and remained on the twenty-eighth day after injury.
Effect of 3.3.3 transplanted Id1 over EPCs on reendothelialization of injured vessels
The re endothelialization rate of Ad-Id1-EPCs transfected group was 68.36 + 4.51%, while the re endothelialization rate of Ad-GFP-EPCs transfected group and untransfected group was 43.1 + 6.59% and 40.5 + 7.82%, respectively, but there was no significant difference between the two groups, but there were significant differences (P0.05) with the Ad-Id1-EPCs transfected group (P0.05), indicating that the transfected Id1 over expressed EPCs could promote the injury of blood at 14d. Endothelialization of the tube.
Effect of 3.3.4 transplanted Id1 over EPCs on local neointimal proliferation of injured vessels
After vascular injury, 14d, Ad-Id1-EPCs transfected mice were injured in the carotid artery, the ratio of middle membrane was 1.08 + 0.15. There was no significant difference between the three groups (P0.05) compared with that of the Ad-GFP-EPCs group (1.16 + 0.14) and the untransfected group (1.15 + 0.17), but there was no significant difference in the middle membrane area of the three groups (P0.05), suggesting that EPCs with the transfer of Id1 did not reduce the vascular loss. The degree of proliferation of the neointima after injury.
4. conclusion:
4.1 the spleen derived EPCs in the resting state of Id1 was low expressed and localized in cytoplasm.
4.2 Id1 affects EPCs proliferation and migration function: over expression of Id1 can promote EPCs proliferation and migration, interfere with Id1, inhibit EPCs proliferation and migration;
4.3 Id1 showed dynamic expression in injured vessels, and 14d was the peak of expression.
4.4 Transplantation of Id1-overexpressing EPCs into carotid artery injury animal models could promote the re-endothelialization of injured vessels 14 days later, but did not significantly inhibit the proliferation of local neointima.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363

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1 任s，

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