rBMSCs/Cav1F92A对PAH大鼠肺血管增殖性病变的影响及其机制

杨红莉，潘丽，徐聪，唐文强，汪磊，夏鹏，陈双峰，王乐信,2，陈海英

(1聊城市人民医院，山东聊城252000；2 School of Biomedical Sciences, Charles Sturt University)

rBMSCs/Cav1F92A对PAH大鼠肺血管增殖性病变的影响及其机制

杨红莉1，潘丽1，徐聪1，唐文强1，汪磊1，夏鹏1，陈双峰1，王乐信1,2，陈海英1

(1聊城市人民医院，山东聊城252000；2 School of Biomedical Sciences, Charles Sturt University)

目的探讨突变型窑蛋白1(Cav1F92A)修饰的大鼠骨髓间充质干细胞(rBMSCs/Cav1F92A)对肺动脉高压(PAH)大鼠肺血管增殖性病变的影响及其可能的机制。方法将40只Wistar大鼠随机分为窑蛋白1(Cav1)组、Cav1F92A组、PAH组及正常对照组，每组10只。Cav1组、Cav1F92A组、PAH组给予1%野百合碱60 mg/kg腹腔注射建立PAH模型，正常对照组腹腔注射等体积生理盐水。建模后2周，Cav1组、Cav1F92A组分别于尾静脉移植rBMSCs/Cav1、rBMSCs/Cav1F92A各1 mL(1×106个/mL)，PAH组不予处理。各组移植后3周处死，分离肺组织。HE染色后显微镜下观察肺血管管腔及血管壁厚度改变，采用实时荧光定量PCR法检测肺组织Bbc3、B细胞易位2(Btg2)、Dab2、过氧化物酶体增生物激活受体(Ppard)、Rock1、富亮氨酸alpha-2糖蛋白1(Lrg1)基因表达，采用Western blotting法检测肺组织内皮素1(ET-1)、平滑肌肌球蛋白重链(Myocardin)蛋白表达。结果与正常对照组比较，PAH组肺血管管腔显著狭窄，几乎闭锁，血管壁明显增生肥厚。Cav1组、Cav1F92A组较PAH组肺血管壁增厚程度减轻，以Cav1F92A组减轻更明显，但肺血管壁仍较正常对照组增厚。与正常对照组比较，PAH组肺组织Bbc3、Btg2 mRNA相对表达量均降低，Dab2、Ppard、Rock1、Lrg1 mRNA相对表达量及ET-1、Myocardin蛋白相对表达量均升高(P均<0.01)。与PAH组比较，Cav1组与Cav1F92A组肺组织Btg2 mRNA相对表达量均升高，Dab2、Ppard、Rock1及Lrg1 mRNA相对表达量均降低，且Cav1F92A组Ppard、Rock1及Lrg1 mRNA相对表达量降低更明显(P<0.05或<0.01)。Cav1F92A组肺组织Bbc3、Btg2 mRNA相对表达量均高于PAH组、Cav1组(P<0.05或<0.01)。与PAH组、Cav1组比较，Cav1F92A组肺组织ET-1、Myocardin蛋白相对表达量均降低(P均<0.01)。结论rBMSCs/Cav1F92A可减轻PAH大鼠的肺血管增殖性病变；通过上调Bbc3、Btg2基因表达，下调Ppard、Dab2、Rock1基因及ET-1、Myocardin蛋白表达而抑制血管平滑肌细胞增殖、纤维化及血管收缩可能是其作用机制。

肺动脉高压；窖蛋白1；内皮型一氧化氮合酶；一氧化氮；骨髓间充质干细胞；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

肺动脉高压(PAH)的标志性病理特征为肺外周小动脉内膜增厚、纤维化及中膜增厚、外膜增生等血管增殖性病变[1]。在PAH发生、发展的病理过程中，肺动脉血管内皮细胞最先受到攻击，并发生病理变化[2]，内皮功能障碍导致一氧化氮合酶(eNOS)催化生成的扩血管物质一氧化氮(NO)合成减少[3]。窖蛋白1(Cav1)是各种信号的调节中枢，是一氧化氮合酶(eNOS)的负调控子，可降低NO的合成。前期研究显示，采用丙氨酸替代92位苯丙氨酸可获得突变型Cav1(Cav1F92A)，其不具有抑制eNOS活性的作用，但可促进NO生成，降低肺动脉压力，对多条信号通路具有调节作用[4,5]。2015年6月～2016年10月，本研究采用Cav1F92A修饰大鼠骨髓间充质干细胞(rBMSCs)，观察其对PAH大鼠肺血管增殖性病变的影响，并探讨其可能的机制。

1 材料与方法

1.1 材料 ①实验动物：成年雄性Wistar大鼠40只，4～6周龄，体质量100～120 g，购自山东大学实验动物中心。将大鼠分笼饲养，自由摄食摄水，昼夜时间比为1∶1。②质粒：突变的Cav1质粒12AAPFVP-F92A-pMA-T、慢病毒包装质粒(psPax2、pRSV Rev、VSV-G)由澳大利亚Charles Sturt University的Padraig Strappe教授馈赠；慢病毒载体骨架pLVX-mCMV-zsgreen购自深圳市百恩维生物科技有限公司。③细胞：rBMSCs由聊城市人民医院中心实验室分离、培养、鉴定[5]；④试剂：PrimeScript®RT试剂盒及 SYBR Premix Ex TaqTMⅡ试剂盒均购自日本Takara公司，anti-内皮素1(ET-1)鼠单克隆抗体购自美国Abcam公司，anti-平滑肌肌球蛋白重链(Myocardin)兔多克隆抗体购自美国Santa Cruz公司。

1.2 慢病毒质粒构建、包装及转导 参照前期研究[6]的方法构建质粒LV-Cav1、LV-Cav1F92A，参照Chen等[4]的方法进行慢病毒质粒包装。rBMSCs培养融合至50%～70%时进行慢病毒转导，分别记为rBMSCs/Cav1、rBMSCs/Cav1F92A。

1.3 PAH大鼠模型建立及分组处理 将40只Wistar大鼠随机分为Cav1组、Cav1F92A组、PAH组及正常对照组，每组10只。Cav1组、Cav1F92A组、PAH组均给予1%野百合碱60 mg/kg腹腔注射建立PAH模型，正常对照组腹腔注射等体积生理盐水。参照前期方法，注射后2周采用MP150 Systems测量肺动脉压力，肺动脉收缩压均>30 mmHg，证实PAH模型建立成功[4]。建模后2周，Cav1组、Cav1F92A组分别于尾静脉移植rBMSCs/Cav1、rBMSCs/Cav1F92A各1 mL(1×106个/mL)，PAH组不予处理。各组移植后3周，腹腔注射4 000 mg/kg水合氯醛进行处死，分离左、右侧肺组织。

1.4 肺血管增殖情况观察 取左下部分肺组织，10%甲醛固定24 h，常规脱水、石蜡包埋，5 μm厚度切片。常规HE染色，40倍显微镜下观察肺血管管腔及血管壁厚度改变等。

1.5 肺组织血管增殖性病变相关基因mRNA表达检测 采用实时荧光定量PCR法检测肺组织Bbc3、B细胞易位2(Btg2)、Dab2、过氧化物酶体增生物激活受体(Ppard)、Rock1、富亮氨酸alpha-2糖蛋白1(Lrg1)基因表达。取新鲜肺组织，采用gentleMACSTM Dissociator进行匀浆，TRIzol试剂提取总RNA，PrimeScript®试剂盒进行RNA反转录，合成cDNA，并去除基因组DNA。PCR引物由上海生工生物工程股份有限公司合成，以GAPDH为管家基因，参照AceQ qPCR SYBR Green Master Mix说明进行PCR扩增。PCR反应体系共20 μL。PCR反应条件：95 ℃预变性 5 min，95 ℃变性10 s，57 ℃退火30 s，扩增40个循环；72 ℃延伸10 min。收集SYBR Green荧光信号，采用ABI 7500检测获取各组Ct值，ΔCt=Ct目的基因-Ct管家基因，ΔΔCt=ΔCt实验组-ΔCt正常对照组，采用2-ΔΔCt法计算各目的基因mRNA相对表达量。每组设置3个复孔，实验重复3次。

1.6 肺组织ET-1、Myocardin蛋白表达检测 采用Western blotting法。采用gentleMACSTM全自动组织处理器将各组大鼠肺组织进行匀浆，加预冷的蛋白裂解液及蛋白酶抑制剂PMSF，冰上裂解30 min；12 000 r/min离心10 min，收集上清煮沸5 min备用。每孔上样15 μg蛋白质，进行10% SDS-PAGE电泳、转膜，5%脱脂奶粉室温封闭1 h。分别加入anti-ET-1(1∶1 000)鼠单克隆抗体和anti-Myocardin(1∶1 000)的兔多克隆抗体，4 ℃孵育过夜；PBS洗涤，加入相应二抗室温孵育1 h。ECL试剂显色，采用AlphaView图象分析系统分析目的条带灰度值。

2 结果

2.1 各组肺血管增殖情况比较 与正常对照组比较，PAH组肺血管管腔显著狭窄，几乎闭锁，血管壁明显增生肥厚。Cav1组、Cav1F92A组较PAH组肺血管壁增厚程度减轻，以Cav1F92A组减轻更明显，但肺血管壁仍较正常对照组增厚。见插页Ⅰ图2。

2.2 各组肺组织血管增殖性病变相关基因mRNA表达比较 见表1。

表1 各组肺组织血管增殖性病变相关基因mRNA表达比较(相对表达量，

注：与正常对照组比较，*P<0.01，#P<0.05；与PAH组比较，△P<0.05，▽P<0.01；与Cav1组比较，▲P<0.05，▼P<0.01。

2.3 各组肺组织ET-1、Myocardin蛋白表达比较 见表2。

表2 各组肺组织ET-1、Myocardin蛋白表达比较(灰度值

注：与正常对照组比较，*P<0.01，#P<0.05；与PAH组比较，△P<0.05；与Cav1组比较，▽P<0.05。

3 讨论

血管平滑肌细胞和内皮细胞在PAH的发生与发展中扮演重要角色。血管平滑肌细胞大量增殖可引起血管壁增厚、管腔压力增大，最终导致肺动脉压力不断升高，而血管内皮细胞损伤是PAH各种病理变化的基础。目前，靶向内皮功能障碍的治疗药物如依前列醇及其衍生物、内皮素受体拮抗剂和磷酸二酯酶5抑制剂等，均可改善PAH患者的临床症状及血流动力学指标，但仍无法治愈PAH[7,8]，因此临床上迫切需要寻找有效治疗PAH的措施。NO主要由血管内皮生成，具有良好的抗炎、抗血小板和血管舒张作用[9]。Cav1主要通过位于92位的苯丙氨酸与eNOS结合发挥负调控作用，进而可降低NO的合成。采用丙氨酸替代92位苯丙氨酸可获得Cav1F92A，不具有抑制eNOS活性的作用，但可以促进NO的产生，从而舒张血管、降低肺动脉压力。本研究结果显示，与正常组比较，PAH组肺血管管腔显著狭窄，几乎闭锁，血管壁明显增生肥厚；Cav1组肺血管壁肥厚较PAH组减轻，可能与移植的rBMSC有关，其管腔仍较Cav1F92A组狭窄；Cav1F92A组管腔较大，血管壁较正常组增厚，但较PAH组和Cav1组明显减轻。说明rBMSCs/Cav1、rBMSCs/Cav1F92A均可减轻PAH大鼠的血管增殖性病变，其中rBMSCs/Cav1F92A的效果更好，可能与其对eNOS无负调控作用有关。

Bbc3与Btg2均为p53信号通路的下游靶基因，Bbc3可诱导细胞周期阻滞，介导细胞凋亡[10]。Btg2是抗增殖相关Btg/Tob基因的家族成员之一，是p53信号通路下游的细胞死亡诱导因子，可通过下调cyclin D1、cyclin E及基质金属蛋白酶9(MMP-9)的表达而抑制细胞增殖、迁移和侵袭[11,12]。Ppard与Dab2均属于Wnt信号通路的调控分子。Ppard在高血压患者中表达升高，上调Ppard表达可促进细胞增殖[13]。Dab2的功能具有细胞特异性，可介导纤维化，其与成纤维细胞生长因子2(FGF2)、Cav1共同作用可促进成纤维细胞的迁移[14]，正向调控转化生长因子β-R(TGF-β-R)通路，促进纤连蛋白表达及细胞黏附、迁移[15]。本研究结果显示，PAH组肺组织Bbc3、Btg2 mRNA相对表达量明显低于正常组，Dab2、Ppard mRNA相对表达量明显高于正常组，而rBMSCs/Cav1F92A移植后上述指标变化发生逆转。说明rBMSCs/Cav1F92A可激活PAH大鼠的p53信号通路、抑制Wnt信号通路，从而抑制细胞增殖及迁移，可能是其减轻PAH大鼠的血管增殖性病变的相关机制之一。

PAH血管病变的主要特点是纤维化，常发生于血管平滑肌细胞。Lrg1是一种新型血管生成性糖蛋白，通过调节内皮细胞TGF-β信号通路促进血管形成，而TGF-β信号通路失调又常伴有纤维化、异常血管生成，并加速心力衰竭进展[16]。研究显示，血清Lrg1水平是诊断特发性PAH的生物标志物之一[17]。ET-1是目前已知作用最强的缩血管活性物质，可促进平滑肌细胞增殖、迁移而引起血管重构。ET-1可激活Rock1，导致肺动脉血管内皮细胞的血管壁增厚，激活的Rock1也可与Ppard相互作用，促进血管平滑肌细胞增殖，导致血管重塑；Rock1还可上调与炎症、血栓形成及纤维化相关的分子，参与细胞黏附、迁移、增殖及凋亡等，在血管痉挛、高血压、肺动脉高压及心力衰竭等疾病的发生、发展中发挥重要作用[17]。Myocardin是维持平滑肌细胞收缩状态的主要辅助因子，可促进血管内皮细胞向平滑肌细胞的转化[7]。本研究结果显示，PAH组Lrg1、Rock1 mRNA及ET-1、Myocardin蛋白表达均明显高于正常对照组，而rBMSCs/Cav1F92A移植可有效降低PAH大鼠肺组织中上述基因或蛋白表达，表明rBMSCs/Cav1F92A可抑制PAH大鼠肺血管平滑肌细胞增殖、迁移及纤维化。

综上所述，rBMSCs/Cav1F92A可减轻PAH大鼠的血管增殖性病变，其机制可能是通过上调Bbc3、Btg2表达，下调Ppard、Dab2、Rock1基因及ET-1、Myocardin蛋白表达，进而抑制血管平滑肌细胞增殖、纤维化及血管收缩。

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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)

国家自然科学基金资助项目(81270104)；山东省自然科学基金资助项目(ZR2016HP33)。

杨红莉(1987-)，女，技师，研究方向为药理学。E-mail: yanghongli2011slky@163.com

陈海英(1969-)，女，副主任技师，研究方向为药理学。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)