转录因子Id1对内皮祖细胞增殖、迁移的影响及在损伤血管修复中作用的研究
发布时间：2018-08-07 22:04
【摘要】： 1.背景与目的: 血管内皮损伤是动脉粥样硬化、高血压、糖尿病、介入治疗后再狭窄等多种血管性疾病共同的病理生理基础,尽早促进受损血管再内皮化、恢复内皮功能为抑制血管损伤不良修复、预防或防治血管再狭窄及血栓形成的有效策略。因此,揭示内皮再生机理,促进内皮细胞有益再生是损伤性血管疾病治疗中亟待解决的问题。既往认为血管损伤主要依靠损伤部位邻近的内皮细胞再生修复,但病损情况下邻近内皮的增殖能力有限。近年研究显示除了血管损伤局部附近内皮细胞再生外,不同来源的血管前体细胞同样是参与损伤内皮修复的重要力量。业已证实,内皮前体细胞即所称的内皮祖细胞(Endothelial precusor cells, EPCs),能“归巢”于血管损伤处定向分化为内皮细胞并通过旁分泌机制促进内皮修复和血管新生。然而,目前对于调控EPCs增殖、迁移等生理功能的机制或某些关键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)属于螺旋-环-螺旋(Helix-loop-helix, HLH)转录因子家族,Id包括Id1-Id4四个亚型,各成员均包括高度保守的HLH结构区,但缺乏碱性DNA结合区。Id与其他碱性HLH蛋白(如E2A,E12,E47,c-Myc)形成异二聚体,从而抑制了这些碱性HLH蛋白与DNA及其他组织特异性碱性LHL转录因子结合,影响特异性蛋白的表达,抑制细胞分化。目前发现,Id参与了包括表皮、肌肉、神经等多种类细胞增殖分化及及肿瘤形成,且Id1与骨髓EPCs动员等密切相关,但Id是否也对EPCs功能有调控作用并参与血管损伤修复尚不清楚。研究结果发现:①Id与血管系统发生密切相关,胚胎发育中Id1-Id4表达呈复杂的时空模式,但Id1、Id3广泛表达且重叠贯穿整个胚胎脑血管系统,Id1/Id3双基因敲除小鼠由于明显的血管畸形、分叉障碍导致胚胎E13.5期死亡;②Id参与肿瘤血管新生:Id在内皮细胞过表达导致血管新生,缺失导致促血管生成基因如FGFR-1的下调,而野生型骨髓内皮前体细胞高表达Id1、Id3,移植后掺合到肿瘤血管床参与血管新生,且Id1、Id3阳性表达与血管密度显著正相关;③体外细胞实验中,Id能促进人脐静脉内皮细胞(HUVEC)的增殖、激活及管样形成,促血管生长因子VEGF、TGF-β则诱导HUVEC及骨髓源EPCs表达Id1和Id3,提示Id1、Id3可能是VEGF等促血管生长作用的下游关键靶点。研究结果提示:Id1可能表达于EPCs,并有调控其增殖、血管新生的功效,推测Id1为决定EPCs增殖的一类开关基因,受上游特殊信号刺激Id1的表达左右着增殖重要基因的转录,在EPCs介导的血管损伤修复中发挥重要作用。 在本课题中,我们将分别从细胞及在体动物水平探讨Id1在EPCs增殖、迁移以及血管损伤修复中的作用。为研究EPCs生理功能的调控机制及EPCs在血管损伤修复中的作用提供实验依据,为深入探讨促进血管损伤后内皮有益再生提供新的思路。 2.方法: 2.1重组腺病毒Ad-Id1的构建 由大鼠组织中提取RNA,经RT-PCR扩增得到目的基因Id1片段,经pMD19T-Simple和AdEasy细菌内同源重组系统构建Id1的病毒过表达载体,即Ad-Id1,通过测序、PCR和酶切鉴定重组病毒Ad-Id1的构建。 2.2 Id1对EPCs增殖、迁移功能的影响 用密度梯度离心法及选择性培养的方法,体外分离、培养小鼠脾源性EPCs,通过细胞形态学、表面分子标志以及Dil-acLDL/FITC-UEA-I双阳性等方法进行鉴定;转染Ad- Id1以及si-RNA-Id1,观察过表达或沉默Id1基因对EPCs增殖、迁移的作用。 2.3观察Id1在血管损伤后局部血管壁的表达及参与血管损伤修复过程的作用 用体重为20g-30g的雄性昆明小鼠复制颈动脉损伤模型,通过荧光定量RT-PCR以及Western blot分别观察Id1在血管损伤修复过程中mRNA和蛋白表达水平的变化;将过表达Id1的EPCs注入颈动脉损伤小鼠尾静脉,14d后观察损伤血管局部再内皮化及新生内膜增殖程度。 3.结果; 3.1.重组腺病毒Ad-Id1的构建 由大鼠组织提取RNA,进行RT-PCR获得含酶切位点的Id1基因全CDS区,将目的基因连接到pMD19T-Simple载体中进行扩增,经酶切、连接、转化等步骤后得到pAdTrack-Id1,与骨架质粒pAdEasy-1在BJ5183细菌内进行同源重组,通过筛选、293T细胞包装后获得重组病毒Ad-Id1,经鉴定、扩增获得高滴度的Ad-Id1病毒颗粒以用于下一步的实验。Ad-Id1的病毒滴度约为1.2×1010 -2.8×1011 pfu/ml。 3.2 Id1对体外培养脾源性EPCs增殖、迁移功能的影响 3.2.1脾源性EPCs鉴定 分离培养的脾源性EPCs经过诱导分化后向内皮细胞表型转变,流式细胞检测Scal-1、VEGFR-2在培养细胞中的阳性率分别为83.5%、57.6%,Dil-acLDL/FITC- UEA-I双阳性细胞约占90%,说明所培养脾源性细胞是EPCs。 3.2.2 Id1在脾源性EPCs的表达情况 Id1在脾源性EPCs呈低水平表达,而当受到血清或VEGF刺激后,Id1的基因及蛋白水平均明显增高;静止状态下Id1定位表达于EPCs细胞浆内。 3.2.3基因转染脾源性EPCs 转染后细胞状态良好,贴壁生长,无变圆、缩小或脱落等病理迹象,经过荧光倒置显微镜、RT-PCR以及Western blot观察发现Ad- Id1的转染效率在第24小时约60%,48-72小时达高峰,约80%。si-RNA-Id1转染后4天,Id1在EPCs的表达被显著抑制,转染率约50%。 3.2.4 Ad- Id1对EPCs增殖的作用 采用MTT法分析发现,Ad- Id1对EPCs增殖显著影响。无论与未转染对照组还是Ad-GFP对照组比较,Ad- Id1对EPCs的增殖有统计学差异(均* P0.05),提示Ad- Id1对EPCs有促进增殖的作用。 3.2.5 Ad- Id1对EPCs迁移的作用 外源性Id1过表达显著促进EPCs迁移。在Ad- Id1作用诱导下,平均每个视野中迁移EPCs的数目从7.1±1.8增加到26.1±2.8,增加了近3倍(* P0.01)。加入Id1封闭性抗体Id1-Ab后,Ad- Id1促迁移作用明显减弱(# P0.05),提示EPCs迁移能力的增强是过表达Id1导致。 3.2.6利用小片段RNA(si-RNA)干扰沉默Ad- Id1对EPCs增殖、迁移的影响 结果表明si-RNA- Id1介导的Id1基因的沉默明显抑制了EPCs的增殖、迁移功能,与未干预组及阴性对照组比较均有显著差异(* p0.05)。 3.3 Id1在血管损伤修复中的表达及作用 3.3.1小鼠颈动脉损伤模型的建立 经损伤血管组织切片HE染色证实,本研究成功复制了小鼠颈动脉损伤模型,镜下观察到血管损伤后7天局部内膜有增生,14天时新生内膜增生明显,到28天新生的内膜几乎堵塞整个血管腔。内膜/中膜比值(IA/MA)14d组为1.30±0.15,28d组为4.10±0.20较损伤7d组的0.28±0.02显著增加(分别为P0.01,P=0.000)。 3.3.2 Id1在血管损伤局部血管壁的表达 免疫组化实验显示Id1在损伤血管的新生内膜、中膜以及外膜组织均有表达。Id1mRNA在正常血管组织低表达,血管损伤后表达迅速上升,114d即达到高峰,之后逐渐下降维持到血管损伤后第28天仍较对照组高。Western blot检测到Id1蛋白表达在血管损伤后第7d开始上调,14d时达到高峰,然后逐渐回落,在损伤后第28天仍有表达。 3.3.3移植过表达Id1的EPCs对损伤血管再内皮化的影响 损伤14d时Ad-Id1-EPCs转染组再内皮化率为68.36±4.51%,而Ad-GFP-EPCs转染组及未转染组再内皮化率分别为43.1±6.59%、40.5±7.82%,两者之间无统计学差异,但与Ad-Id1-EPCs转染组比较均存在明显差异(P0.05),说明转染Id1过表达的EPCs可促进第14d时损伤血管再内皮化。 3.3.4移植过表达Id1的EPCs对损伤血管局部新生内膜增殖的影响 血管损伤后第14d,Ad-Id1-EPCs转染组小鼠损伤颈动脉内、中膜比值为1.08±0.15,结果与Ad-GFP-EPCs组(1.16±0.14)及未转染组(1.15±0.17)比较三组之间比较无明显差异(P0.05),而三组中膜面积均无明显差异(P0.05),提示转移植表达Id1的EPCs未减轻血管损伤后新生内膜的增殖程度。 4.结论: 4.1 Id1在静止状态的脾源性EPCs呈低表达,定位于细胞浆内; 4.2 Id1影响EPCs增殖、迁移功能:过表达Id1可促进EPCs的增殖、迁移,干扰Id1则抑制EPCs的增殖、迁移; 4.3 Id1在损伤血管局部呈动态表达,14d为其表达高峰期; 4.4移植过表达Id1的EPCs到颈动脉损伤动物模型,可促进14d后损伤血管再内皮化,但未明显抑制局部新生内膜的增殖。
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363
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