楊紅莉，潘麗，徐聰，唐文強，汪磊，夏鵬，陳雙峰，王樂信,2，陳海英

(1聊城市人民醫(yī)院，山東聊城252000；2 School of Biomedical Sciences, Charles Sturt University)

rBMSCs/Cav1F92A對PAH大鼠肺血管增殖性病變的影響及其機制

楊紅莉1，潘麗1，徐聰1，唐文強1，汪磊1，夏鵬1，陳雙峰1，王樂信1,2，陳海英1

(1聊城市人民醫(yī)院，山東聊城252000；2 School of Biomedical Sciences, Charles Sturt University)

目的探討突變型窯蛋白1(Cav1F92A)修飾的大鼠骨髓間充質干細胞(rBMSCs/Cav1F92A)對肺動脈高壓(PAH)大鼠肺血管增殖性病變的影響及其可能的機制。方法將40只Wistar大鼠隨機分為窯蛋白1(Cav1)組、Cav1F92A組、PAH組及正常對照組，每組10只。Cav1組、Cav1F92A組、PAH組給予1%野百合堿60 mg/kg腹腔注射建立PAH模型，正常對照組腹腔注射等體積生理鹽水。建模后2周，Cav1組、Cav1F92A組分別于尾靜脈移植rBMSCs/Cav1、rBMSCs/Cav1F92A各1 mL(1×106個/mL)，PAH組不予處理。各組移植后3周處死，分離肺組織。HE染色后顯微鏡下觀察肺血管管腔及血管壁厚度改變，采用實時熒光定量PCR法檢測肺組織Bbc3、B細胞易位2(Btg2)、Dab2、過氧化物酶體增生物激活受體(Ppard)、Rock1、富亮氨酸alpha-2糖蛋白1(Lrg1)基因表達，采用Western blotting法檢測肺組織內(nèi)皮素1(ET-1)、平滑肌肌球蛋白重鏈(Myocardin)蛋白表達。結果與正常對照組比較，PAH組肺血管管腔顯著狹窄，幾乎閉鎖，血管壁明顯增生肥厚。Cav1組、Cav1F92A組較PAH組肺血管壁增厚程度減輕，以Cav1F92A組減輕更明顯，但肺血管壁仍較正常對照組增厚。與正常對照組比較，PAH組肺組織Bbc3、Btg2 mRNA相對表達量均降低，Dab2、Ppard、Rock1、Lrg1 mRNA相對表達量及ET-1、Myocardin蛋白相對表達量均升高(P均<0.01)。與PAH組比較，Cav1組與Cav1F92A組肺組織Btg2 mRNA相對表達量均升高，Dab2、Ppard、Rock1及Lrg1 mRNA相對表達量均降低，且Cav1F92A組Ppard、Rock1及Lrg1 mRNA相對表達量降低更明顯(P<0.05或<0.01)。Cav1F92A組肺組織Bbc3、Btg2 mRNA相對表達量均高于PAH組、Cav1組(P<0.05或<0.01)。與PAH組、Cav1組比較，Cav1F92A組肺組織ET-1、Myocardin蛋白相對表達量均降低(P均<0.01)。結論rBMSCs/Cav1F92A可減輕PAH大鼠的肺血管增殖性病變；通過上調(diào)Bbc3、Btg2基因表達，下調(diào)Ppard、Dab2、Rock1基因及ET-1、Myocardin蛋白表達而抑制血管平滑肌細胞增殖、纖維化及血管收縮可能是其作用機制。

肺動脈高壓；窖蛋白1；內(nèi)皮型一氧化氮合酶；一氧化氮；骨髓間充質干細胞；大鼠

Abstract:ObjectiveTo investigate the effect of Cav1F92Amodified rat bone marrow mesenchymal stem cells (rBMSCs/Cav1F92A) on pulmonary vascular proliferation lesions in rats with pulmonary arterial hypertension (PAH) and to explore its possible mechanism.MethodsForty Wister rats were randomly divided into the Cav1, Cav1F92A, PAH, and normal control groups with 10 in each. PAH was induced by intraperitoneal injection of 1% monocrotaline (MCT) (60 mg/kg) in adult male Wistar rats in the Cav1, Cav1F92A, and PAH groups. Meanwhile, rats in the control group were injected with the same volume of normal saline. At week 2 after PAH models were established, 1 mL rBMSCs/Cav1 and 1 mL rBMSCs/Cav1F92A(1×106/mL) were transplantated into the rats of the Cav1 and Cav1F92Agroups by tail vein injection, while rats in the PAH group were not treated. Rats in each group were sacrificed and the lung tissues were dissected after 3 weeks of transplantation. The changes of pulmonary vascular lumen and vessel wall thickness were observed under microscope by HE staining. The gene expression of Bbc3, B cell translocation of lung tissue 2 (Btg2), Dab2, peroxisome proliferator activated receptor (Ppard), and Rock1 and leucine rich alpha-2 glycoprotein 1 (Lrg1) in the lung tissues was detected by using real-time quantitative PCR. The protein expression of endothelin 1 (ET-1) and smooth muscle myosin heavy chain (Myocardin) was investigated by Western blotting.ResultsCompared with the normal control group, the pulmonary vascular lumen became narrow, nearly closed, and the vascular wall became hyperplastic and hypertrophic significantly in the PAH group. Compared with the PAH group, the thickening degree of pulmonary vascular wall was reduced in the Cav1 and Cav1F92Agroups but more obviously decreased in the Cav1F92Agroup, but the pulmonary vascular wall was thicker than that in the normal control group. Compared with the normal control group, the relative mRNA expression of Bbc3 and Btg2 in the lung tissues of the PAH group decreased, but the mRNA expression of Dab2, Ppard, Rock1, and Lrg1 relative, and the protein expression of ET-1 and Myocardin increased (allP<0.01). Compared with the PAH group, the Bbc3 and Btg2 mRNA expression increased but Dab2, Ppard, Rock1 and Lrg1mRNA expression decreased in the Cav1 and Cav1F92Agroups, especially in the Cav1F92Agroup (P<0.05 orP<0.01); the Bbc3 and Btg2 mRNA expression in the lung tissues was higher than that in the PAH and Cav1 groups (P<0.05 orP<0.01). Compared with the PAH and Cav1 groups, the protein expression of ET-1 and Myocardin in the lung tissues decreased in the Cav1F92Agroup (bothP<0.01).ConclusionrBMSCs/Cav1F92Acan effectively alleviate vascular proliferative lesions in PAH rats by up-regulating the expression of Bbc3 and Btg2, Ppard, Dab2, and Rock1, and down-regulating the expression of ET-1 and Myocardin, and thus inhibiting the vascular smooth muscle cell proliferation, fibrosis, and vascular contraction.

Keywords: pulmonary arterial hypertension; Caveolin-1; endothelial nitric oxide synthase; nitric oxide; bone marrow mesenchymal stem cells; rats

肺動脈高壓(PAH)的標志性病理特征為肺外周小動脈內(nèi)膜增厚、纖維化及中膜增厚、外膜增生等血管增殖性病變[1]。在PAH發(fā)生、發(fā)展的病理過程中，肺動脈血管內(nèi)皮細胞最先受到攻擊，并發(fā)生病理變化[2]，內(nèi)皮功能障礙導致一氧化氮合酶(eNOS)催化生成的擴血管物質一氧化氮(NO)合成減少[3]。窖蛋白1(Cav1)是各種信號的調(diào)節(jié)中樞，是一氧化氮合酶(eNOS)的負調(diào)控子，可降低NO的合成。前期研究顯示，采用丙氨酸替代92位苯丙氨酸可獲得突變型Cav1(Cav1F92A)，其不具有抑制eNOS活性的作用，但可促進NO生成，降低肺動脈壓力，對多條信號通路具有調(diào)節(jié)作用[4,5]。2015年6月～2016年10月，本研究采用Cav1F92A修飾大鼠骨髓間充質干細胞(rBMSCs)，觀察其對PAH大鼠肺血管增殖性病變的影響，并探討其可能的機制。

1 材料與方法

1.1 材料 ①實驗動物：成年雄性Wistar大鼠40只，4～6周齡，體質量100～120 g，購自山東大學實驗動物中心。將大鼠分籠飼養(yǎng)，自由攝食攝水，晝夜時間比為1∶1。②質粒：突變的Cav1質粒12AAPFVP-F92A-pMA-T、慢病毒包裝質粒(psPax2、pRSV Rev、VSV-G)由澳大利亞Charles Sturt University的Padraig Strappe教授饋贈；慢病毒載體骨架pLVX-mCMV-zsgreen購自深圳市百恩維生物科技有限公司。③細胞：rBMSCs由聊城市人民醫(yī)院中心實驗室分離、培養(yǎng)、鑒定[5]；④試劑：PrimeScript?RT試劑盒及 SYBR Premix Ex TaqTMⅡ試劑盒均購自日本Takara公司，anti-內(nèi)皮素1(ET-1)鼠單克隆抗體購自美國Abcam公司，anti-平滑肌肌球蛋白重鏈(Myocardin)兔多克隆抗體購自美國Santa Cruz公司。

1.2 慢病毒質粒構建、包裝及轉導 參照前期研究[6]的方法構建質粒LV-Cav1、LV-Cav1F92A，參照Chen等[4]的方法進行慢病毒質粒包裝。rBMSCs培養(yǎng)融合至50%～70%時進行慢病毒轉導，分別記為rBMSCs/Cav1、rBMSCs/Cav1F92A。

1.3 PAH大鼠模型建立及分組處理 將40只Wistar大鼠隨機分為Cav1組、Cav1F92A組、PAH組及正常對照組，每組10只。Cav1組、Cav1F92A組、PAH組均給予1%野百合堿60 mg/kg腹腔注射建立PAH模型，正常對照組腹腔注射等體積生理鹽水。參照前期方法，注射后2周采用MP150 Systems測量肺動脈壓力，肺動脈收縮壓均>30 mmHg，證實PAH模型建立成功[4]。建模后2周，Cav1組、Cav1F92A組分別于尾靜脈移植rBMSCs/Cav1、rBMSCs/Cav1F92A各1 mL(1×106個/mL)，PAH組不予處理。各組移植后3周，腹腔注射4 000 mg/kg水合氯醛進行處死，分離左、右側肺組織。

1.4 肺血管增殖情況觀察 取左下部分肺組織，10%甲醛固定24 h，常規(guī)脫水、石蠟包埋，5 μm厚度切片。常規(guī)HE染色，40倍顯微鏡下觀察肺血管管腔及血管壁厚度改變等。

1.5 肺組織血管增殖性病變相關基因mRNA表達檢測 采用實時熒光定量PCR法檢測肺組織Bbc3、B細胞易位2(Btg2)、Dab2、過氧化物酶體增生物激活受體(Ppard)、Rock1、富亮氨酸alpha-2糖蛋白1(Lrg1)基因表達。取新鮮肺組織，采用gentleMACSTM Dissociator進行勻漿，TRIzol試劑提取總RNA，PrimeScript?試劑盒進行RNA反轉錄，合成cDNA，并去除基因組DNA。PCR引物由上海生工生物工程股份有限公司合成，以GAPDH為管家基因，參照AceQ qPCR SYBR Green Master Mix說明進行PCR擴增。PCR反應體系共20 μL。PCR反應條件：95 ℃預變性 5 min，95 ℃變性10 s，57 ℃退火30 s，擴增40個循環(huán)；72 ℃延伸10 min。收集SYBR Green熒光信號，采用ABI 7500檢測獲取各組Ct值，ΔCt=Ct目的基因-Ct管家基因，ΔΔCt=ΔCt實驗組-ΔCt正常對照組，采用2-ΔΔCt法計算各目的基因mRNA相對表達量。每組設置3個復孔，實驗重復3次。

1.6 肺組織ET-1、Myocardin蛋白表達檢測 采用Western blotting法。采用gentleMACSTM全自動組織處理器將各組大鼠肺組織進行勻漿，加預冷的蛋白裂解液及蛋白酶抑制劑PMSF，冰上裂解30 min；12 000 r/min離心10 min，收集上清煮沸5 min備用。每孔上樣15 μg蛋白質，進行10% SDS-PAGE電泳、轉膜，5%脫脂奶粉室溫封閉1 h。分別加入anti-ET-1(1∶1 000)鼠單克隆抗體和anti-Myocardin(1∶1 000)的兔多克隆抗體，4 ℃孵育過夜；PBS洗滌，加入相應二抗室溫孵育1 h。ECL試劑顯色，采用AlphaView圖象分析系統(tǒng)分析目的條帶灰度值。

2 結果

2.1 各組肺血管增殖情況比較 與正常對照組比較，PAH組肺血管管腔顯著狹窄，幾乎閉鎖，血管壁明顯增生肥厚。Cav1組、Cav1F92A組較PAH組肺血管壁增厚程度減輕，以Cav1F92A組減輕更明顯，但肺血管壁仍較正常對照組增厚。見插頁Ⅰ圖2。

2.2 各組肺組織血管增殖性病變相關基因mRNA表達比較 見表1。

表1 各組肺組織血管增殖性病變相關基因mRNA表達比較(相對表達量，

注：與正常對照組比較，*P<0.01，#P<0.05；與PAH組比較，△P<0.05，▽P<0.01；與Cav1組比較，▲P<0.05，▼P<0.01。

2.3 各組肺組織ET-1、Myocardin蛋白表達比較 見表2。

表2 各組肺組織ET-1、Myocardin蛋白表達比較(灰度值

注：與正常對照組比較，*P<0.01，#P<0.05；與PAH組比較，△P<0.05；與Cav1組比較，▽P<0.05。

3 討論

血管平滑肌細胞和內(nèi)皮細胞在PAH的發(fā)生與發(fā)展中扮演重要角色。血管平滑肌細胞大量增殖可引起血管壁增厚、管腔壓力增大，最終導致肺動脈壓力不斷升高，而血管內(nèi)皮細胞損傷是PAH各種病理變化的基礎。目前，靶向內(nèi)皮功能障礙的治療藥物如依前列醇及其衍生物、內(nèi)皮素受體拮抗劑和磷酸二酯酶5抑制劑等，均可改善PAH患者的臨床癥狀及血流動力學指標，但仍無法治愈PAH[7,8]，因此臨床上迫切需要尋找有效治療PAH的措施。NO主要由血管內(nèi)皮生成，具有良好的抗炎、抗血小板和血管舒張作用[9]。Cav1主要通過位于92位的苯丙氨酸與eNOS結合發(fā)揮負調(diào)控作用，進而可降低NO的合成。采用丙氨酸替代92位苯丙氨酸可獲得Cav1F92A，不具有抑制eNOS活性的作用，但可以促進NO的產(chǎn)生，從而舒張血管、降低肺動脈壓力。本研究結果顯示，與正常組比較，PAH組肺血管管腔顯著狹窄，幾乎閉鎖，血管壁明顯增生肥厚；Cav1組肺血管壁肥厚較PAH組減輕，可能與移植的rBMSC有關，其管腔仍較Cav1F92A組狹窄；Cav1F92A組管腔較大，血管壁較正常組增厚，但較PAH組和Cav1組明顯減輕。說明rBMSCs/Cav1、rBMSCs/Cav1F92A均可減輕PAH大鼠的血管增殖性病變，其中rBMSCs/Cav1F92A的效果更好，可能與其對eNOS無負調(diào)控作用有關。

Bbc3與Btg2均為p53信號通路的下游靶基因，Bbc3可誘導細胞周期阻滯，介導細胞凋亡[10]。Btg2是抗增殖相關Btg/Tob基因的家族成員之一，是p53信號通路下游的細胞死亡誘導因子，可通過下調(diào)cyclin D1、cyclin E及基質金屬蛋白酶9(MMP-9)的表達而抑制細胞增殖、遷移和侵襲[11,12]。Ppard與Dab2均屬于Wnt信號通路的調(diào)控分子。Ppard在高血壓患者中表達升高，上調(diào)Ppard表達可促進細胞增殖[13]。Dab2的功能具有細胞特異性，可介導纖維化，其與成纖維細胞生長因子2(FGF2)、Cav1共同作用可促進成纖維細胞的遷移[14]，正向調(diào)控轉化生長因子β-R(TGF-β-R)通路，促進纖連蛋白表達及細胞黏附、遷移[15]。本研究結果顯示，PAH組肺組織Bbc3、Btg2 mRNA相對表達量明顯低于正常組，Dab2、Ppard mRNA相對表達量明顯高于正常組，而rBMSCs/Cav1F92A移植后上述指標變化發(fā)生逆轉。說明rBMSCs/Cav1F92A可激活PAH大鼠的p53信號通路、抑制Wnt信號通路，從而抑制細胞增殖及遷移，可能是其減輕PAH大鼠的血管增殖性病變的相關機制之一。

PAH血管病變的主要特點是纖維化，常發(fā)生于血管平滑肌細胞。Lrg1是一種新型血管生成性糖蛋白，通過調(diào)節(jié)內(nèi)皮細胞TGF-β信號通路促進血管形成，而TGF-β信號通路失調(diào)又常伴有纖維化、異常血管生成，并加速心力衰竭進展[16]。研究顯示，血清Lrg1水平是診斷特發(fā)性PAH的生物標志物之一[17]。ET-1是目前已知作用最強的縮血管活性物質，可促進平滑肌細胞增殖、遷移而引起血管重構。ET-1可激活Rock1，導致肺動脈血管內(nèi)皮細胞的血管壁增厚，激活的Rock1也可與Ppard相互作用，促進血管平滑肌細胞增殖，導致血管重塑；Rock1還可上調(diào)與炎癥、血栓形成及纖維化相關的分子，參與細胞黏附、遷移、增殖及凋亡等，在血管痙攣、高血壓、肺動脈高壓及心力衰竭等疾病的發(fā)生、發(fā)展中發(fā)揮重要作用[17]。Myocardin是維持平滑肌細胞收縮狀態(tài)的主要輔助因子，可促進血管內(nèi)皮細胞向平滑肌細胞的轉化[7]。本研究結果顯示，PAH組Lrg1、Rock1 mRNA及ET-1、Myocardin蛋白表達均明顯高于正常對照組，而rBMSCs/Cav1F92A移植可有效降低PAH大鼠肺組織中上述基因或蛋白表達，表明rBMSCs/Cav1F92A可抑制PAH大鼠肺血管平滑肌細胞增殖、遷移及纖維化。

綜上所述，rBMSCs/Cav1F92A可減輕PAH大鼠的血管增殖性病變，其機制可能是通過上調(diào)Bbc3、Btg2表達，下調(diào)Ppard、Dab2、Rock1基因及ET-1、Myocardin蛋白表達，進而抑制血管平滑肌細胞增殖、纖維化及血管收縮。

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Effect of rBMSCs/Cav1F92Aon pulmonary vascular proliferation lesions in PAH rats

YANGHongli1,PANLi,XUCong,TANGWenqiang,WANGLei,XIAPeng,CHENShuangfeng,WANGLexin,CHENHaiying

(1LiaochengPeople′sHospital,Liaocheng252000,China)

國家自然科學基金資助項目(81270104)；山東省自然科學基金資助項目(ZR2016HP33)。

楊紅莉(1987-)，女，技師，研究方向為藥理學。E-mail: yanghongli2011slky@163.com

陳海英(1969-)，女，副主任技師，研究方向為藥理學。E-mail： hychenmay5@126.com

10.3969/j.issn.1002-266X.2017.32.004

R543.2

A

1002-266X(2017)32-0013-04

2016-11-21)