大隱靜脈源性血管平滑肌細胞表型轉化的研究進展

嚴力 綜述 褚海波 審校

（中國人民解放軍第八十九醫(yī)院 普外中心，山東 濰坊 261021）

大隱靜脈曲張（great saphenous varicose vein，GSVV）是下肢常見疾病之一，以靜脈瓣功能不全、靜脈反流、靜脈擴張和迂曲、管腔內靜脈壓力增高為主要臨床特征，可并發(fā)靜脈血栓形成、淺表血栓性靜脈炎和靜脈性潰瘍[1-2]。大隱靜脈曲張發(fā)生率為20%～40%，老年人為高發(fā)人群[3]。多年來，盡管許多學者[4-5]致力于大隱靜脈曲張發(fā)病機制的研究，但一直尚無定論。研究[6-7]顯示，靜脈壁中細胞和細胞外基質成分的改變，包括內膜增生、平滑肌細胞功能失調、膠原和彈力纖維比例的變化、基質金屬蛋白酶（matrix metalloproteinases，MMPs）及其組織抑制劑（tissue inhibitor of metalloproteinase，TIMPs）的失衡，可能是導致靜脈壁松弛、薄弱，繼而靜脈膨張、靜脈瓣關閉不全和反流的主要因素。生物組織中含有多種具有特定表型的細胞群，在組織穩(wěn)態(tài)中依據(jù)各自的作用相互間調節(jié)，通過細胞微環(huán)境中的信號維持這些細胞靜息與激活之間的平衡。近年來，在控制細胞命運和功能的微環(huán)境刺激中，機械力的重要性逐漸受到關注[8]?，F(xiàn)已明確，外部壓力（如流體靜力壓、拉伸張力或鞘壓）和細胞內細胞骨架張力能驅動轉錄調節(jié)改變[9]。此外，血管平滑肌細胞（vascular smooth muscle cells，VSMCs）的相對動態(tài)環(huán)境，還涉及多種細胞內外生物化學分子以及機械力的變化：包括生長因子、炎癥因子、血管活性因子、血流機械力以及活性氧等因素。鑒于靜脈高壓是靜脈曲張形成的一個重要因素，研究機械力在靜脈曲張病理形成中所起的作用已成為熱門課題。本文就大隱靜脈源性VSMCs表型轉化研究進行歸納敘述，旨在為尋找防治靜脈曲張相關的藥物作用靶點奠定理論基礎。

1 VSMCs表型轉化的概念及特點

1989年，Baumbach等[10]提出“血管重塑”的概念，由此人們逐漸認識到靜脈疾病的病理生理過程不僅是管壁形態(tài)結構的改變，VSMCs表型轉化才是血管重塑的細胞學基礎和關鍵環(huán)節(jié)。與骨骼肌和心肌細胞不同，靜脈源性VSMCs表型具有分化可逆性和多樣性的特點。在胚胎發(fā)育過程中，VSMCs由未分化表型逐漸轉化為具有成熟特征的分化表型。當血管受損或VSMCs受到生長因子、機械作用、血管活性物質以及血流動力學等因素刺激時，VSMCs又從分化型轉化為去分化型，并獲得增殖能力，對這一演變過程稱之為表型轉化（phenotypic transformation）。VSMCs是一種高度特異性細胞，其主要功能為收縮和血管張力調節(jié)，以控制血壓和血流[11]。根據(jù)細胞結構和功能的不同，VSMCs有兩種表型，即收縮型（分化型）和合成型（去分化型或未分化型）。收縮型VSMCs，分化程度高，呈紡錘狀，胞漿內肌絲豐富，高爾基復合體和線粒體較少，增殖和遷移能力差，其主要功能是維持血管壁的彈性和收縮血管。合成型VSMCs內含大量的高爾基復合體、粗面內質網(wǎng)，肌絲含量少，合成和分泌功能旺盛，增殖和遷移能力強，并能產(chǎn)生大量的細胞外基質，其主要功能是合成分泌基質蛋白，并參與到血管壁重塑和損傷修復[12]。

2 VSMCs表型轉化標志物

當VSMCs受到外界刺激后，可通過激活多種信號通路來激發(fā)VSMCs表型轉化，以適應靜脈高壓和缺氧對血管壁的影響。檢測VSMCs表型轉化的標記物種類繁多，一般可分為收縮型標記物和合成型標記物，收縮性標記物主要有平滑肌α-肌動蛋白（α-SM-actin，α-SMA）、平滑肌22α（smooth muscle 22α，SM22α）、平滑肌肌球蛋白重鏈（smooth muscle-myosin heavy chain，SMMHC）、平滑肌蛋白（smoothelin）、以及H-鈣調素結合蛋白（caldesmon），合成型標記物主要有骨橋蛋白（osteopontin，OPN）、上皮調節(jié)蛋白（epiregulin）等[13-18]。

2.1 收縮型表型標志物

α-SMA是一種維持VSMCs形態(tài)和收縮的重要細胞骨架蛋白，在收縮型VSMCs中優(yōu)勢表達，是VSMC分化早期特異性標志物；SM22α屬細胞骨架的結構成分，但不是控制VSMCs收縮的必需蛋白；SM-MHC則是在胚胎形成時期VSMCs特異性表達的一種標志蛋白，且SM-MHC在非SMCs細胞中不表達[19]。因此，SM-MHC被認為是鑒別收縮型VSMCs的最常用標志物之一。Smoothelin表達下調則提示收縮型VSMCs向合成型VSMCs轉化，常用于協(xié)同SM-MHC區(qū)分收縮型VSMCs和合成型VSMCs[20]。

2.2 合成型表型標志物

合成型VSMCs的標志物包括OPN和Epiregulin。OPN是一種富含唾液酸磷酸化和糖基化修飾的基質糖蛋白。OPN是VSMCs表型轉化的起始促進因子，其表達與VSMCs的增殖密和遷移密切相關。Epiregulin是表皮生長因子家族的成員之一，可促進VSMCs的增殖。Takahashi等[21]研究顯示，Epiregulin是VSMCs去分化的主要自分泌和旁分泌因子，可通過趨化因子以及ERK和p38MAPK等信號通路來調控VSMCs的增殖和表型轉化，從而影響到“血管重塑”的病理生理過程。

VSMCs具有高度可塑性，并能在不同的表型狀態(tài)下生存[22]。VSMCs由收縮型調節(jié)至合成型依賴于環(huán)境改變，即增殖和遷移活性增加，收縮性喪失，細胞外基質異常[23-24]。此外，VSMCs不同表型標志物的表達受多種因素的調節(jié)如轉化生長因子β（transforming grow th factor β，TGF-β）、血小板衍生生長因子（platelet derived growth factor，PDGF）、miRNA等。Ha等[25]觀察大鼠胸主動脈VSMCs表型轉化，發(fā)現(xiàn)收縮型VSMCs α-SMA和SM22α呈陽性表達。Hao等[26]研究發(fā)現(xiàn)，SM-MHC表達受TGF-β1信號通路的促進，維持平滑肌收縮表型，Gareri等[27]則通過PDGF刺激VSMCs表型轉化，使得SM-MHC表達大量下降。Feng等[28]研究顯示，在體外血管收縮素（angiotensin II，Ang-II）可誘導VSMCs骨源性表型轉化和鈣化，同時發(fā)現(xiàn)α-SMA和SM22α標記物下調，OPN標記物上調。由此可見，在生理和病理狀態(tài)下，SMCs參與血管重塑，不同的標記物反映不同的VSMCs表型，且表型變異和可逆性轉化是一個復雜和動態(tài)的過程[29-31]。

3 細胞增殖、遷移與表型轉化

VSMCs表型轉化是血管重塑的細胞學基礎。成熟的VSMCs為收縮和分化表型，細胞的增生和合成能力低下，可表達多種收縮蛋白、離子通路、細胞收縮功能分子信號[32-33]。在病理狀態(tài)下，VSMCs由收縮表型轉化為合成和去分化表型，其特征是收縮能力喪失，細胞增生、遷移和基質分泌異常[34]。動物實驗[35-36]表明，移植大隱靜脈管腔狹窄的主要原因是中膜和外膜的SMCs遷移至內膜，導致新生內膜增生。機械力和炎癥的協(xié)調可導致VSMCs增殖和遷移，其中炎性介子可通過白介素1β（interleukin 1β，IL-1β）和白介素18（interleukin 18，IL-18）信號通路誘導增殖和遷移[37]。Zhang等[38-39]研究發(fā)現(xiàn)，腫瘤壞死因子α（tumor necrosis factor α，TNF-α）可抑制絲裂素活化蛋白激酶磷酸酶（mitogen-activated protein kinase phosphatase，MAKP）炎性通路，降低炎性反應。在刺激過程中，VSMCs表型由靜止收縮型轉化為促炎反應型，SMCs向內膜遷移，提示VSMCs增殖和遷移與炎癥相關。動物實驗顯示，血管細胞黏附分子1（vascular cell adhesion molecule-1，VCAM-1）或VCAM-1 siRNA表達能阻止單核/巨噬細胞募集反應，抑制VSMCs增殖、遷移和新生內膜形成[40-41]。在血管重塑過程中，VSMCs收縮表型和合成表型存在本質性差異（即標記物表達、細胞形態(tài)和活性）。合成型VSMCs源于中膜去分化收縮型VSMCs和外膜分化成纖維細胞及未分化間質干細胞。合成型VSMCs收縮器（肌絲，致密體和斑）少，合成器（粗面內質網(wǎng)，高爾基體）增多[42]。諸多因素（物理、機械、缺氧、激素、氧化應激、基因、鈣離子等）影響VSMCs表型，而VSMCs的增殖和遷移改變取決于VSMCs表型。李源等[43]發(fā)現(xiàn)，源于曲張靜脈的VSMCs增殖和遷移能力比正常靜脈增加，提示VSMCs表型轉化影響細胞功能。有學者[44-45]認為，VSMCs在分化和去分化之間表型轉化是一種可逆性變化，并伴隨著細胞與細胞、細胞基質黏附網(wǎng)絡形態(tài)和功能的改變。因此，如何在分子水平調節(jié)VSMCs的增殖與遷移，控制細胞表型的轉化，可視為分子通路靶標研究的發(fā)展趨勢[46-51]。

4 MMPs與表型轉化

MMPs是一種依賴于鋅離子的內肽酶，具有降解細胞外基質（extracellular matrix，ECM）蛋白作用。MMPs可與細胞膜上的生物活性分子相互作用，并能調節(jié)偶聯(lián)G蛋白受體和細胞信號。MMPs參與多種生理過程，并影響細胞增殖、遷移、變異。MMPs還涉及細胞凋亡、免疫反應、組織修復、血管生成[52]。MMPs家族依據(jù)消化底物的不同分為膠原酶類，明膠酶類，基質溶解素類、膜型金屬蛋白酶類和未分類[53]。MMPs有助于VSMCs增殖和遷移，遷移可促進血管新生內膜增生（即內皮細胞和SMCs通過MAPK信號激活，刺激生長因子、細胞因子和MMPs的分泌）[54]。VSMCs表型變化決定MMPs代謝異常。合成表型收縮型喪失，ECM產(chǎn)生異常，VSMCs遷移增加；收縮表型低蛋白含量和增殖率，具有獨特的收縮蛋白和信號分子[55]。動物實驗表明，MMP-2和MMP-9在不同靜脈層表達有所差異。MMP-2表達主要位于外膜，MMP-9表達則位于內皮細胞、中膜SMCs、外膜微血管。與此同時，炎癥可促進MMP-2和MMP-9表達[56-57]。人體外細胞培養(yǎng)顯示，異常VSMCs其MMP-2和MMP-9呈高表達[55,58]。研究[59]發(fā)現(xiàn)，抵抗素可誘導MMP-2和MMP-9表達（mRNA和蛋白水平），抗MMP-2和MMP-9 IgG可抑制抵抗素誘導VSMCs遷移。TIMP-1優(yōu)先結合MMP-9，TIMP-2優(yōu)先結合MMP-2，抵抗素抑制TIMPs表達；WNT1誘導信號通道蛋白1（WNT1 inducible signaling pathway protein 1，WISPI）可促進SMCs內MMPs和TIMPs高表達[60]；氫可降低p38MAPK活性導致移植靜脈MMP-2和MMP-9低表達，抑制VSMCs遷移[61]；在增生的血管內膜中，MMP-3 mRNA和蛋白呈高表達[62]；OPN和MMPs具有密切的關聯(lián)性，這與動脈壓對移植靜脈刺激的適應性有關[63]。VSMCs釋放MMPs降解ECM，并產(chǎn)生TIMPs，因此，MMPs和TIMPs的平衡決定ECM的動態(tài)平衡[64-65]。動物實驗顯示，小鼠移植靜脈TIMP-1呈高表達。這一結果提示，TIMP-1可抑制MMPs，阻止斑塊發(fā)展，并增加其穩(wěn)定性[66]。結締組織生長因子是一種異常的纖維化介質，它能通過結締組織生長因子信號通道調節(jié)ECM的合成，從而改變靜脈管壁纖維化的進程[67]?？梢?，MMPs與SMCs表型轉化關系密切，在VSMCs發(fā)生重構過程中，MMPs和TIMPs通過分子信號通道調節(jié)發(fā)揮重要的生理和病理生理功能。

5 細胞凋亡與表型轉化

細胞凋亡是細胞內有規(guī)律的自我消亡過程，細胞死亡的一種生理形式，受誘導基因（p53、bclxs、bax）、抑制基因（Bcl-2、bcl-xl、mcl-1）及參與基因（c-myc、c-fos）的調控[68]。細胞凋亡的作用是維持組織內環(huán)境的穩(wěn)定性，以減少細胞的更新。細胞凋亡包括內源性和外源性兩條通道。內源性通道又稱線粒體通道，調節(jié)細胞凋亡啟動蛋白（bax或Bcl-2）及特異胱門蛋白酶（caspase）刺激線粒體釋放細胞色素C進入胞漿與凋亡蛋白酶活化因子1（APAF-1）結合引發(fā)細胞凋亡。外源性通道又稱跨膜通道，在細胞凋亡信號的刺激下，F(xiàn)asL和TNF-α作用相應的受體導致細胞凋亡[69]。Whiteley等[70]獲取人曲張大隱靜脈標本，采用免疫組化和免疫熒光法標記p53，發(fā)現(xiàn)曲張靜脈SMCs凋亡上調，并與炎性標記物呈正相關。研究發(fā)現(xiàn)，膜聯(lián)蛋白A2過表達和IncRNA低表達可促進SMCs增殖與遷移，并減少SMCs凋亡[50]。誘導VSMCs缺氧模型顯示，VSMCs增殖與遷移增加，但與細胞凋亡無相關性[71]。在缺氧狀態(tài)下，金屬硫蛋白可阻止VSMCs凋亡，這一結果有可能為靜脈曲張的靶標治療提供理論依據(jù)[72]。許多學者[1,73]認為，VSMCs凋亡與年齡和性別密切相關。＞50歲和女性細胞凋亡明顯增加，故研究VSMCs凋亡與表型轉化應考慮年齡和性別因素。文獻[74]報道，炎性因子可增加VSMCs增殖與遷移，并經(jīng)p38MAPK-HSPS27分子通道誘導細胞凋亡。同源異型盒蛋白過表達可促進VSMCs增殖與遷移及抗凋亡能力，上調OPN可有利于VSMCs表型轉化[30]。細胞自體吞噬作用可抑制肌餓誘導VSMCs凋亡，而異常自體吞噬作用可調節(jié)VSMCs的功能[75]。小窩蛋白3能調節(jié)VSMCs收縮和合成型的轉型，抑制細胞凋亡[76]。由此可見，VSMCs凋亡的變化是建立在表型變化的基礎上，表型的異常必定影響細胞的凋亡。

6 表觀遺傳學調控與表型轉化

VSMCs的表型轉化受多種因素影響，其中，表觀遺傳學調控機制在VSMCs表型轉化中所起到的重要作用日益受到人們重視。表觀遺傳通常被定義為不涉及DNA核苷酸序列改變的基因表達和調控的可遺傳修飾，通常是由于環(huán)境因素而引發(fā)改變，主要包括DNA甲基化、組蛋白修飾、非編碼RNA以及染色質重塑等。研究表明，表觀遺傳對VSMCs表型轉化的調控主要是通過對其表型標志基因（例如：α-SMA、SM22α、SM-MHC、OPN等）表達的調控來實現(xiàn)[34,77-78]。Hiltunen等[79]的早期研究通過高效液相色譜法檢測體內VSMCs染色體5-甲基胞嘧啶含量發(fā)現(xiàn)，動脈粥樣硬化模型VSMCs表型轉化過程中呈現(xiàn)全基因組的低甲基化狀態(tài)；同樣在體外實驗，細胞增殖過程中collagen type VX α1基因的低甲基化誘導相應基因高表達進而調控VSMCs的表型轉化并影響動脈粥樣硬化的發(fā)展[80]。近年來，更多的實驗研究發(fā)現(xiàn)，DNA的異常甲基化可通過特定的基因修飾來調控VSMCs的表型轉化并影響到相關血管疾病進程。Jiang等[34]研究發(fā)現(xiàn)靜脈曲張VSMCs中OPN基因啟動子的低甲基化可能是誘導OPN高表達的重要因素，提示異常的表觀遺傳修飾參與了VSMCs的表型轉化并導致新生血管內膜增厚進而影響靜脈曲張的發(fā)生與發(fā)展。Ali等[81]證實腫瘤壞死因子α（tumor necrosis factor-α，TNT-α）可誘導SMCs收縮型基因（α-SMA、SM-MHC）啟動子甲基化并通過Krupper樣因子4（Krupper like factor-4，KLF-4）調節(jié)通路來誘導大鼠腦SMCs表型變化。組蛋白修飾多發(fā)生于細胞生長發(fā)育期，通過調控血清反應因子（serum response factor，SRF）及其輔助因子與染色質模板的結合來改變外環(huán)境，從而影響VSMCs分化過程[82-83]。由于組蛋白修飾的VSMCs限制性位點位于VSMCs基因染色質的CArG box序列，SRF及其輔助因子與VSMCs染色質啟動子CArG box DNA序列相互反應是引起VSMCs在生長發(fā)育和表型轉化過程中的信號通路關鍵環(huán)節(jié)。此外，有研究[84]表明，高脂飲食可誘導DNA甲基轉移酶1（DNA methyltransferase 1，DNMT-1）負性調控因子miR-152下調，進而導致主動脈VSMCs雌激素受體α基因（Estrogen receptor α gene，ER-α）高甲基化，而染色質重塑亦被發(fā)現(xiàn)在VSMCs分化過程中起到關鍵作用[77]。與此同時，一些新的調控靶點如TET-2（ten-eleven translocation 2）以及YAP（yesassociated protein）相關研究的出現(xiàn)進一步完善了VSMCs表型轉化與血管性疾病發(fā)生發(fā)展的機制探索[85-86]。由此可見，VSMCs的可塑性與細胞外部環(huán)境和表觀遺傳調控機制密切相關，然而，不同于DNA序列的改變，許多表觀遺傳的改變是可逆的，因此，如何從表觀遺傳調控水平去調節(jié)并控制VSMCs表型轉化，可作為相應血管類疾病靶向治療的研究思考方向。

7 結 語

綜上所述，大隱靜脈源性VSMCs表型轉化是血管重塑的細胞學基礎。VSMCs表型轉化除了與其基因表達譜改變有關外，還受細胞骨架結構與功能的影響。設想建立人正常靜脈VSMCs和曲張靜脈VSMCs細胞單層培養(yǎng)系統(tǒng)，從VSMCs增殖、遷移、黏附、衰老、骨架、MMPs和TIMPs分泌和細胞凋亡方面研究曲張大隱靜脈來源VSMCs的生物學變化特征，有助于初步證實曲張大隱靜脈、淺表血栓性靜脈炎及缺氧誘導正常靜脈VSMCs從收縮型向合成型轉化并獲得增生、遷移和分泌大量細胞外基質的能力。

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