【作者】 方立；

【导师】 谢秀梅；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 第一章内皮祖细胞与血管平滑肌细胞共培养条件下内皮祖细胞对血管平滑肌细胞增殖的影响研究背景：近年来大量研究提示内皮祖细胞(endothelial progenitor cell, EPCs)能够延缓高血压、冠心病、血管成形术后再狭窄等血管增殖性疾病血管重塑的进程。鉴于血管平滑肌细胞(vascular smooth muscle cells, VSMCs)的增殖是形成高血压、冠心病、血管成形术后再狭窄等心血管疾病的共同细胞病理基础之一,本实验拟从细胞水平探讨共培养体系下EPCs对大鼠VSMCs增殖的影响。方法：采用3%明胶沉降红细胞及密度梯度离心法分离人脐血单个核细胞,利用内皮生长培养基(Endothelial growth medium-2,EGM-2,含EPCs分化所需各种生长因子)进行培养,诱导单个核细胞贴壁并向EPCs分化；Dil-ac-LDL和FITC-UEA-1荧光双染色鉴定正在分化的EPCs;流式细胞仪和间接免疫荧光法检测EPCs干细胞分子标志CD34／CD133以及内皮细胞分子标志FLK-1/VWF/CD31/ VE-cadherin/CD 105/CD 146;采用Transwell培养板建立早期EPCs和VSMCs共培养模式,20%FBS诱导VSMCs增殖,利用BrdU标记和蛋白定量法检测血管平滑肌细胞DNA和蛋白合成能力,观察共培养条件下EPCs对VSMCs增殖的影响。结果：从人脐血单个核细胞成功分离培养EPCs,早期EPCs和VSMCs共培养12,24,48和72h时,血管平滑肌细胞DNA和蛋白合成能力均较对照组明显降低(P<0.05)；与此类似,流式细胞术结果显示共培养组VSMCs细胞周期中S期细胞所占百分率较对照组显著降低(P<0.05),而细胞周期中G1期细胞所占百分率均相应高于对照组(P<0.05),其中均以48h抑制效果最明显。结论：共培养条件下早期EPCs能够抑制血管平滑肌细胞DNA和总蛋白的合成,其机制可能与早期EPCs通过阻滞细胞周期进程抑制VSMCs的增殖有关。第二章内皮祖细胞对AngⅡ诱导的血管平滑肌细胞增殖与表型转化的抑制作用研究背景：VSMCs表型转化是高血压、冠心病和血管成形术后再狭窄等心血管疾病VSMCs增殖和迁移的关键性起始步骤。病理条件下VSMCs的增殖和表型转化受到多种血管活性物质和细胞因子的调控,其中AngⅡ诱导的VSMCs增殖和表型转化在心血管疾病的发生、发展中起着非常重要的作用。为了进一步阐明EPCs对VSMCs病理性增殖和表型转化的抑制作用及其机制,本研究拟采用AngⅡ刺激VSMCs后,观察内皮祖细胞条件培养基对VSMCs增殖、表型转化及其信号转导通路的影响。方法：分别制备早期EPCs条件培养基(E-EPC-CM)、晚期EPCs条件培养基(L-EPC-CM)以及人脐静脉内皮细胞条件培养基(HUVEC-CM)后,采用BrdU标记法、蛋白定量、MTT以及流式细胞术分析E-EPC-CM、L-EPC-CM、HUVEC-CM对AngⅡ诱导的VSMCsDNA合成能力、细胞总蛋白含量、细胞存活率、细胞周期进程以及VSMCs收缩表型标志基因平滑肌α-肌动蛋白(α-smootn musle actin,α-SM-actin)和合成表型标志基因骨桥蛋白(osteopontin, OPN)表达变化的影响；进一步采用RT-PCR和Western-blot观察E-EPC-CM、L-EPC-CM、HUVEC-CM对AngⅡ诱导的VSMCs MAPK (P38、JNK、ERK活化)和NF-κB(P65活化)信号通路以及原癌基因c-myc、c-fos表达的影响。结果：AngⅡ(10-6mmol/L)诱导VSMCs增殖48h后,血管平滑肌细胞DNA合成能力、细胞总蛋白含量、细胞存活率均较对照组明显增加(P<0.05)；细胞周期中S期细胞所占百分率较对照组显著增加,G1期细胞所占百分率均相应较对照组减少(P<0.05);α-SM-actin mRNA和蛋白表达明显减少,而OPN mRNA和蛋白表达明显增加(P<0.05),提示VSMCs从收缩表型向合成表型转化；P38、JNK. ERK、P65活化以及原癌基因c-myc、c-fos的表达均较对照组显著增加(P<0.05)。E-EPC-CM、L-EPC-CM、HUVEC-CM预处理后能够显著抑制AngⅡ诱导的VSMCs DNA合成能力、细胞总蛋白含量、细胞存活率的增加以及阻滞VSMCs从Gl期向S期的转化；并且抑制VSMCs从收缩表型向合成表型转化,其中均以E-EPC-CM的抑制效果最明显(P<0.05)。同样E-EPC-CM也抑制了AngⅡ诱导的P38、JNK、ERK.P65的活化以及c-myc、c-fos的表达(P<0.05)。结论：EPCs能够抑制AngⅡ诱导的VSMCs病理性增殖和表型转化,其机制可能与其抑制MAPK和NF-κB信号通路的活化以及原癌基因c-myc、c-fos的表达有关。第三章降钙素基因相关肽介导内皮祖细胞抑制血管平滑肌细胞的增殖与表型转化研究背景：EPCs许多生物学功能主要都是通过其旁分泌功能而实现的,研究表明降钙素基因相关肽(calcitonin gene-related peptide, CGRP)能够显著抑制AngⅡ等诱导的VSMCs增殖和表型转化,已有研究证实EPCs(主要是早期EPCs)能够合成并分泌CGRP。前一章研究已证实EPCs可抑制AngⅡ诱导的VSMCs增殖与表型转化,那么EPCs通过旁分泌释放的CGRP是否参与这一过程值得进一步探讨。因此,本研究拟初步探讨CGRP在EPCs抑制AngⅡ诱导的VSMCs增殖与表型转化过程中的作用和可能的分子机制。方法：采用ELISA和RT-PCR检测EPCs和脐静脉内皮细胞CGRP的表达情况,对E-EPC-CM采取CGRP抗体封闭和CGRP受体阻断策略后,观察E-EPC-CM对AngⅡ诱导的VSMCs的增殖、表型转化及其MAPK、NF-κB信号转导通路和原癌基因c-myc、c-fos表达的影响。结果：早期EPCs能够表达并分泌高浓度的CGRP,并且显著高于晚期EPCs和HUVEC; CGRP阻断后,E-EPC-CM对AngⅡ诱导的VSMCs DNA合成能力、细胞总蛋白含量、细胞存活率、细胞周期进程以及表型转化的的抑制作用均较对照组明显降低(P<0.05)；并且对MAPK(P38. JNK、ERK)、NF-κB(P65)信号转导通路的活化以及原癌基因c-myc、c-fos表达的抑制能力也较对照组明显降低(P<0.05)。结论：EPCs通过旁分泌CGRP抑制AngⅡ诱导的VSMCs增殖与表型转化。更多还原

【Abstract】 Chapter 1 Effect of endothelial progenitor cell on the proliferation of co-cultured vascular smooth muscle cellsBackground Previous studies have demonstrated EPCs can delay the progress of vascular remodeling in blood vessel proliferating diseases such as hypertension, coronary artery disease, and vascular restenosis after stent deployment. It has been proposed that the proliferation and phenotype transformation of vascular smooth muscle cells(VSMCs) is the common basis of cell pathology for vessel proliferating diseases.Thus the present study aimed to explore the effect of EPCs on the proliferation of VSMCs in a VSMCs/EPCs co-culture system in vitro.Methods Mononuclear cells were isolated from fresh cord blood by 3%Gelatin and density gradient centrifugation. Isolated mononuclear cells were cultured in EBM-2 medium supplemented with 20%FBS, VEGF, bFGF and other growth factors. Flow cytometry and indirect immunofluorescence revealed that EPCs expressed hematopoietic progenitor cell-surface antigens CD34 and CD 133 as well as endothelial cell-surface antigens FLK-1, VWF, FLK-1, VE-cadherin, CD 146, CD31 and CD 105. Fluorescence microscopy showed that adherent EPCs are positive for Dil-ac-LDL uptaking and FITC-UEA-1 binding. The co-culture system of EPCs and VSMCs was established by Transwell permeable support,20%fetal bovine serum was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system the DNA synthesis ability, total protein level and cell cycle of VSMCs was determined by BrdU marking method, protein quantitation and flow cytometry after co-culture for 6,12,24,48 and 72 h.Results After co-culture for 12,24,48 and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with control group(P<0.05). Flow cytometry showed the percentage of S phase of VSMCs in VSMCs/EPCs co-culture group significantly decreased and the percentage of G1 phase increased markedly compared with control group(P< 0.05), The maximal inhibitory effect was obtained at the time of 48 h.Conclusion The early EPCs could inhibit the proliferation of VSMCs in vitro co-culture.Chapter 2 The effect of endothelial progenitor cells on angiotensin II induced proliferation and phenotype transformation of cultured rat vascular smooth muscle cellsBackground It has been proposed that phenotype transformation of VSMCs is pivotal for the proliferation for cardiovascular diseases such as hypertension, coronary artery disease, and vascular restenosis after stent deployment. Under pathological conditions, proliferation and phenotype transformation of VSMCs was regulated by a variety of vasoactive substances and cytokines. In order to further elucidate the inhibitory effect and its mechanisms of EPCs on the proliferation and phenotype transformation of VSMCs, the present study investigate the effect of pretreatment with EPC-CM on Ang II induced the proliferation, phenotype transformation and the MAPK and NF-κB signaling pathways in VSMCs.Methods After preparating early endothelial progenitor cell conditioned medium (E-EPC-CM), late endothelial progenitor cell conditioned medium (L-EPC-CM) and human umbilical vein endothelial cell conditioned medium (HUVEC-CM), the effect of E-EPC-CM, L-EPC-CM and HUVEC-CM on Ang II-induced DNA synthesis, total protein expression, cell survival and cycle progress was assessed by BrdU incorporation, total protein content, MTT assays and flow cytometry. RT-PCR and Western-blot were performed to analyze the effect of E-EPC-CM, L-EPC-CM and HUVEC-CM on AngⅡ-induced the expression of a-SM-actin and osteopontin in VSMCs. Moreover, the investigators further investigated the effect of E-EPC-CM on the AngⅡinduced phosphorylations of ERK, JNK, p38 MAPKs/NF-κB p65 (or nuclear translocation of p65) and the expressions of proto-oncogene c-myc and c-fos.Results The DNA synthesis, total protein contents and cell survival obviously increased in AngⅡ(10-6 mM) 48 h induced VSMCs compared with control group, while pretreatment with E-EPC-CM, L-EPC-CM and HUVEC-CM resulted in significant inhibitions in DNA synthesis, total protein content, and cell survival compared with the control group (P<0.05);The percentage of S phase cells significantly increased, while the percentage of cells in G1 phase decreased remarkably compared with control group, pretreatment with E-EPC-CM, L-EPC-CM, or HUVEC-CM led to sustained decrease in the percentage of cells in S phase and sustained increase in the percentage of cells in G1 phase after 48 h of stimulation with AngⅡ(P<0.05); The expression of theα-SM-actin mRNA and protein was significantly decreased and the osteopontin mRNA and protein was markerly increased compared with control group, suggesting that VSMCs was changed from contractile to synthesize type by angiotensinⅡ, pretreatment with E-EPC-CM, L-EPC-CM and HUVEC-CM could inhibit significantly the down-regulation of a-SM-actin expression and the up-regulation of osteopontin expression by AngⅡ(P<0.05). E-EPC-CM showed strongest inhibitory effect among all treatment groups(P<0.05). Western-blot showed that AngⅡtime dependently induced phosphorylation of p38, JNK, ERK/NF-κB p65 and significantly increased the expressions of c-myc and c-fos after AngⅡstimulation in VSMCs, while pretreatment with E-EPC-CM showed marked inhibitory effect on the AngⅡinduced phosphorylations of these MAPKs/NF-κB p65(nuclear translocation of p65) and the expressions of proto-oncogene c-myc and c-fos (P<0.05).Conclusion EPCs may inhibit AngⅡinduced proliferation and phenotype transformation of VSMCs through inactivating MAPKs and NF-κB signaling pathways as well as by reducing the expressions of proto-oncogene c-myc and c-fos. Chapter 3 The inhibitory effect of calcitonin gene-related peptide released from endothelial progenitor cells on proliferation and phenotype transformation of vascular smooth muscle cellsBackground Many biological functions of EPCs mainly attributed to cytokines by paracrine secretion, Previous studies showed that calcitonin gene-related peptide (CGRP) could significantly inhibit the proliferation and phenotype transformation of VSMCs induced by angiotensinⅡ. Moreover, studies demonstrated that early EPCs could express and secrete CGRP. In previous charpter, it was confirmed EPCs could inhibit the proliferation and phenotype transformation of VSMCs induced by angiotensinⅡ.According to CGRP released by EPCs by paracrine manner, we proposed that CGRP may be involved in this process.Methods The release and expression of CGRP was determined by ELISA and RT-PCR. E-EPC-CM was pre-incubated with functional blocking antibodies against CGRP for 1h or VSMCs was preteated with CGRP837(CGRP receptor antagonist) for 1h before VSMCs were pretreated with CM for 30 min. DNA synthesis ability, total protein levels, cell survival, MAPK/NF-κB signal transduction and expression of c-myc and c-fos of VSMCs induced by AngⅡ(10-6mol/L) were detected to assess the role of CGRP in AngⅡ-induced proliferation of VSMCs.Results The level of CGRP was significantly increased after 48 h and was higher than that observed in the L-EPC-CM and HUVEC-CM group.After pretreatment with anti-CGRP antibody and CGRP837 (CGRP receptor inhibitor), inhibitory effect of E-EPC-CM on DNA synthesis ability, total protein levels, cell survival, cycle progress and phenotype transformation of VSMCs induced by Ang II decreased obviously compared with the control group(P<0.05). Likewise, pretreatment with anti-CGRP antibody and CGRP837, in turn, partly attenuated the decreased activity of p38, JNK and ERK MAPKs and NF-κB p65(nuclear translocation of p65) induced by E-EPC-CM treatment in a time-dependent manner and partly cancelled the decreased expression of c-myc and c-fos induced by E-EPC-CM treatment (P<0.05).Conclusion The inhibitory effect of EPCs on proliferation and phenotype transformation of VSMCs may be achieved by the paracrine effect of CGRP.更多还原