【作者】 郭炜；

【导师】 林娜；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2011， 硕士

【摘要】 目的：研究比较清络饮和温络饮对RA滑膜血管新生的干预作用并探索相关机制,为指导RA临床的合理应用提供实验依据,也为中医络病学说的科学内涵研究提供方法学参考。方法：SD大鼠清络饮、温络饮水煎液连续灌胃五次后,末次灌胃后1h腹主动脉采血分离血清,制得清络饮含药血清(QX)温络饮含药血清(WX)。分设以下组：正常鼠血清组(Con)、IL-1β诱导正常鼠血清组(Mod)、QX(5%、10%、20%)组, WX(5%、10%、20%)组。30%乙醇回流提取清络饮、温络饮制得清络饮醇提物(QL)、温络饮醇提物(WL)。分设以下组：空白组(Con)、IL-1β诱导组(Mod)、IL-1β诱导+QL(2、4、8 mg/ml)组、IL-1β+WL(2、4、8、16、32 mg/ml)组。将不同浓度的清络饮、温络饮分别与IL1-β诱导的RA-HFLS及未诱导的HUVEC共孵育,采用MTS比色法检测细胞增殖活性,以观察清、温络饮对RA-HFLS和HUVEC增殖的影响；通过Transwell双室培养板观察清、温络饮RA-HFLS和HUVEC趋化性迁移的影响；采用细胞外基质主要成分之一FN包被96孔培养板,考察清、温络饮对RA-HFLS和HUVEC细胞与细胞外基质黏附作用的影响；通过基质胶包被的96孔板来观察清、温络饮对RA-HFLS和HUVEC管腔形成能力的影响;以及通过在体实验观察清、温络饮对小鼠基质胶栓子中血管形成的影响；ELISA(?)去检测清、温络饮对RA-HFLS分泌的血管新生相关细胞因子(VEGF、Ang1、Ang2、TNFα、IL-17)及HUVEC血管新生相关受体蛋白VEGFR2和Tie2表达水平的影响的影响。结果1.清、温络饮对RA-HFLS增殖、迁移、黏附及血管新生相关因子的影响1.1.清、温络饮对IL1-β诱导的RA-HFLS增殖的影响与空白对照组相比,IL-1β可明显提高细胞的增殖活性;QX组对IL-1β诱导的RA-HFLS增殖无明显影响；WX组随着浓度的增大,抑制作用逐渐增强,Wx20可显著抑制RA-HFLS的增殖(P<0.05);QL在较低浓度2～8 mg/ml即可抑制RA-HFLS的增殖,而WL在高剂量16～32 mg/ml组才可显著抑制RA-HFLS的增殖(P<0.01或P<0.05),且呈现剂量依赖性：同时TP10、50组亦可剂量依赖性的抑制RA-HFLS的增殖(P<0.01)1.2.清、温络饮对RA-HFLS迁移的影响与空白对照组相比,VEGF可明显提高RA-HFLS细胞的迁移数量；随着QX浓度增大,QX20组可显著抑制其迁移(P<0.05)随着Wx浓度增大,Wx10,20组均可显著抑制其迁移(P<0.05或P<0.01);同时QL可剂量依赖性的抑制RA-HFLS细胞的迁移(P<0.05或P<0.01)；WL组随浓度增大WL8-32组均可显著抑制其迁移。TP组随浓度增大,可剂量依赖性的抑制RA-HFLS的迁移。1.3.清、温络饮对RA-HFLS黏附的影响与空白组相比,RA-HFLS对FN有较强的黏附能力(P<0.01)；同时IL-1β诱导可显著增加RA-HFLS对FN的黏附(P<0.05);QX,WX随浓度的增大,对RA-HFLS黏附能力均无显著影响。QL, WL随浓度的增大,QL4、QL8, WL16、WL32组均可显著降低RA-HFLS黏附能力(P<0.05或P<0.01)。TP浓度的增大,TP10、50组可显著降低RA-HFLS黏附能力(P<0.01)。1.4.清、温络饮对RA-HFLS分泌的血管新生相关因子(TNFα、IL-17、VEGF、Ang1、Ang2)的影响(1)对炎性因子TNFα、IL-17水平的影响与空白对照组相比,IL1-β诱导组可增加RA-HFLS上清中TNFα、IL-17水平,QX对TNFα的影响无显著性差异,Qx20能显著降低IL-17水平,而Wx20组较IL1-β诱导组TNFα、IL-17水平显著降低(P<0.05或P<0.01)；QL4～8、WL16～32组可显著抑制TNFα、IL-17的表达(P<0.05或P<0.01)TP可剂量依赖性的抑制RA-HFLS分泌IL-17,而TP10、50组对TNFα的抑制作用具有显著性差异(P<0.05或P<0.01)。(2)对血管新生相关因子VEGF, Ang1, Ang2的影响与空白对照组相比,IL1-β诱导组可增加RA-HFLS上清中VEGF、Ang1、Ang2水平,QX、WX对VEGF、Ang1、Ang2的影响无显著性差异。QL组对RA-HFLS分泌Ang2无明显影响,QL4～8组可显著抑制上清中VEGF、Ang1水平(P<0.05或P<0.01);WL8～32组亦可显著抑制上清中Ang1水平,而只有WL32组可明显抑制VEGF、Ang2的表达。TP组可剂量依赖性的降低VEGF、Ang1水平(P<0.05或P<0.01)。只有TP50组Ang2水平显著降低(P<0.05)。2.清、温络饮对HUVEC增殖,迁移,黏附,管腔形成及血管新生相关受体蛋白VEGFR2和Tie2表达水平的影响2.1.清、温络饮对HUVEC增殖的影响浓度增大,Qx10和QX20组均可显著抑制HUVEC增殖；Wx组剂量依赖性的抑制其增殖(P<0.01), QL、WL组随着浓度增大,QL2、QL4, WL16、WL32均可显著抑制HUVEC增殖(P<0.01),但是同剂量组相比,QL较WL对HUVEC增殖的抑制作用强。TP10、50组均可显著抑制RA-HFLS的增殖(P<0.01)。2.2.清、温络饮对HUVEC迁移的影响与空白对照组相比,VEGF可明显提高HUVEC细胞的迁移数量；随着QX、WX浓度增大,QX20、WX20组可显著抑制其迁移(P<0.05或P<0.01);QL组可剂量依赖性的抑制其迁移(P<0.05或P<0.01)；随着WL浓度增大WL8-32组的迁移作用明显降低,而TP组可剂量依赖性的抑制RA-HFLS的迁移。2.3.清、温络饮对HUVEC黏附的影响与空白组相比,HUVEC对FN有较强的黏附能力(P<0.01)；同时IL-1β诱导可显著增加HUVEC对FN的黏附(P<0.05); QX, WX随浓度的增大,对HUVEC黏附能力均无显著影响。而QL可剂量依赖性的抑制HUVEC的黏附(P<0.05或P<0.01),随着WL浓度的增大,WL16、WL32可显著降低HUVEC黏附能力(P<0.05或P<0.01)。同时TP组随着浓度的增大,均可显著降低HUVEC黏附能力(P<0.01)。2.4.清、温络饮对HUVEC管腔形成的影响与空白对照组相比,VEGF诱导可显著增加HUVEC在基质胶上的管腔形成,QL、WL、TP组随着浓度的增加均可剂量依赖性的抑制HUVEC的管腔形成(P<0.01)。2.5.清、温络饮对HUVEC血管新生相关受体蛋白VEGFR2和Tie2表达水平的影响与空白对照组相比QX, WX均可剂量依赖性的促进HUVEC的VEGFR2、Tie2的表达(P<0.01), QL, WL组随浓度增大,Tie2的表达水平均呈上升趋势,除WL2组外,均具有显著性差异(P<0.05或P<0.01); QL8、WL16～32组可显著促进VEGFR2的表达(P<0.01)：TP组与空白对照组相比TP10、TP50可显著上调Tie2的表达水平(P<0.01),而只有高剂量的TP才可显著促进VEGFR2的表达。3.清、温络饮30%乙醇提取物对在体小鼠基质胶栓子中血管形成的影响与空白对照组相比,VEGF诱导组见基质胶内部可见大量血管内皮细胞浸润,且有管样结构形成,说明VEGF有促进血管新生的作用；QL8, WL32, TP50组较VEGF诱导组相比,血管内皮细胞浸润均明显减少。4.结论根据国内外的最新研究进展,借助体外培养的RA-HFLS和HUVEC细胞模型以及在体的小鼠基质胶栓子模型成功模拟了实验性RA滑膜血管新生病理过程,并以此为基础得到如下结论：(1)清、温络饮均具有显著的RA滑膜血管新生抑制作用,但清络饮的作用显著强于温络饮。(2)清、温络饮对RA滑膜炎症的抑制作用可能是通过抑制RA-HFLS的增殖、迁移和黏附及炎症因子TNFα、IL-17和促血管新生因子VEGF、Ang1的分泌来实现的；而对血管新生的抑制作用则可能与其对HUVEC增殖、迁移、黏附的抑制以及对血管新生相关受体Tie2、VEGFR2水平的有效调控有关。(3)清络饮与温络饮二者对RA滑膜血管新生的作用环节和靶点相同,但清络饮作用强度显著高于同浓度的温络饮,而阳性对照药雷公藤甲素抗血管新生作用明确,可显著抑制上述滑膜血管新生的主要病理环节和靶点。(4)清、温络饮30%醇提物对滑膜炎症和血管生成作用与整体动物实验较为一致,而清、温络饮含药血清的作用在本实验中表现较弱。更多还原

【Abstract】 OBJECTIVEExplore the role of Qingluoyin and Wenluoyin in RA angiogenesis and compare the differences between them,reveal mechanism of the effect, provide the experimental basis for clinical application for the treatment of RA.METHODSSD rats were continuous taken Qingluoyin and Wenluoyin by intragastric administration for five times, one hour later, abdominal aortic blood serum were separated. Qingluoyin containing medicine serum (QX) and Wenluo yin containting medicated serum (WX) was obtained. There are five experiental groups:normal mouse serum group (Con), IL-1 beta induced normal mouse serum group (Mod), QX (5%,10%, and 20%) group, WX (5%,10%, and 20%) group.30% ethanol reflux extraction of Qingluoyin and Wenluoyin, made Qingluoyin ethanol extract (QL), Qingluoyihn ethanol extract (WL). Consisted of the following groups:the blank group (Con), IL-1 beta induction group (Mod), IL-1 beta induction+QL (2,4,8 mg/ml) group, IL-1 beta+WL (2,4,8,16,32 mg/ml) group. TP group is divided into three Concentrations 1,10,50 ng/ml.RA-HFLS and HUVEC are inbubated With different Concentrations of Qingluoyin and Wenluoyin. Cell proliferative activity are detected by the incubation of MTS, to observe the effect of Qingluoyin and Wenluoyin on the proliferative of RA-HFLS and HUVEC; the influence of Qingluoyin and Wenluoyin on the migration of RA-HFLS and HUVEC are observed through the Transwell inserts; the effect of Qingluoyin and Wenluoyin on HFLS and HUVEC cells with the extracellular matrix adhesion are observed by using the extracellular matrix FN, one of main component, enveloped 96 well board; the role of Qingluoyin and Wenluoyin on RA-HFLS and HUVEC lumen formation ability are observed through the matrix gel enveloped 96 well board. In vivo Qingluoyin and Wenluoyin extracts have the influence on the angiogenesis in mice matrigel plug; Meanwhile ELISA test show Qingluoyin and Wenluoyin influence the expression level of angiogenic relevant cell factor (VEGF, Ang1, Ang2, TNF alpha, IL 17,) and HUVEC angiogenesis related receptor proteins VEGFR2 and Tie2.RESULTS:1. Effect on RA-HFLS proliferation, migration, adhesion and angiogenesis related factors (TNF alpha, IL-17, VEGF, Angl, Ang2)1.1. Effect on the proliferation of IL1-beta induced RA-HFLS of Qingluoyin and WenluoyinCompared with the Control group, IL-1 beta can obviously improve cell proliferation activity; QX group has no significant effect on IL-1 beta induced RA-HFLS proliferation; with the increased of Concentration,inhibition strengthen gradually, WX20 can significantly inhibit proliferation of RA-FLS (P<0.05). QL in lower Concentration 2-8 can inhibit the proliferation and WL in high doses 16-32 group just can significantly inhibit RA-FLS proliferation (P<0.01 or P<0.05), and shows the dose-dependence; Meanwhile TP10,50 group also inhibit the proliferation RA-HFLS,ands show dose-dependent (P<0.01)1.2. Effect on the migration of RA-HFLS of Qingluoyin and WenluoyinCompared with the Control group, VEGF can obviously increase the number of RA-HFLS cell migrated; With the increased Concentration,QX, QX20 group can significantly inhibit its migration (P<0.05); With the increased Concentration, WX10,20 group significantly inhibit its migration (P<0.05 or P<0.01); Meanwhile QL can inhibit the migration of RA-HFLS,show dose-dependent (P<0.05 or P<0.01); WL group with Concentration of WL8-32 group significantly inhibit the migration, with the increased Concentration, TP group inhibit the migration of RA-HFLS,show dose-dependent.1.3. Effect on RA HFLS adhesion of Qingluoyin and Wenluoyin.Compared with the Control group RA-FLS have strong adhesion ability to FN (P< 0.01); Meanwhile IL-1 beta induced RA-HFLS can significantly boost adhesion to FN (P < 0.05). QX, WX did show dose-dependent on the ability of adhesion. QL4 QL8, WL16, WL32. group significantly reduce the ability of adhesion (P<0.05 or P<0.01). At the same time, TP10,50 group, can significantly reduce reduce the ability of adhesion(P< 0.01). 1.4. Effect on RA-HFLS secreted angiogenic related factors (TNF alpha, IL-17, VEGF, Angl, Ang2) of Qingluoyin and WenluoyinCompared with the Control group, group inducted with IL1-beta can increase TNF alpha in RA-HFLS supernatant, QX did not. compared with IL1-beta induced group, WX20 significantly reduced TNF alpha level (P< 0.05). QL4,8, WL16,32 groups can significantly inhibit the secrete of TNF alpha (P< 0.05 or P<0.01), TP10,50 also inhibit the secrete of TNF alpha (P<0.05 or P<0.01).Compared with the Control group, group induced IL1-beta can significantly raise IL-17 level in RA-HFLS supernatant. QL4,8, WL16,32 group can significantly inhibit expression of IL-17 (P< 0.05 or P< 0.01), IL-17 level in TP groups are significantly reduced(P<0.05 or P<0.01)Compared with the Control group, group induced IL1-beta can increase VEGF in RA-HFLS supernatant. There is no significant differences on QX group and WX group. QL4, 8, WL32 group can significantly inhibit VEGF level in supernatant (P<0.05) TP group can inhibit VEGF level in supernatant, and show dose-dependent (P<0.05 or P<0.01).Compared with the Control group, group induced IL1-beta can increase Ang1 level in RA-HFLS supernatant, There is no significant differences on QX group and WX group. QL4,8, WL8,16,32 groups can significantly inhibit Angl level in supernatant (P < 0.05 or P< 0.01) and Ang1 level in TP group were significantly lower (P<0.05 or P< 0.01).Compared with the Control group, group induced IL1-beta can significantly increase Ang2 level in RA-HFLS supernatant, There is no significant differences on QX group and WX group. Meanwhile QL group had no significant effect on Ang2 RA-FLS secretion, but WL32 group can significantly inhibite the Ang2 express (P< 0.05). Along with the increase of Concentration TP50 group, Ang2 were significantly reduced (P< 0.05).2. Effect on proliferation, migration, adhesion, lumen formation of HUVEC and the expression of angiogenesis related receptor proteins VEGFR2 and Tie22.1. Effect on HUVEC proliferation of Qingluoyin and WenluoyinQX10 and QX20 group significantly inhibit proliferation of HUVEC; WX inhibit the proliferation,and show dose-dependent(P<0.01)QL2, QL4, WL16, WL32 all can significantly inhibit proliferation of HUVEC (P< 0.01), but compared with the same dosage group of WL, QL has strong inhibition effect. Meanwhile TP10,50 group significantly inhibit the proliferation of RA-HFLS(P< 0.01).2.2. Effect on migration of HUVEC of Qingluoyin and WenluoyinCompared with the Control group, VEGF can obviously increase quantity of HUVEC cells migrated; QX20, WX20 group can significantly inhibit its migration (P<0.05 or P< 0.01); QL group inhibit its migration,and show dose-dependent (P<0.05 or P<0.01); WL8～32 obviously inhibit the migration. TP group inhibit RA-HFLS migration,and show dose-dependent.2.3. Effect on adhesion of HUVEC of Qingluoyin and WenluoyinCompared with the group, HUVEC has stronger adhesion ability to FN (P<0.01); Meanwhile IL-1 beta induction can significantly increase HUVEC adhesion of FN (P< 0.05). QX, WX with increased Concentration, there were no significant influence on HUVEC adhesion ability. And QL can dose-dependently inhibit HUVEC adhesion (P< 0.05 or P<0.01), along with the increase of WL Concentration, WL16, WL32 can significantly reduce HUVEC adhesion ability (P<0.05 or P<0.01). And with the increase of Concentration of TP group, all can significantly reduce HUVEC adhesion ability (P<0.01).2.4. Effect on tube formation ability of Qingluoyin and WenluoyinCompared with the Control group, VEGF induced can significantly increase HUVEC formation in the lumen Matrigel, QL, WL, TP group, with the increase of Concentratio,can dose-dependently inhibit HUVEC formation the lumen. (P<0.01).2.5. Effect on the expression level of angiogenic related receptor proteins Tie2 and VEGFR2 on HUVEC of Qingluoyin and Wenluoyin(1)VEGFR2 levelCompared with the Control group, WX QX dose-dependently promote VEGFR2 expression of the HUVEC (P<0.01), meanwhile QL2 group significantly inhibit the expression of VEGFR2 (P<0.05), QL8 promote VEGFR2 expression. And WL2 can down regulation its expression, with Concentration increases, TP1,10 group were significantly lower VEGFR2 level (P<0.05), along with the increase of dose, in TP50 group VEGFR2 express are on the rise.(2) Tie2 levelCompared with the Control group, QX, WX dose-dependently promote VEGFR2 expression of HUVEC (P<0.01), QL, WL group, except WL2 group, with the increase of Concentration, Tie2 expression level all shows ascendant trend, [P< 0.05 or P< 0.01); with TP Concentration increases, down regulation transforms to up regulation3. Effect on angiogenesis in the mice matrigel plug of Qingluoyin and Wenluoyin. Compared with the Control group, through the observation, in VEGF induction groups, the amount of endothelial cell infiltrates in internal matrix glue, and a tube sample structure formation, explain that VEGF have promote angiogenesis role; QL8, WL32 TP50 group, compared to VEGF induction group, vascular endothelial cells infiltration are significantly less.更多还原