刘蕾,马德宾,史亮,孙文武
LIU Lei,MA Debin,SHI Liang,SUN Wenwu

• 1:中国人民解放军北部战区总医院呼吸内科

摘要(Abstract)：

目的探讨Shh/Twist1对低氧诱导的大鼠肺动脉内皮细胞间质转化及血管重构的影响及机制。方法将原代分离的大鼠肺动脉内皮细胞分为正常对照组(不予低氧处理)和低氧诱导的肺动脉高压(PAH)细胞模型(3%O_2低氧处理3 d),PAH细胞模型又分为模型组、Shh抑制组(造模前采用Shh抑制剂预处理细胞)及siRNA-Twist1组(造模前采用siRNA-Twist1预处理细胞);于3%O2培养细胞3 d时,采用划痕实验检测细胞的迁移能力,小管形成实验检测细胞的成管能力,Real-time PCR法检测细胞的CD31、vWF、α-SMA mRNA的表达,Western blot法检测细胞的Shh、Twist1、血管内皮生长因子(VEGF)和基质金属蛋白酶2(MMP2)蛋白的表达。结果与模型组比较,Shh抑制组和siRNA-Twist1组的迁移和成管能力均降低,差异有统计学意义(P<0.05);Real-time PCR及Western blot结果显示,与模型组比较,Shh抑制组和siRNA-Twist1组α-SMA mRNA及VEGF、MMP2蛋白的表达水平降低,CD31、vWF mRNA的表达升高,差异有统计学意义(P<0.05)。结论抑制Shh/Twist1表达能显著降低大鼠肺动脉内皮细胞的内皮间质转化及血管重构能力。
Objective To investigate the effect and mechanism of Shh/Twist1 on hypoxia-induced pulmonary artery endothelial cell mesenchymal transition and vascular remodeling.Methods Primary rat pulmonary artery endothelial cells were isolated, Shh inhibitor( Cyclopamine) or siRNA-Twist1 were used for intervention, then hypoxia-induced pulmonary( PAH) cell model was established.Wound Healing detected cell migration ability, tubule formation experiment detected cell tube formation ability, Real-time PCR detected CD31,vWF,α-SMA mRNA expression, Western blot detected Shh, Twist1, VEGF, MMP2 protein expression.Results Compared with the model group,Cyclopamine and siRNA-Twist1 treatment reduced cell migration and tube formation ability(P<0. 05). Real-time PCR and Western blot results showed that the inhibition of the expression of Shh and Twist1 were significantly reduced the expression of α-SMA mRNA and VEGF, MMP2 protein, and upregulate the expression of CD31 and vWF mRNA were significantly reduced(P< 0. 05).Conclusion Inhibiting the expression of Shh/Twist1 can significantly reduce the endothelial-mesenchymal transition and vascular remodeling of pulmonary artery endothelial cells.

关键词(KeyWords)： 肺动脉高压;肺动脉内皮细胞;缺氧;内皮间质转化;血管重构;Shh;Twist1
pulmonary arterial hypertension(PAH);pulmonary artery endothelial cells;hypoxia;endothelial-to-mesenchymal transition(EndMT);vascular remodeling;sonic hedgehog(Shh);Twist1

Abstract:

Keywords:

基金项目(Foundation): 辽宁省自然科学基金指导计划项目（20180550385）

作者(Author): 刘蕾,马德宾,史亮,孙文武
LIU Lei,MA Debin,SHI Liang,SUN Wenwu

DOI: 10.19367/j.cnki.2096-8388.2022.01.008

参考文献(References)：

• [1]莫连芹，黄栋，王予川，等．血浆脑钠肽及血尿酸在评估儿童肺动脉高压中的价值[J]．贵州医科大学学报，2018, 43(3):320-323.
• [2]牛志浩，刘庆，刘俊峰，等．间充质干细胞条件培养基对炎症介导的肺动脉血管平滑肌细胞增殖调控的研究[J]．贵州医科大学学报，2018, 43(6):641-645.
• [3] ZHANG H, LIN Y, MA Y, et al. Protective effect of hydrogen sulfide on monocrotaline-induced pulmonaryarterial hypertension via inhibition of the endothelial mesenchymal transition[J]. International Journal of Molecular Medicine, 2019, 44(6):2091-2102.
• [4]张家豪．基质金属蛋白酶2和9在角膜新生血管调节机制中的作用研究进展[J]．中华实验眼科杂志，2020, 38(10):900-904.
• [5] GHOULEH I A, SAHOO S, MEIJLES D N, et al. Endothelial nox1 oxidase assembly in human pulmonary arterial hypertension; driver of gremlin1-mediated proliferation[J]. Clinical Science, 2017,131(15):2019-2035.
• [6] LI Z, CHEN B, DONG W, et al. MKL1 promotes endothelial-to-mesenchymal transition and liver fibrosis by activating TWIST1 transcription[J]. Cell Death&Disease,2019, 10(12):899.
• [7] SUZUKI T, CARRIER E J, TALATI M A, et al. Isolation and characterization of endothelial-to-mesenchymal transition-cells in pulmonary arterial hypertension[J]. American Journal of Physiology Lung Cellular&Molecular Physiology, 2017,314(1):118-126.
• [8] ADESINA S E, WADE B E, BIJLI K M, et al. Hypoxia inhibits expression and function of mitochondrial thioredoxin 2 to promote pulmonary hypertension[J]. Am J Physiol Lung Cell Mol Physiol, 2017, 312(5):599-608.
• [9] DAVID C, IRWIN,NOZIKGRAYCK E et al. A potential role for reactive oxygen species and the HIF-1α–VEGF pathway in hypoxia-induced pulmonary vascular leak[J].Free Radical Biology&Medicine, 2009,47(1):55-61.
• [10]RAMU B, HOUSTON B A, TEDFORD R J. Pulmonary vascular disease:hemodynamic assessment and treatment selection-focus on group II pulmonary hypertension[J].Current Heart Failure Reports, 2018,15(2):81-93.
• [11]唐铭．双氢青蒿素通过抑制肺血管重构调控肺动脉高压的分子机制研究[D]．重庆：重庆医科大学，2020.
• [12]张卉，蔺艳军，毛承誉，等．硫化氢可通过改善肺动脉内皮间质转化减轻野百合碱诱导的大鼠肺动脉高压[J]．中国临床医学，2019,26(5):721-726.
• [13]邱浚，周建庆．内皮-间质转化与心血管疾病的研究进展[J]．浙江医学，2021, 43(5):559-562.
• [14]FANG D, KITAMURA H. Cancer stem cells and epithelial-mesenchymal transition in urothelial carcinoma:possible pathways and potential therapeutic approaches[J].International Journal of Urology, 2018,25(1):7-17.
• [15]郑小芳，胡慧佳，李跃，等．川芎嗪减轻博莱霉素诱导的大鼠肺纤维化的作用机制研究[J]．浙江医学，2019, 41(14):1468-1472.
• [16] FATEHIAGDAM M, VATANKHAH M A, PANAHIZADEH R, et al. Efficacy of metformin and chemotherapeutic agents on the inhibition of colony formation and Shh/Gli1 pathway:metformin/docetaxel versus metformin/5-fluorouracil[J]. Drug Research, 2020, 71(11):17-25.
• [17]MAMMOTO T, MUYLEART M, KONDURI G G, et al.Twist1 in hypoxia-induced pulmonary hypertension through transforming growth factor-β-smad signaling[J].American Journal of Respiratory Cell&Molecular Biology, 2018, 58(2):194.
• [18]ZHANG B O, NIU W, DONG H Y, et al. Hypoxia induces endothelial-mesenchymal transition in pulmonary vascular remodeling[J]. International Journal of Molecular Medicine, 2018, 42(1):270-278.
• [19]LIU J, WANG W, WANG L, et al. IL-33 Initiates vascular remodelling in hypoxic pulmonary hypertension by up-regulating HIF-1αand VEGF expression in vascular endothelial cells[J]. Ebiomedicine, 2018, 33:196-210.
• [20]ZOU J, LIU K C, WANG W P, et al. Circular RNA COL1A2 promotes angiogenesis via regulating miR-29b/VEGF axis in diabetic retinopathy[J]. Life Sciences,2020, 256:117888.
• [21]赵小亚，江如钞，肖青．MMP2介导Sonic hedgehog信号通路对糖尿病内皮祖细胞功能的影响[J]．广州医科大学学报，2021, 49(1):1-6.
• [22]赵海燕，王晓非．miR-181a-5p对缺氧诱导的大鼠肺动脉高压的影响[J]．中国医科大学学报，2020, 49(2):129-133.
• [23]LU J L, DONG, FAN W J. Twist1/2 activates MMP2expression via binding to its promoter in colorectal cancer[J]. European Review for Medical&Pharmacological Sciences, 2018,22(23):8210-8219.
• [24] NEDJADI T, SALEM N, KHAYYAT D, et al. Sonic hedgehog expression is associated with lymph node invasion in urothelial bladder cancer[J]. Pathology&Oncology Research, 2019, 25(3):1067-1073.
• [25] SHI S, SUN J, MENG Q, et al. Sonic hedgehog promotes endothelial differentiation of bone marrow mesenchymal stem cells via VEGF-D[J]. Journal of Thoracic Disease, 2018, 10(9):5476-5488.
• [26]KOO Y J, KIM T J, MIN K J, et al. CXCL11 mediates TWIST1-Induced angiogenesis in epithelial ovarian cancer[J]. Tumor Biology, 2017, 39(5):101042831770622.

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