【作者】 程江涛；

【导师】 高传玉；

【作者基本信息】 郑州大学 ， 内科学博士， 2017， 博士

【摘要】 研究背景我国不同地区之间经济发展不平衡,目前仍处于发展中行列。目前肺动脉高压（Pulmonary Arterial Hypertension,PAH）靶向治疗药物未能列入国家基本医疗保险药品目录,患者经济负担沉重。我国PAH患者接受靶向药物治疗的比例较低,约为20%,即使在部分经济发达地区,PAH靶向药物的覆盖率仅能达到75%。PAH是一种高致死性心肺血管疾病,主要特征是肺血管重构引起阻力进行性升高,最终导致患者右心衰竭而死亡,临床上主要表现为呼吸困难、乏力、头晕、胸痛、咯血等,与祖国医学“肺胀”、“心衰病”类似。PAH的药物治疗主要是对右心功能不全和肺动脉原位血栓形成的治疗,即吸氧、利尿、强心和抗凝治疗,以及肺血管扩张剂的靶向药物治疗。通过治疗,可明显得改善患者的症状,提高生活质量,而且延长生存时间,但靶向药物价格昂贵、耐药发生率较高、副作用较大,中医药能克服以上缺点,因此,开展中医药对肺动脉高压的防治研究具有十分重要的实际意义。研究目的观察参附益心颗粒对PAH大鼠血流动力学及血管新生的影响,并进一步了解PAH大鼠血流动力学与血管新生之间的关系;探讨参附益心颗粒含药血清对大鼠骨髓源性晚期内皮祖细胞的增殖、迁移及旁分泌的影响;运用GO功能分析和KEGG信号通路分析,初步建立PAH调控网络模型,为药物治疗PAH提供新思路和新靶点。材料与方法1.随机将30只雄性Wistar大鼠分为空白组、模型组和参附益心颗粒组,每组10只。模型组和参附益心颗粒组皮下注射野百合碱（60mg/kg）建立PAH模型,空白组给予皮下注射等量的生理盐水。用改良后的右心导管技术测定各组大鼠药物灌胃28天后的肺血流动力学参数,计算肺动脉平均压,心输出量（CO）,心指数（CI）。处死测量其右心室肥厚指数,并用RV/（LV+S）的比值表示。最后取其右下肺组织测定肺微血管密度（Microvessel density,MVD）。2.随机将60只雄性Wistar大鼠分为模型组50只和空白对照组（K组）10只,模型组耳后颈背部皮下注射野百合碱进行肺动脉高压造模,K组给予皮下注射等量的生理盐水。模型组再随机分为:生理盐水组（N组）、PI3K抑制剂组（Y组）、参附益心颗粒低剂量组（D组）、参附益心颗粒中剂量组（Z组）、参附益心颗粒高剂量组（G组）,处理制备含药血清。用各组含药血清的培养液对晚期内皮祖细胞进行培养,24 h后用流式细胞仪对其进行检测,并用ModFitLT软件分析得出各细胞周期情况;取处于对数生长期的晚期内皮祖细胞,用Transwell小室法检测各组细胞迁移数目;用ELISA测空白组、模型组和参附益心颗粒组的各生长因子（Ang-II、IGF、HGF及VEGF）。3.利用基因本体数据库（GO）和京都基因与基因组百科全书数据库（KEGG）对PH相关基因TGF、ECM、HGF、STAT、VEGF等五种基因标志物进行GO功能分析和KEGG信号通路分析,初步建立PAH调控网络模型。研究结果1.肺血流动力学与血管新生结果:与空白组相比,模型组RVSP、RVDP、mPAP、RV/（LV+S）及PVR均有明显增高（P<0.05）,而CO和CI则显著降低（P<0.05）。而参附益心颗粒组较模型组,RVSP、RVDP、mPAP、RV/（LV+S）及PVR均显著降低（P<0.05）,而CO和CI则显著增高（P<0.05）。提示参附益心颗粒对野百合碱诱导的PAH大鼠血流动力学指标有改善作用,模型组的MVD与空白组相比,明显降低,（P<0.05）,而参附益心颗粒组的MVD显著高于模型组（P<0.05）,参附益心颗粒可以增加PAH肺动脉高压大鼠的MVD。2.细胞增殖实验结果:与Y组相比,K组、N组、D组、Z组、G组细胞周期中G₁期有所减少（P<0.05）,G₂+S期及S期有所增加（P<0.05）,与K组相比,D组、Z组、G组细胞周期中G₁期有所减少（P<0.05）,G₂+S期及S期有所增加（P<0.05）;与N组对比,D组、Z组、G组细胞周期中G₁期有所减少（P<0.05）,G₂+S期及S期有所增加（P<0.05）;D组、Z组、G组细胞周期中G₁期有所减少（P<0.05）,G₂+S期及S期有所增加（P<0.05）,说明细胞各周期的分布在一定剂量范围内呈剂量依赖性;细胞迁移实验结果:与Y组相比,K组、N组、D组、Z组、G组细胞迁移数目均增加（P<0.05）;与K组相比,D组、Z组、G组细胞迁移数目均显可见明显增加（P<0.05）;D组、Z组、G组细胞迁移数目均有所增加且在一定剂量范围内呈剂量依赖性;ELISA检测结果:与空白组相比,模型组Ang-II、IGF、HGF及VEGF均有明显增高（P<0.05）;同样,与模型组相比,参附益心颗粒组Ang-II、IGF、HGF及VEGF也均有明显增高（P<0.05）。3.结合GO功能分析和KEGG信号通路分析结果,初步建立了由TGF、ECM、HGF、STAT、VEGF五个相关基因及其他相关基因组成的PAH初级调控网络模型。研究结论1.参附益心颗粒可改善野百合碱诱导的PAH大鼠的血流动力学指标,促进血管新生。2.参附益心颗粒可促进晚期内皮祖细胞的增殖及迁移,促进其旁分泌,为深入研究参附益心颗粒的作用机制提供了相关的实验数据。3.初步建立了PAH初级调控网络模型,为进一步研究参附益心颗粒治疗PAH的作用路径提供依据。更多还原

【Abstract】 BackgroundChina is a developing country in which the economic development is uneven between regions.Currently,since targeted drug of pulmonary hypertension(PH)is not listed in National Basic Medical Care Insurance Medicine Catalogue,patients bear a heavy economic burden.In China,only about 20% PH patients receive targeted drug therapy,and even in some well-developed cities and regions,the rate of using PAH targeted drugs just reaches 75%.PAH is a highly fatal disease in heart,lung and blood vessel featured with resistance progressive increase by pulmonary vascular remodeling which eventually results in death from right heart failure,and its clinical manifestations include dyspnea,fatigue,dizziness,chest pain,hemoptysis,and so on,which are similar to "lung distension" and “heart failure” in the traditional medicine.Traditional drug therapies for PAH mainly treat right ventricular dysfunction and primary thrombus formation in pulmonary artery,namely,targeted drug therapy for oxygen inhalation,diuresis,cardiac and anticoagulation as well as pulmonary vascular dilatation.Though targeted drugs can not only significantly improve symptoms and life quality of patients,but also extend their life span,they are expensive and have high incidenceof drug resistance and strong side effects.Therefore,it is of great practical significance to study the prevention and treatment of PAH by traditional Chinese medicine which can overcome the above disadvantages.ObjectiveThis study is to observe the effect of the Shenfuyixin granule on hemodynamics and angiogenesis in PAH rats and further explore the relationship between hemodynamics and angiogenesis in PH rats;to investigate the effect of Shenfuyixin granule on the proliferation and migration of bone marrow-derived late endothelial progenitor cells in rats;to establish a primary PH regulatory network through GO function analysis and KEGG signaling pathway analysis,thereby providing new ideas and novel targets for drug therapy against PAH.Material and methods1.Thirty male Wistar rats were randomly divided into blank group,model group and Shenfuyixin granule group,with 10 rats in each group.And rats in model group and Shenfuyixin granule group were hypodermically injected with monocrotaline(60mg/kg)to establish PAH model.The parameters of pulmonary hemodynamics in rats after 28-day drug gavage were determined by modified right heart catheterization and the mean pulmonary arterial pressure was calculated.Rats were sacrificed to measure their right ventricular hypertrophy indexes which were represented by RV/(LV+S).Finally,the right lower lung tissues were taken to measure the microvessel density(MVD).2.Sixty male Wistar rats were randomly divided into model group(50 rats)and blank group(group K,10 rats).And rats in model group were hypodermically injected with monocrotaline in the neck and back to establish the PH model.Then,rats in model group were randomly divided into five groups,saline group(group N),PI3 K inhibitor group(group Y),low-dose Shenfuyixin granule group(group D),medium-dose Shenfuyixin granule group(group Z)and high-dose Shenfuyixingranule group(group G),and their serums containing medicine were prepared.The late endothelial progenitor cells were cultured in serum containing medicine of each group for 24 hours and detected by flow cytometry,and then the ModFitLT software was employed to analyze the cell cycle situations.Cells at logarithmic growth phase were obtained to examine the number of migrating cells by Transwell method;the levels of Ang-II,IGF,HGF and VEGF in blank group,model group and Shenfuyixin granule group were detected by ELISA.3.GO and KEGG databases were used to perform GO analysis and KEGG signaling pathway analysis on TGF,ECM,HGF,STAT and VEGF genes and a primary PAH regulatory network was established.Results1.Shenfuyixin granule has significant effect on the hemodynamic index of PAH rat induced by monocrotaline.The MVDs of Shenfuyixin granule group and model group were both significantly higher than that of blank group(P<0.05),and that in Shenfuyixin granule group was dramatically higher than that in model group(P<0.05),which indicated that the intervention of Shenfuyixin granule prevented the MVD of lung tissue in PH rat from reduction.2.Cell proliferation experimental results: compared with group Y,cells at G1 phase in group K,group N,group D,group Z and group G decreased(P<0.05)while at G2/M and S phases increased(P<0.05);compared with group K,cells at G1 phase in group D,group Z and group G decreased(P<0.05)while at G2/M and S phases increased(P<0.05);compared with group N,cells at G1 phase in group D,group Z and group G decreased(P<0.05)while at G2/M and S phases increased(P<0.05);and these above results indicated that the distribution of cells in different phase of cell cycles was dose-dependent within certain dose range.Cell migration experimental results: compared with group Y,the number of migrating cells in group K,group N,group D,group Z and group G rose(P<0.05);compared with group K,the number of migrating cells in group D,group Z and group G notably rose(P<0.05);from the results described above,the numbers of migrating cells in group D,group Z andgroup G all rose in a dose-dependent manner within certain dose range.ELISA test:compared with blank group,the levels of Ang-II,IGF,HGF and VEGF in model group were elevated remarkably(P<0.05);similarly,compared with model group,the levels of Ang-II,IGF,HGF and VEGF in Shenfuyixin granule were also elevated significantly(P<0.05).3.Through GO function analysis and KEGG signaling pathway analysis,the primary PAH regulatory network based on TGF,ECM,HGF,STAT,VEGF and other related genes was established.Conclusions1.Shenfuyixin granule can significantly improve the hemodynamic indexes and symptoms of PH rat induced by monocrotaline and promote angionesis.2.Shenfuyixin granule can promote the survival,proliferation and migration of late endothelial progenitor cells and improved their paracrine effect,providing reliable experimental data for further study on mechanism of Shenfuyixin granule.3.The primary PAH regulatory network was established,which lays foundation for further exploration on pathway of Shenfuyixin granule in treating PAH.更多还原