【作者】 马宇滢；

【导师】 王文健； 潘永福； 张新民；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2006， 博士

【摘要】 背景外周血、脐带血和骨髓中存在能分化为成熟内皮细胞，参与成体动物的新血管形成的一群功能性前体细胞，这类细胞被称为内皮祖细胞（endothelial progenitor cell）。当发生血管损伤时，内皮祖细胞从骨髓释放入外周血，迁移至受损部位，掺入血管壁中，分化为成熟内皮细胞，修复受损血管，参与血管新生，改善甚至恢复缺血组织的血供。通过移植含有内皮祖细胞的骨髓成分或经体外扩增的内皮祖细胞，可以治疗缺血性疾病。外周血内皮祖细胞的数量和功能还可用来评估心血管疾病的危险性和预后，用药物等干预手段提高外周血内皮祖细胞的数量和功能，可能是防治心血管疾病的有效的新方法。不论是为提高自体内皮祖细胞移植的疗效，还是探索防治心血管疾病的新方法，研究药物增加在体或离体内皮祖细胞的数量、提高其功能都具有重要的意义。中药红景天及其有效成分红景天苷和丹参及其有效成分丹酚酸B对心血管系统有保护作用，它们有可能对内皮祖细胞也有调控作用。目的观察红景天苷和丹酚酸B对体外培养的人内皮祖细胞的增殖、迁移及凋亡的影响。如果确有影响，则进一步探讨可能的机制。希望通过本实验的研究，能够初步明了红景天苷和丹酚酸B对体外培养的人内皮祖细胞的影响，为今后研究更多中药及中药组分对内皮祖细胞的影响打下基础。以这些研究为基础，有望研制开发一些具有临床应用价值的药物，将骨髓内皮祖细胞动员到受损部位，为防治心血管疾病提供一种新的策略。方法1，红景天甙类与丹酚酸B对大鼠心肌梗死模型的血管新生作用3月龄雄性SD大鼠，结扎左冠状动脉前降支制作心肌梗死模型。随机分为7组，每组10只，分别为红景天甙类大剂量组(10g·kg^-1)；中剂量组(5g·kg^-1)；小剂量组(2.5g·kg^-1)；丹酚酸B(1g·kg^-1)；西药对照组舒降之：(3.3mg·kg^-1)；假手术组（生理盐水）；模型组（生理盐水）；每日灌胃一次，共14天。14天后处死取材。梗死面积检测：垂直于左室长轴切取0.3cm厚的含梗死区的左心室，常规固定，石腊包埋，沿长轴面切片（4μm厚），苏木精—伊红（HE）染色，IMS细胞图像分析系统、医学图像分析软件测量相同心室面积中心肌梗死面积。心肌CD34表达量检测：常规免疫组化染色，IMS细胞图像分析系统、医学图像分析软件，对包括梗死区和梗死周围区心肌CD34的表达量进行定量分析。2，人内皮祖细胞的分离、培养与鉴定采用Ficoll密度梯度离心的方法，分离健康志愿者外周血中的单个核细胞，以每孔1.5*10⁶个细胞的密度，800ml内皮祖细胞培养基的体积种植于预先包被Fibronectin的24孔培养板中。培养至第四天，换液，吸去悬浮细胞。继续培养至第七天，对贴壁细胞作Dil-acLDL与FITC-UEA-1 Lectin双标记，以鉴定是否为内皮祖细胞。用流式细胞术分析细胞表面CD34与VEGF-receptor 2的表达情况。对培养至第七天的细胞，用0.25％胰蛋白酶消化传代，以每孔3*10⁴个细胞的密度，100μl培养基的体积种植于预先包被过Fibronectin的96孔板中。3，红景天苷与丹酚酸B对人内皮祖细胞增殖的影响采用XTT比色法检测细胞增殖情况。首先根据XTT作用时间与XTT检测的OD值之间的关系确定XTT最适反应时间，然后根据细胞密度与XTT检测的OD值之间的关系确定最适细胞种植密度，再根据药物作用时间与XTT检测的OD值之间的关系确定最佳药物作用时间，最后观察4μg／ml、8μg／ml和16μg／ml的红景天苷以及5μg／ml、10μg／ml、20μg／ml和100μg／ml丹酚酸B对内皮祖细胞增殖的影响。4，红景天苷与丹酚酸B对人内皮祖细胞迁移的影响采用Transwell系统检测细胞的迁移。对Transwell小室多孔滤膜的上、下面均包被Fibronectin，0.25％的胰蛋白酶消化培养至第七天的细胞，以每孔3*10⁴的细胞密度，100μl体积种植于Transwell小室中。下室中加入内皮细胞培养基600μl，培养24h。在Transwell小室中加入8μg／ml红景天苷或10μg／ml丹酚酸B，下室中加入VEGF使终浓度为500ng／ml，继续培养12h。对Transwell小室多孔滤膜的下面进行固定、染色、封片，每张膜由独立观察者在高倍镜下随机选取9个视野，计数细胞，取平均值。5，红景天苷与丹酚酸B对人内皮祖细胞凋亡的影响采用流式细胞术分析DNA含量来反应细胞凋亡。以无血清，无细胞因子的M199培养基来诱导内皮祖细胞凋亡，加入8μg／ml红景天苷或10μg／ml丹酚酸B培养24h。用1mmol／L EDTA／PBS消化细胞，以流式细胞固定液固定细胞，PI染色，做凋亡细胞核PI标记后的流式细胞术分析。6，红景天苷与丹酚酸B促人内皮祖细胞增殖机制的初步探讨应用PI3K特异性抑制剂LY294002和Wortmannin以及MEK1特异性抑制剂PD98059观察内皮祖细胞的增殖情况。分组：对照组不加任何药物：8μg／ml红景天苷组；红景天苷+10μM LY294002组；红景天苷+10μM Wortmannin组；红景天苷+10μM PD98059组；10μg／ml丹酚酸B组；丹酚酸B+10μM LY294002组；丹酚酸B+10μM Wortmannin组；丹酚酸B+10μM PD98059组。作用24h，检测XTT光密度值。结果1，红景天甙类与丹酚酸B对大鼠心肌梗死模型的血管新生作用红景天甙类各剂量组、丹酚酸B能减少梗死面积，各P值均＜0.01。甙类大剂量组、丹酚酸B组与西药对照组舒降之的疗效一致，各P值均＞0.05。甙类各剂量组之间比较，大剂量组的疗效优于小剂量组，P＜0.01，与中剂量组无差别，P＞0.05。对红景天甙类来说，虽无典型的剂量效应关系，但仍以大剂量为佳。红景天甙类各剂量组与丹酚酸B均能提高CD34表达量，各值均＜0.05或0.01；表明红景天甙类和丹酚酸B对心肌梗死后的血管新生有促进作用，且疗效与舒降之一致；这几组成分之间互相比较，疗效无差异，各P值均＞0.05。2，人内皮祖细胞的分离、培养与鉴定用密度梯度离心法分离人外周血单个核细胞，在体外培养七天，贴壁细胞呈梭形，能同时摄取Dil-acLDL与结合FITC-UEA-1 Lectin，具有内皮细胞特征。部分细胞表达CD34和VEGFR。传代后的细胞出现栅栏样排列。3，红景天苷与丹酚酸B对人内皮祖细胞增殖的影响不同工作浓度（4μg／ml、8μg／ml和16μg／ml）的红景天苷对体外培养的内皮祖细胞都有促进增殖的作用，P＜0.01，没有剂量效应关系，P＞0.05。10μg／ml、20μg／ml的丹酚酸B对内皮祖细胞的增殖有促进作用，P＜0.01，10μg／ml比20μg／ml的效果更好，P＜0.05；5μg／ml的浓度对内皮祖细胞的增殖无影响，P＞0.05；100μg／ml的浓度对内皮祖细胞的增殖表现出抑制作用，P＜0.01。4，红景天苷与丹酚酸B对人内皮祖细胞迁移的影响内皮祖细胞在下室中VEGF的吸引下，穿过多孔滤膜的小孔，黏附于包被了Fibronectin的膜的下面，呈梭形、长条形或多角形，排列无规则。红景天苷和丹酚酸B对内皮祖细胞的迁移有促进作用，P＜0.05；而红景天苷和丹酚酸B在促进内皮祖细胞迁移方面的作用相同，P＞0.05。5，红景天苷与丹酚酸B对人内皮祖细胞凋亡的影响用无血清、无VEGF、bFGF、IGF-1、EGF等细胞因子的营养的培养条件下，未加药物的对照组内皮祖细胞细胞凋亡率较高，而红景天苷能够减少细胞凋亡的百分比，P＜0.01；丹酚酸B也能减少细胞凋亡，P＜0.05。红景天苷和丹酚酸B对内皮祖细胞的凋亡有抑制作用，而丹酚酸B的作用弱于红景天苷，P＜0.05。6，红景天苷与丹酚酸B促人内皮祖细胞增殖机制的初步探讨PI3K抑制剂LY294002和Wortmannin能够拮抗红景天苷和丹酚酸B对内皮祖细胞的促增殖作用，P＜0.01；Wortmannin的拮抗作用使内皮祖细胞的增殖降低到对照组水平，P＞0.05；LY294002甚至低于对照组水平，P＜0.01。MAPK抑制剂PD98059同样能够拮抗红景天苷和丹酚酸B对内皮祖细胞的促增殖作用，P＜0.05；这种拮抗作用使内皮祖细胞的增殖也降低到对照组水平，P＞0.05。本实验说明，红景天苷和丹酚酸B对内皮祖细胞的促增殖作用，可能通过PI3K和MAPK两条信号转导途径实现。结论红景天苷和丹酚酸B对体外培养的人内皮祖细胞有促增殖、促迁移和抗凋亡作用，其促增殖作用可能通过PI3K和MAPK两条信号转导途径实现。红景天甙类和丹酚酸B对心肌梗死大鼠的促血管新生效应可能与这种作用有关。更多还原

【Abstract】 BackgroundPeripheral blood, umbilical cord blood and bone marrow contain a subtype of functional progenitor cells which can differentiate into mature endothelial cells, participate in new vessel formation in postnatal subjects. Thus, these cells are termed as endothelial progenitor cells （EPCs）. When vessel injury occurs, EPCs are released from bone marrow, migrate to the site of injury, incorporate into the vessel wall, differentiate into mature endothelial cells, repair the injured vessel, participate the neovascularization and restore or improve the blood flow of the ischemic tissue. Transplantation of bone marrow components containing EPCs or EPCs expanded in vitro may be a potent treatment for ischemic diseases. The number and function of EPCs in peripheral blood may also be used to estimate the risk of cardiovascular diseases and predict the prognosis of such diseases. Pharmacological intervention to enhance the number and function of EPCs in peripheral blood could be a new method to prevent and treat cardiovascular diseases. So it’s important to search drugs which can increase the number of EPCs and improve their functions in vivo or in vitro therefore to improve the effect of autologous EPCs transplantation or explore a new way to prevent and cure cardiovascular diseases.Chinese herb Rhodiola sacra and its active component Salidrosid along with Salvia miltiorrhiza and it’s active component Salvianolic acid B show protective effects on cardiovascular system. They might have the potential to regulate EPCs.AimWe plan to observe the effects of Salidrosid and Salvianolic acid B on proliferation, migration and apoptosis of EPCs cultured in vitro. If they do have effects on EPCs, we would further study the possible mechanism. We hope tounderstand the effects of these two components on EPCs in vitro and make a fundament for future researches of other Chinese herbs’ effects on EPCs. Based upon these researches, we hope to develop some valuable drugs to mobilize EPCs to sites of injury and provide a new strategy to prevent and cure ischemic diseases.Methods1, Neovascularization of myocardial infarction rat model by glycoside components of Rhodiola sacra and Salvianolic acid BMyocardial infarction rat model was established by ligating the left coronary descendent anterior artery. Rats were divided randomly to 7 groups with 10 rats in each group: high/medium/low dose glycoside components of Rhodiola sacra(10g·kg^-1, 5g·kg^-1 and 2.5g·kg^-1, respectively) ; Salvianolic acid B (1g·kg^-1); simvastatin (3.3mg·kg^-1) ; Sham （saline） and model （saline）. Rats were fed once per day and killed after 14 days’ treatment.Measurement of infarct size: 0.3cm thick infarct parts of left ventricles were cut off along the vertical axis. They were fixed, embedded and stained with HE regularly. The infarct size was measured by IMS image analyzing system.Measurement of expression of CD34 in myocardium: regular immunohistochemistry method was used and quantitative analysis of expression of CD34 in infarct and surrounding areas was performed by IMS image analyzing system2, Isolation, culture and identification of human EPCsTotal mononuclear cells （MNCs） were isolated from peripheral blood of healthy human volunteers by density gradient centrifugation. MNCs （1.5 × 10⁶/well） were plated in 0.8 ml endothelial culture medium on fibronectin coated 24-well plates. After 4 days of culture, medium was changed and suspended cells were aspirated away. Up to the 7^th day after isolation, adherent cells were stained with Dil-acLDL and FITC-UEA-1 Lectin to be identified whether they were EPCs. FACS analysis of surface antigen CD34 and VEGFR-2 was performed. The 7th day cells were digested by 0.25% trypsin and 3 × 10⁴ cells/well were planted on fibronectin coated 96-well plates.3, Effects of Salidrosid and Salvianolic acid B on proliferation of human EPCsXTT chromatometry was used to assess the proliferation of EPCs. First we decided the optimal reaction time of XTT by analyze the correlation between XTT reactiontime and corresponding OD value. Then we decided the optimal planting cell density by analyze the correlation between cell density and corresponding XTT OD value. The optimal drug intervention time was decided accordingly. Finally, we observed the effects of 4μg/ml, 8μg/ml, 16μg/ml Salidrosid and 5μg/ml, 10μg/ml, 20μg/ml, 100μg/ml Salvianolic acid B on proliferation of human EPCs.4, Effects of Salidrosid and Salvianolic acid B on migration of human EPCs Transwell system was used to test the cell migration. Fibronectin was coated onboth the upper and down sides of the porous membranes of Transwell inserts. Cells cultured for 7 days were trypsinized and 3 × 10⁴ cells were plated in the upper chamber of each inserts. 600 μ l culture medium was added into the lower chamber. After 24h culturing, 8μg/ml Salidrosid and 10μg/ml Salvianolic acid B were added to the upper chamber and VEGF to the lower chamber reaching the concentration of 500ng/ml. After another 12h culturing, the down sides of the porous membranes of Transwell inserts was fixed, stained and mounted. Independent investigators evaluated the number of cells of each membrane by counting 9 randomly selected high-power fields and the mean value was calculated.5, Effects of Salidrosid and Salvianolic acid B on apoptosis of human EPCsFACS analysis of DNA contents was used to reflect cell apoptosis. EPC apoptosis was induced by depletion of serum and growth factors. 8μg/ml Salidrosid and 10μg/ml Salvianolic acid B were added to the M199 medium. After 24h inducing, cells were digested by 1 mmol/L EDTA/PBS, fixed with fixation solution and stained by PI. Apoptosis was then assessed by FACS.6, Study of the mechanism of pro-proliferation effects by Salidrosid and Salvianolic acid B on EPCsPI3K inhibitor LY294002, Wortmannin and MEK1 inhibitor PD98059 were used. Groups were: control; 8μg/ml Salidrosid; Salidrosid +10μM LY294002; Salidrosid + 10μM Wortmannin; Salidrosid+10μM PD98059; 10μg/ml Salvianolic acid B; Salvianolic acid B +10μM LY294002; Salvianolic acid B+10μM Wortmannin; Salvianolic acid B+ 10μM PD98059. After 24h culturing, cell proliferation was tested by XTT chromatometry.Results1, Neovascularization of myocardial infarction rat model by glycoside components of Rhodiola sacra and Salvianolic acid BDifferent dose intervention of glycoside components of Rhodiola sacra and Salvianolic acid B all could decrease the infarct size, P<0.01. The effects of high dose glycoside components of Rhodiola sacra and Salvianolic acid B were same in magnitude, P>0.05 and were as strong as simvastatin, P>0.05. While the typical dose-effect relationship was not found in glycoside components of Rhodiola sacra, High dose was better.Different dose intervention of glycoside components of Rhod Aiola sacra and Salvianolic acid B all could increase CD34 expression, P<0.05 or 0.01 and the effects were as strong as simvastatin, P>0.05.2, Isolation, culture and identification of human EPCsHuman PMNC were isolated by density gradient centrifugation. After 7 days culture, adherent cells were spindle shaped. They could take Dil-acLDL and bind with FITC-UEA-1 Lectin at the same time which was a character of endothelial cells. Some cells express CD34 and VEGFR. The passaged cells appeared to be bar-like alignment.3, Effects of Salidrosid and Salvianolic acid B on proliferation of human EPCs Different concentration （4μg/ml、 8μg/ml and 16μg/ml） intervention of Salidrosid allcould increase the proliferation of EPCs in vitro, P<0.01. There was no dose-effect relationship, P>0.05. 10μg/ml、 20μg/ml Salvianolic acid B also could increase the proliferation of EPCs, P<0.01, 10μg/ml was better than 20μg/ml, P<0.05. At 5μg/ml, Salvianolic acid B had no impact, P>0.05, while 100μg/ml could suppress the proliferation of EPCs, P<0.01.4, Effects of Salidrosid and Salvianolic acid B on migration of human EPCsEPCs moved through the pores of Transwell inserts by the attraction of VEGF in the lower chamber and attached on the Fibronectin-coated lower sides of the inserts. These migrated cells appeared to be spindle, sliver or polygon shaped and arranged irregularly. Salidrosid and Salvianolic acid B both could improve the migration of EPCs, P<0.05. And the effects were similar in magnitude, P>0.05.5, Effects of Salidrosid and Salvianolic acid B on apoptosis of human EPCsUnder cultural conditions without serum, VEGF, bFGF, IGF-1 and EGF, the control group had high apoptosis rate. While Salidrosid could reduce the apoptosis rate, P<0.01. Salvianolic acid B also could reduce the rate, P<0.05. Salidrosid and Salvianolic acid B both could suppress the apoptosis of EPCs. The impact of Salvianolic acid B was weaker than Salidrosid, P<0.05.6, Study of the mechanism of pro-proliferation effects by Salidrosid and Salvianolic acid B on EPCsPI3K inhibitorLY294002 and Wortmannin could antagonize the effects of Salidrosid and Salvianolic acid B on proliferation of human EPCs, P<0.01; The antagonistic effect of Wortmannin reduced the proliferation of EPCs to the level of the control group, P>0.05; LY294002’s was even lower than the control one, P<0.01. MAPK inhibitor PD98059 had the similar antagonistic effcts, P<0.05. It reduced the proliferation of EPCs to the level of the control group, P>0.05. The study showed that Salidrosid and Salvianolic acid B’s impacts on the proliferation of EPCs in vitro were partly via PI3K and MAPK signaling pathways.ConclusionSalidrosid and Salvianolic acid B can improve the proliferation and migration of human EPCs in vitro and suppress EPC apoptosis as well. These effects are partly via PI3K and MAPK signaling pathways. The influence of improving neovascularization in myocardial infarct rats by glycoside components of Rhodiola sacra and Salvianolic acid B may be related to these effects.更多还原