三甲氧基二苯乙烯的合成及其致肺动脉平滑肌细胞凋亡的研究
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摘要
目的：①对以虎杖为原料提取的ReS,再经甲基化反应合成的TMS进行结构鉴定和药代动力学测定。②检测TMS对PASMCs生长抑制和致凋亡作用。
方法：①用紫外吸收光谱检测、红外吸收光谱检测、1H-NMR谱检测、13C-NMR图谱检测、质谱检测等方法对TMS进行结构鉴定。②经大鼠测定TMS口服后的生物利用度,肠道吸收特点,体内组织分布和体内排泄。③经小鼠测定TMS的急性毒性。④利用大鼠肺动脉制备PASMCs。⑤用MTT法测定TMS对PASMCs增殖的影响。⑥流式Annexin V/PI法测定TMS对PASMCs致凋亡的影响。⑦用免疫组化方法检测TMS干预PASMCs对Bax、Bcl-2表达的影响。⑧用原位杂交法检测TMS干预PASMCs对bax、bcl-2mRNA;表达的影响。
结果：①TMS的紫外吸收图谱为λmax(MeOH):318nm,306.2nm,217.8nm,红外光谱解析为1):2999,2935,2836,1591,1511,1456,'H-NMR谱解析显示结构含有3个甲氧基,13C-NMR图谱解析为17个碳信号,并且其结构中含有对称的结构片段。质谱图谱解析为ESI-MS m/z:271[M+H]+,256[M+H-CH3]+,241[256-CH3]+,表明分子量为270,推测分子式为C17H18O3,结构中含有甲基。样品元素分析得分子结构式可用C17H18O3表示,结构式为因此可确定化合物为反-3,4',5-三甲氧基二苯乙烯。②TMS绝对生物利用度为45.4%,在小肠上段有较好吸收,通过粪便、胆汁排泄,在各组织中均有分布,MTD约5.85g/kg。③TMS干预PASMCs24h后经MTT法测定显示对其生长具有显著的抑制作用,其强度呈剂量依赖性改变,较同剂量的Res作用高10多倍。A、B、C、D、E、F、G、H组的生长抑制率(%)分别为4.07±2.12,6.54±4.78,23.74±7.07,16.75±5.34,9.35±4.26,17.81+6.03,8.81士5.16,6.78±5.58。④TMS干预PASMCs24h后经流式Annexin V/PI法检测显示对其细胞凋亡百分率(%)显著高于TNF-a干预组,其作用程度呈剂量依赖型改变,并且较Res高10多倍。A、B、C、D、E、F、G、H组的细胞凋亡百分率(%)分别为2.63+0.74,3.54±0.81,18.79±4.15,11.68±5.35,5.77±4.62,12.47±5.06,6.33+4.8,4.11+3.59。⑤TMS干预PASMCs24h后经免疫组化法检测显示其对细胞表达Bax阳性的百分率呈剂量依赖性升高,而Bcl-2阳性细胞百分率呈剂量依赖性减少,其改变较Res提高10多倍。A、B、C、D、E、F、G、H各组Bcl-2/Bax比值分别为0.99±0.24,1.07±0.22,0.74±0.37,0.84±0.41,0.92±0.32,0.86±0.45,1.02±0.42,1.12±0.51。⑥TMS干预PASMCs24h后经原位杂交法检测其对细胞呈现bcl-2mRNA阳性的百分率呈剂量依赖性减少,而bax mRNA阳性的百分率呈剂量依赖性升高,其改变较Res提高10多倍。A、B、C、D、E、F、G、H各组bcl-2mRNA/bax mRNA的比值分别为1.00±0.14,1.09±0.11,0.90±0.43,0.97±0.44,1.05±0.50,0.95±0.42,1.03±0.56,1.09±0.53。
结论：①TMS的分子结构式可用C17H1803表示,结构式为,是反-3,4',5-三甲氧基二苯乙烯。②TMS的生物利用度为45%。③TMS干预PASMCs,抑制细胞增殖,作用比Res强10多倍并且呈剂量依赖性；致凋亡作用比Res强20倍。TMS对PASMCs生长具有显著的抑制作用,其强度呈剂量依赖性改变,较同剂量的Res作用高10多倍。④TMS通过下调Bcl-2的产生和上调Bax致凋亡。
OBJECTIVE:To synthesize3,5,4'-trimethoxystilbene (TMS) by methylation of resveratrol (Res), a natural compound extracted from polygonum cuspidatum, to identify the chemical structure of TMS, to test its pharmacokinetics, and to determine the effects of TMS on the growth inhibition and apoptosis in pulmonary artery smooth muscle cells (PASMCs).
METHODS:The chemical structure of TMS was analyzed by UV-and IR-absorption spectrometry,1H-NMR and13C-NMR spectroscopy and mass spectrometry. We measured the bioavailability, the characteristics of intestinal absorption, and the distribution of TMS in body and excretions of SD rats after oral administration of TMS. Th acute toxicity of TMS in mice was tested. PASMCs were prepared from pulmonary artery of SD rats. The proliferation of PASMCs after treatment was determined by MTT assay. The apoptosis of PASMCs after treatment was determined by flow cytometry. The intervention of TMS on protein expression of Bax and Bcl-2in PASMCs was detected immunohistochemically. The intervention of TMS on in mRNA expression of bax and bcl-2in PASMCs after treatment was analyzed by in situ hybridization assay.
RESULTS:The UV absorption map of TMS showed λmax(MeOH) at318,306.2, and217.8nm. Analysis of infrared spectrum of TMS showed IRvmaxKBr/cm at2999,2935,2836,1591,1511and1456/cm. The1H-NMR map showed that the synthetic product contained three hydroxy groups, while13C-NMR map showed17carbon signals and some symmetrical structural fragments. Electospray ionization mass spectrometry of the product showed m/z peaks corresponded to271[M+H]+,256[M+H-CH3]+and241[256-CH3]+; the implied relative molecular weight is270and the implied molecular formula is C17H18O3. These data confirm the product is3,5,4'-trimethoxystilbene. The absolute bioavailability of TMS was45.4%. TMS was well absorbed in the upper small intestine; it was excreted in stool and bile and distributed into several tissues. The maximal tolerance dose (MTD) of TMS was5.85g/kg. MTT assay showed TMS inhibited the proliferation of PASMCs in a dose-dependent manner. The extent of growth inhibition in A-H groups were (4.07±2.12)%,(6.54±4.78)%,(23.74±7.07)%,(16.75±5.34)%,(9.35±4.26)%,(17.81±6.03)%,(8.81±5.16)%and (6.78±5.58)%respectively. Flow cytometry showed the extent of apoptosis in PASMCs (after being treated with TMS for24h) was significantly higher than that in PASMCs treated only with TNF-a. The apoptosis rates of A-H groups were (2.63±0.74)%,(3.54±0.81)%,(18.79±4.15)%,(11.68±5.35)%,(5.77±4.62)%,(12.47±5.06)%,(6.33±4.8)%and (4.11±3.59)%respect ively. Immunohistochemistry assay showed the protein expression of Baxin PASMCs (after being treated with TMS for24h) increased while the protien expression of Bcl-2decreased. The effect stimulated by TMS was10times stronger than that by Res. The ratio ofBcl-2/Bax of A-H group were(0.99±0.24)%,(1.07±0.22)%,(0.74±0.37)%,(0.84±0.41)%,(0.92±0.32)%,(0.86±0.45)%,(1.02±0.42)%and (1.12±0.51)%respectively. In situ hybridization technic showed the mRNA expression of bax in PASMCs (after being treated with TMS for24h) increased while the mRNA expression of bcl-2mRNA decreased in a dose-dependent manner. Compared with Res, the effect stimulated by TMS increased10times. The ratio of bcl-2/bax of A-H group were (1.00±0.14)%,(1.09±0.11)%,(0.90±0.43)%,(0.97±0.44)%,(1.05±0.50)%,(0.95±0.42)%,(1.03±0.56)%and (1.09±0.53)%respectively.
CONCLUSION:We have confirmed our synthetic product as3,5,4'-trimethoxystilbene (TMS),with the molecular formula of C17H18O3and appropriate molecular weight and absorption and NMR spectra. The bioavailability of TMS was to45%. TMS strongly inhibited the proliferation of PASMCs in a dose-dependent manner and the effect of inhibition of TMS was10times stronger than that of Res. Apoptosis of PASMCs could be induced by TMS, extent of which was10-fold of that by Res. TMS induced apoptosis of PASMCs by down-regulation of Bcl-2expression and up-regulation of Bax expression.
方法：①用紫外吸收光谱检测、红外吸收光谱检测、1H-NMR谱检测、13C-NMR图谱检测、质谱检测等方法对TMS进行结构鉴定。②经大鼠测定TMS口服后的生物利用度,肠道吸收特点,体内组织分布和体内排泄。③经小鼠测定TMS的急性毒性。④利用大鼠肺动脉制备PASMCs。⑤用MTT法测定TMS对PASMCs增殖的影响。⑥流式Annexin V/PI法测定TMS对PASMCs致凋亡的影响。⑦用免疫组化方法检测TMS干预PASMCs对Bax、Bcl-2表达的影响。⑧用原位杂交法检测TMS干预PASMCs对bax、bcl-2mRNA;表达的影响。
结果：①TMS的紫外吸收图谱为λmax(MeOH):318nm,306.2nm,217.8nm,红外光谱解析为1):2999,2935,2836,1591,1511,1456,'H-NMR谱解析显示结构含有3个甲氧基,13C-NMR图谱解析为17个碳信号,并且其结构中含有对称的结构片段。质谱图谱解析为ESI-MS m/z:271[M+H]+,256[M+H-CH3]+,241[256-CH3]+,表明分子量为270,推测分子式为C17H18O3,结构中含有甲基。样品元素分析得分子结构式可用C17H18O3表示,结构式为因此可确定化合物为反-3,4',5-三甲氧基二苯乙烯。②TMS绝对生物利用度为45.4%,在小肠上段有较好吸收,通过粪便、胆汁排泄,在各组织中均有分布,MTD约5.85g/kg。③TMS干预PASMCs24h后经MTT法测定显示对其生长具有显著的抑制作用,其强度呈剂量依赖性改变,较同剂量的Res作用高10多倍。A、B、C、D、E、F、G、H组的生长抑制率(%)分别为4.07±2.12,6.54±4.78,23.74±7.07,16.75±5.34,9.35±4.26,17.81+6.03,8.81士5.16,6.78±5.58。④TMS干预PASMCs24h后经流式Annexin V/PI法检测显示对其细胞凋亡百分率(%)显著高于TNF-a干预组,其作用程度呈剂量依赖型改变,并且较Res高10多倍。A、B、C、D、E、F、G、H组的细胞凋亡百分率(%)分别为2.63+0.74,3.54±0.81,18.79±4.15,11.68±5.35,5.77±4.62,12.47±5.06,6.33+4.8,4.11+3.59。⑤TMS干预PASMCs24h后经免疫组化法检测显示其对细胞表达Bax阳性的百分率呈剂量依赖性升高,而Bcl-2阳性细胞百分率呈剂量依赖性减少,其改变较Res提高10多倍。A、B、C、D、E、F、G、H各组Bcl-2/Bax比值分别为0.99±0.24,1.07±0.22,0.74±0.37,0.84±0.41,0.92±0.32,0.86±0.45,1.02±0.42,1.12±0.51。⑥TMS干预PASMCs24h后经原位杂交法检测其对细胞呈现bcl-2mRNA阳性的百分率呈剂量依赖性减少,而bax mRNA阳性的百分率呈剂量依赖性升高,其改变较Res提高10多倍。A、B、C、D、E、F、G、H各组bcl-2mRNA/bax mRNA的比值分别为1.00±0.14,1.09±0.11,0.90±0.43,0.97±0.44,1.05±0.50,0.95±0.42,1.03±0.56,1.09±0.53。
结论：①TMS的分子结构式可用C17H1803表示,结构式为,是反-3,4',5-三甲氧基二苯乙烯。②TMS的生物利用度为45%。③TMS干预PASMCs,抑制细胞增殖,作用比Res强10多倍并且呈剂量依赖性；致凋亡作用比Res强20倍。TMS对PASMCs生长具有显著的抑制作用,其强度呈剂量依赖性改变,较同剂量的Res作用高10多倍。④TMS通过下调Bcl-2的产生和上调Bax致凋亡。
OBJECTIVE:To synthesize3,5,4'-trimethoxystilbene (TMS) by methylation of resveratrol (Res), a natural compound extracted from polygonum cuspidatum, to identify the chemical structure of TMS, to test its pharmacokinetics, and to determine the effects of TMS on the growth inhibition and apoptosis in pulmonary artery smooth muscle cells (PASMCs).
METHODS:The chemical structure of TMS was analyzed by UV-and IR-absorption spectrometry,1H-NMR and13C-NMR spectroscopy and mass spectrometry. We measured the bioavailability, the characteristics of intestinal absorption, and the distribution of TMS in body and excretions of SD rats after oral administration of TMS. Th acute toxicity of TMS in mice was tested. PASMCs were prepared from pulmonary artery of SD rats. The proliferation of PASMCs after treatment was determined by MTT assay. The apoptosis of PASMCs after treatment was determined by flow cytometry. The intervention of TMS on protein expression of Bax and Bcl-2in PASMCs was detected immunohistochemically. The intervention of TMS on in mRNA expression of bax and bcl-2in PASMCs after treatment was analyzed by in situ hybridization assay.
RESULTS:The UV absorption map of TMS showed λmax(MeOH) at318,306.2, and217.8nm. Analysis of infrared spectrum of TMS showed IRvmaxKBr/cm at2999,2935,2836,1591,1511and1456/cm. The1H-NMR map showed that the synthetic product contained three hydroxy groups, while13C-NMR map showed17carbon signals and some symmetrical structural fragments. Electospray ionization mass spectrometry of the product showed m/z peaks corresponded to271[M+H]+,256[M+H-CH3]+and241[256-CH3]+; the implied relative molecular weight is270and the implied molecular formula is C17H18O3. These data confirm the product is3,5,4'-trimethoxystilbene. The absolute bioavailability of TMS was45.4%. TMS was well absorbed in the upper small intestine; it was excreted in stool and bile and distributed into several tissues. The maximal tolerance dose (MTD) of TMS was5.85g/kg. MTT assay showed TMS inhibited the proliferation of PASMCs in a dose-dependent manner. The extent of growth inhibition in A-H groups were (4.07±2.12)%,(6.54±4.78)%,(23.74±7.07)%,(16.75±5.34)%,(9.35±4.26)%,(17.81±6.03)%,(8.81±5.16)%and (6.78±5.58)%respectively. Flow cytometry showed the extent of apoptosis in PASMCs (after being treated with TMS for24h) was significantly higher than that in PASMCs treated only with TNF-a. The apoptosis rates of A-H groups were (2.63±0.74)%,(3.54±0.81)%,(18.79±4.15)%,(11.68±5.35)%,(5.77±4.62)%,(12.47±5.06)%,(6.33±4.8)%and (4.11±3.59)%respect ively. Immunohistochemistry assay showed the protein expression of Baxin PASMCs (after being treated with TMS for24h) increased while the protien expression of Bcl-2decreased. The effect stimulated by TMS was10times stronger than that by Res. The ratio ofBcl-2/Bax of A-H group were(0.99±0.24)%,(1.07±0.22)%,(0.74±0.37)%,(0.84±0.41)%,(0.92±0.32)%,(0.86±0.45)%,(1.02±0.42)%and (1.12±0.51)%respectively. In situ hybridization technic showed the mRNA expression of bax in PASMCs (after being treated with TMS for24h) increased while the mRNA expression of bcl-2mRNA decreased in a dose-dependent manner. Compared with Res, the effect stimulated by TMS increased10times. The ratio of bcl-2/bax of A-H group were (1.00±0.14)%,(1.09±0.11)%,(0.90±0.43)%,(0.97±0.44)%,(1.05±0.50)%,(0.95±0.42)%,(1.03±0.56)%and (1.09±0.53)%respectively.
CONCLUSION:We have confirmed our synthetic product as3,5,4'-trimethoxystilbene (TMS),with the molecular formula of C17H18O3and appropriate molecular weight and absorption and NMR spectra. The bioavailability of TMS was to45%. TMS strongly inhibited the proliferation of PASMCs in a dose-dependent manner and the effect of inhibition of TMS was10times stronger than that of Res. Apoptosis of PASMCs could be induced by TMS, extent of which was10-fold of that by Res. TMS induced apoptosis of PASMCs by down-regulation of Bcl-2expression and up-regulation of Bax expression.
引文
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[1]Rubin LJ. Pulmonary Arterial Hypertension. The Proceedings of the American Thoracic Society,2006,3:111-115.
[2]HoeperMM, Rubin LJ. Update in Pulmonary Hypertension 2005. Am J Respir Crit CareMed,2006,173:4992-505.
[3]Galie N, Torbicki A. The task force on diagnosis and treatment of pulmonary arterial hypertension of the European society of cardiology.Guidelines on diagnosis and treatment of pulmonary arterial hypertension. European Heart Journal,2004,25:2243-2278.
[4]Si monneau G, Galie N, Rubin L J, et al. Clinical classification of pulmonary hypertensi on [J]. J Am Coll Cardiol,2004,43 (1):5S-12S.
[5]张烜,张奉春,董怡。结缔组织病中肺动脉高压临床特点分析。中华风湿病学杂志,1999,3：5-7.
[6]6柳志红,秦春常欧洲心脏病学会肺动脉高压指南解读中国实用内科杂志2007年第04期.
[7]Badesch DB, Abman SH, Ahearn GS. Medical therapy for pulmonary arterial hypertension:ACCP evidence-based clinical practice guidelines. Chest,2004,126(1 Suppl):35S-62S.
[8]Nazzareno Galie, Adam Torbicki. The task force on diagnosis and treatment of pulmonary arterial hypertension of the European society of cardiology. Guidelines on diagnosis and treatment of pulmonary arterial hypertension. European Heart Journal (2004), 25:2243-2278.
[9]Thomson J R,Machado RD,Pauciulo M, et al. Sporadic primary pul-monary hypertension is associated with germline mutations of the gene-encoding BMPR Ⅱ,a receptor of the TGF-B family[J]. J Med Genet,2000,37 (10):741-745.
[10]Morse J H,Deng Z,Knowles JA. Genetic aspects of pulmonary arterial hypertension[J]. Ann Med,2001,33 (9):596-603.
[11]陆立鹤,荆志成,邹玉宝,等.骨形成蛋白Ⅱ型受体基因R491W突变导致家族性原发性肺动脉高压[J].中华循环杂志,2002,17(5)：380-382.
[12]Cool CD, Stewart J S,Werahera P, et al. Three dimensional reconst ruction of pulmonary arteries in plexiform pulmonary hypertensionusing cellspecificarkers:evidence for a dynamic and heterogeneous process of pulmonary endot helial cell growt h[J]. Am J Pat hol,1999,155 (2):411-419.
[13]Trembath RC, Thomson J R,Machado RD,et al. Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic talangiectasia[J]. N Engl J Med,2001,345 (5):325-334.
[14]Eddahibi S,Humbert M,Fadel E,et al. Serotonint ransporter overexpression is responsible for pulmonary artery smoot h muscle cell hyperplasia in primary pulmonary hypertension[J]. J Clin Invest,2001,108 (8):1141-1150.
[15]Eddahibi S, Hanoun N,Lanfumey L, et al. At tenuated hypoxic pulmonary hypertension in mice lackingt he 54-hydroxyt ryptaminet ransporter gene[J]. J Clin Invest,2000,105 (11):1555-1562.
[16]Long L,MacLean MR,J effery TK,et al. Serotonin increases susceptibility to pulmonary hypertension in BMPR 2-deficient mice[J]. Circ Res,2006,98 (6):818-827.
[17]Williams LA, Egner W, Hart DN. Isolation and function of human dendritic cells. Int Rev Cytol,1994,153:41-103.
[18]Min WP, Gorczynski R, Huang XY, et al. Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival. J Immunol,2000,164:161-167.
[19]Jenkins MK, Chen CA, Jung G, et al. Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody. J Immunol,1990,144:16-22.
[20]Roncarolo MQ Levings MK, Traversari C. Differentiation of T regulatory cells by immature dendritic cells. J Exp Med,2001,193:5-9.
[21]Fairchild PJ, Waldmann H. Dendritic cells and prospects for transplantation tolerance. Curr Opin Immunol,2000,12:528-535.
[22]Vendetti S, Chai JG, Dyson J, et al. Anergic T cells inhibit the antigen-presenting function of dendritic cells. J Immunol,2000,165:1175-1181.
[23]. Rissoan MC, Soumelis V, Kadowaki N, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science,1999,283:1183-1186.
[24]Perros F, Dorfmuller P, Souza R, et al. Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension [J]. European Respiratory Journal, 2007,29:462-468.
Miyata M, Sakuma F, Youshimura A, et al. Pulmonary hypertension in rats:role of bromodeoxyuridine positive mononuclear cells and x lveolar macrophages。Int Arch Allery Immunol,1995,108:286
[26]Bartram U, Speer CP. The role of transforming growth factor beta in lung development and disease. Chest,2004,125:745-765.
[27]Lundblad LK* Thompson-Figueroa J, Leclair T, et al. Tumor necrosis factor-alpar overexpression in lung disease:a single cause behind a complex phenotype. AM J Respir Cnt Care Med,2005,171:1363-1370
[28]Sime PJ, Marr RA, Gauldie D, et al. Transfer of tumor necrosis factor-alpha to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming groeth factor-betal and myofibroblasts. AM J Pathol,1998,153:825-832.
[29]Iwasaki T, Takahashi T,Shimizu H, et al. Increased pulmonary hehe oxygenase-1 and delta-aminolevulinate synthase expression in monocrotaline-induced pulmonary hypertension. Curr Neurovasc Res,2005,2:133-139
[30]李梦涛,卢杰,王迁等,衣那西普在野百合碱诱导的大鼠肺动脉高压模型中作用的初步探讨。中华风湿病学杂志,2009,12(13)：804-807
[31]张晓,林莉,张光峰等。肿瘤坏死因子及转化生长因子在肺间质病变发病机制中的作用。中华风湿病学杂志,2010,1(14)：13-16
[32]Gauldie J, Bonniaud P, Sime P,et al. TGF-beta, Smad3 and the process of progressive fibrosis. Biochem Soc Trans,2007,35(Pt4):661-664
[33]Pantelidis P, McGrath DS, Southcott AM, et al. Tumour necrosis factor-beta production infibosing alveolitis is macrophage subset specific. Respir Res.2001,2:365-372
[34]Balabanian K, Foussat A, Dorfmuller P, et al. CX3C Chemokine Fractalkine in Pulmonary Arterial Hypertension [J].American Journal ofRespiratory and Critical Care Medicine,2002,165:1419-1425
[35]Kitaura H,UozumiN, TohmiM, et al. Roles of nitric oxide as a vas odilator in neurovascular coup ling ofinouse s omatosens ory cortex[J]. N eurosci Res,2007,59 (2): 160-171.
[36]Fisher K A, Serlin DM,Wils on KC, et al. Sarcoidosis-ass ociated pulmonary hypertensi on:Outcome with 1 ong-ter m epop r ostenol treatment[J].Chest,2006,130 (5):1481-1488
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[1]Rubin LJ. Pulmonary Arterial Hypertension. The Proceedings of the American Thoracic Society,2006,3:111-115.
[2]HoeperMM, Rubin LJ. Update in Pulmonary Hypertension 2005. Am J Respir Crit CareMed,2006,173:4992-505.
[3]Galie N, Torbicki A. The task force on diagnosis and treatment of pulmonary arterial hypertension of the European society of cardiology.Guidelines on diagnosis and treatment of pulmonary arterial hypertension. European Heart Journal,2004,25:2243-2278.
[4]Si monneau G, Galie N, Rubin L J, et al. Clinical classification of pulmonary hypertensi on [J]. J Am Coll Cardiol,2004,43 (1):5S-12S.
[5]张烜,张奉春,董怡。结缔组织病中肺动脉高压临床特点分析。中华风湿病学杂志,1999,3：5-7.
[6]6柳志红,秦春常欧洲心脏病学会肺动脉高压指南解读中国实用内科杂志2007年第04期.
[7]Badesch DB, Abman SH, Ahearn GS. Medical therapy for pulmonary arterial hypertension:ACCP evidence-based clinical practice guidelines. Chest,2004,126(1 Suppl):35S-62S.
[8]Nazzareno Galie, Adam Torbicki. The task force on diagnosis and treatment of pulmonary arterial hypertension of the European society of cardiology. Guidelines on diagnosis and treatment of pulmonary arterial hypertension. European Heart Journal (2004), 25:2243-2278.
[9]Thomson J R,Machado RD,Pauciulo M, et al. Sporadic primary pul-monary hypertension is associated with germline mutations of the gene-encoding BMPR Ⅱ,a receptor of the TGF-B family[J]. J Med Genet,2000,37 (10):741-745.
[10]Morse J H,Deng Z,Knowles JA. Genetic aspects of pulmonary arterial hypertension[J]. Ann Med,2001,33 (9):596-603.
[11]陆立鹤,荆志成,邹玉宝,等.骨形成蛋白Ⅱ型受体基因R491W突变导致家族性原发性肺动脉高压[J].中华循环杂志,2002,17(5)：380-382.
[12]Cool CD, Stewart J S,Werahera P, et al. Three dimensional reconst ruction of pulmonary arteries in plexiform pulmonary hypertensionusing cellspecificarkers:evidence for a dynamic and heterogeneous process of pulmonary endot helial cell growt h[J]. Am J Pat hol,1999,155 (2):411-419.
[13]Trembath RC, Thomson J R,Machado RD,et al. Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic talangiectasia[J]. N Engl J Med,2001,345 (5):325-334.
[14]Eddahibi S,Humbert M,Fadel E,et al. Serotonint ransporter overexpression is responsible for pulmonary artery smoot h muscle cell hyperplasia in primary pulmonary hypertension[J]. J Clin Invest,2001,108 (8):1141-1150.
[15]Eddahibi S, Hanoun N,Lanfumey L, et al. At tenuated hypoxic pulmonary hypertension in mice lackingt he 54-hydroxyt ryptaminet ransporter gene[J]. J Clin Invest,2000,105 (11):1555-1562.
[16]Long L,MacLean MR,J effery TK,et al. Serotonin increases susceptibility to pulmonary hypertension in BMPR 2-deficient mice[J]. Circ Res,2006,98 (6):818-827.
[17]Williams LA, Egner W, Hart DN. Isolation and function of human dendritic cells. Int Rev Cytol,1994,153:41-103.
[18]Min WP, Gorczynski R, Huang XY, et al. Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival. J Immunol,2000,164:161-167.
[19]Jenkins MK, Chen CA, Jung G, et al. Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody. J Immunol,1990,144:16-22.
[20]Roncarolo MQ Levings MK, Traversari C. Differentiation of T regulatory cells by immature dendritic cells. J Exp Med,2001,193:5-9.
[21]Fairchild PJ, Waldmann H. Dendritic cells and prospects for transplantation tolerance. Curr Opin Immunol,2000,12:528-535.
[22]Vendetti S, Chai JG, Dyson J, et al. Anergic T cells inhibit the antigen-presenting function of dendritic cells. J Immunol,2000,165:1175-1181.
[23]. Rissoan MC, Soumelis V, Kadowaki N, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science,1999,283:1183-1186.
[24]Perros F, Dorfmuller P, Souza R, et al. Dendritic cell recruitment in lesions of human and experimental pulmonary hypertension [J]. European Respiratory Journal, 2007,29:462-468.
Miyata M, Sakuma F, Youshimura A, et al. Pulmonary hypertension in rats:role of bromodeoxyuridine positive mononuclear cells and x lveolar macrophages。Int Arch Allery Immunol,1995,108:286
[26]Bartram U, Speer CP. The role of transforming growth factor beta in lung development and disease. Chest,2004,125:745-765.
[27]Lundblad LK* Thompson-Figueroa J, Leclair T, et al. Tumor necrosis factor-alpar overexpression in lung disease:a single cause behind a complex phenotype. AM J Respir Cnt Care Med,2005,171:1363-1370
[28]Sime PJ, Marr RA, Gauldie D, et al. Transfer of tumor necrosis factor-alpha to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming groeth factor-betal and myofibroblasts. AM J Pathol,1998,153:825-832.
[29]Iwasaki T, Takahashi T,Shimizu H, et al. Increased pulmonary hehe oxygenase-1 and delta-aminolevulinate synthase expression in monocrotaline-induced pulmonary hypertension. Curr Neurovasc Res,2005,2:133-139
[30]李梦涛,卢杰,王迁等,衣那西普在野百合碱诱导的大鼠肺动脉高压模型中作用的初步探讨。中华风湿病学杂志,2009,12(13)：804-807
[31]张晓,林莉,张光峰等。肿瘤坏死因子及转化生长因子在肺间质病变发病机制中的作用。中华风湿病学杂志,2010,1(14)：13-16
[32]Gauldie J, Bonniaud P, Sime P,et al. TGF-beta, Smad3 and the process of progressive fibrosis. Biochem Soc Trans,2007,35(Pt4):661-664
[33]Pantelidis P, McGrath DS, Southcott AM, et al. Tumour necrosis factor-beta production infibosing alveolitis is macrophage subset specific. Respir Res.2001,2:365-372
[34]Balabanian K, Foussat A, Dorfmuller P, et al. CX3C Chemokine Fractalkine in Pulmonary Arterial Hypertension [J].American Journal ofRespiratory and Critical Care Medicine,2002,165:1419-1425
[35]Kitaura H,UozumiN, TohmiM, et al. Roles of nitric oxide as a vas odilator in neurovascular coup ling ofinouse s omatosens ory cortex[J]. N eurosci Res,2007,59 (2): 160-171.
[36]Fisher K A, Serlin DM,Wils on KC, et al. Sarcoidosis-ass ociated pulmonary hypertensi on:Outcome with 1 ong-ter m epop r ostenol treatment[J].Chest,2006,130 (5):1481-1488
[37]chcar RO. Yung G L, Saffer H, et al. Morphologic changes in exp lantedlungs after prostacyclin therapy for pulmonary hypertensi on [J]. Eur J Med Res,2006,11 (5):203 -207
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