【作者】 宋颖；

【导师】 杨向红；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 内皮祖细胞(endothelial progenitor cells, EPCs)是胚胎发育期中胚层卵黄囊血岛的成血管细胞向内皮细胞分化过程中的一个过渡阶段。胎儿出生后EPCs主要存在于骨髓,在某些情况下可以被动员到外周血,归巢到靶组织参与生理或(和)病理性血管生成。内源性因素如缺血、缺氧、损伤、应激和外源性因素如细胞因子VEGF、GM-CSF、G-CSF、药物等可促进EPC的动员。VEGF作用于小鼠后,循环血液中EPCs大量增加,并且增殖和迁移能力都得到了增加。随着对EPCs研究的深入,研究者发现EPCs在缺血性疾病、创伤愈合、肿瘤血管生成等诸多方面发挥着重要作用。其基本过程为骨髓中的EPCs被动员、迁移至外周血中,归巢、募集于缺血组织,进而分化为内皮细胞发挥功能。实体肿瘤的生长依赖于肿瘤新血管的形成,因此,通过抑制新血管的形成以抑制肿瘤的生长和转移成为肿瘤治疗的一种新的重要靶点和策略。既往认为肿瘤血管来源于局部血管内皮细胞的迁移和增殖,但针对肿瘤血管内皮细胞的抗肿瘤血管生成治疗疗效并不尽如人意,而且也不能很好的解释肿瘤血管生成拟态等肿瘤血管生成特性。现在研究显示EPC参与的血管发生过程在肿瘤血管生成中起重要作用,因此,EPCs有可能成为抗肿瘤治疗的另一个靶点。榄香烯(elemene)是从中药温莪术(经鉴定为我国特有新种定名为温郁金)挥发油中提取的抗癌有效成分。p-榄香烯是榄香烯的主要活性成分,有关p-榄香烯具体作用机制的研究尚处于起步阶段。体外研究发现β-榄香烯可阻滞多种肿瘤细胞从S期进入G2/M期,抑制其增殖[1],下调肺腺癌和喉鳞状细胞癌等癌细胞VEGF-C、VEGFR-3、β-EGF蛋白的表达水平,降低肿瘤微血管密度。本实验目的是研究p-榄香烯对肿瘤细胞上清液诱导的EPCs增殖等活性有何影响,进而探讨其能否抑制EPCs在肿瘤血管发生中的作用。实验方法一.EPCs的分离、培养、纯化与鉴定采用Ficoll密度梯度离心法结合差速贴壁筛选法从大鼠骨髓分离EPCs:收集培养4d的EPCs,用激光共聚焦显微镜鉴定,CD133和vWF双染阳性细胞为正在分化的EPCs.二、四唑氮蓝还原反应(MTT)检测细胞增殖率消化收集贴壁EPCs:以5×103/孔的细胞数接种于96孔板中,每孔体积200μl；放入37℃、5%C02的孵育箱内培养；待细胞基本融合,无血清DMEM作用12h,加入40%体积分数的肿瘤细胞上清液作用12h后,分别加入含有0μg/ml,5μg/ml, 10μg/ml,20μg/mlβ-榄香烯的DMEM培养液,作用48h后检测。每孔加入20μl MTT(5mg/ml)孵育4h,吸除孔内液体,每孔加入150μlDMSO,微量振荡器震荡10min,置酶标仪测OD490值。三.Matrigel检测EPCs形成管腔的能力消化收集贴壁EPCs；以5×105/孔的细胞数接种于涂有Matrigel的24孔板中；在40%体积分数的肿瘤细胞上清液作用12h后,分别加入含有Oμg/ml,5μg/ml, 10μg/ml,20μg/mlβ-榄香烯的DMEM培养液,继续培养48h。在倒置显微镜下观察管腔形成情况,每孔随机选取5个视野计数管腔数,并取平均值。四、碘化丙啶(PI)标记流式细胞仪检测EPCs细胞周期取生长5d的EPCs,40%体积分数的肿瘤细胞上清液作用12h后加入含有0μg/ml,5μg/ml,10μg/ml,20μg/mlβ-榄香烯的DMEM培养液作用48h,胰酶消化吹打,制成细胞悬液,转移至离心管中,1000转/min,离心10分钟,PBS清洗2次。弃上清,用4℃预冷的75%乙醇固定,4℃过夜。1000转/min,离心10分钟,PBS清洗1次,弃上清,加入终浓度为200μg/ml的RNase A,37℃孵育1h；再加入浓度为20μg/ml的PI溶液,4℃避光染色30分钟,流式细胞仪分析EPCs细胞周期。五.Annexin-V/FITC法检测EPCs凋亡率取生长5d的EPCs,40％体积分数的肿瘤细胞上清液作用12h后加入含有0μg/ml,5μg/ml,10μg/ml,20μg/mlβ-榄香烯的DMEM培养液作用48h,胰酶消化吹打,制成细胞悬液,转移至离心管中,1000转/min,离心10分钟,PBS清洗2次,弃上清,按试剂盒说明书分别加入配制好的FITC及Annexin-V,避光孵育10分钟后上流式细胞仪检测分析。六、RT-PCR检测EPCsVEGF及VEGFR.2的mRNA表达水平Trizol法提取各组EPCs总RNA,按试剂盒说明书进行反转录、PCR反应,最后取5μlPCR反应液进行琼脂糖凝胶电泳,采用凝胶成像分析系统半定量分析,确认结果并拍照。七、统计学分析应用SPSS13.0软件进行统计学分析,实验结果采用方差分析,P<0.05表示差异有显著性。实验结果一、EPCs的培养与鉴定原代培养24h后,细胞大部分呈小圆形；差速贴壁筛选后,培养1d可见少量细胞贴壁；3d后细胞开始伸展成梭形或纺锤形；5d有细胞集落形成；7d后细胞继续保持贴壁状态,生长旺盛；大约14d融合成内皮细胞典型的铺路石样形态。CD133.vWF荧光双染阳性为正在分化的EPCs.二、β-榄香烯对肿瘤细胞上清液诱导的EPCs增殖率的影响不同浓度(5.10.20μg/ml)β-榄香烯作用48h及20μg/mlβ-榄香烯作用不同时间(12、24、48h)后,与对照组相比,细胞增殖率明显被抑制(P<0.01)。三、β-榄香烯对肿瘤细胞上清液诱导的EPCs管腔形成能力的影响不同浓度(5、10、20μg/ml)β-隘香烯作用48h及20μg/mlp-榄香烯作用不同时间(12、24、48h)后,与对照组相比,EPCs形成管腔的数量明显减少(P<0.01)。四、β-榄香烯对肿瘤细胞上清液诱导的EPCs凋亡的影响β-榄香烯浓度为5μg/ml时,与对照组相比,EPCs凋亡率升高无统计学差异,当β-榄香烯浓度上升为10μg/ml时EPCs凋亡率明显升高(P<0.05)；20μg/mlβ-榄香烯作用12h时,与对照组相比,EPCs凋亡率升高无统计学差异,当作用时间延长为24h、48h后,EPCs凋亡率明显升高(P<0.05)。五、p-榄香烯对肿瘤细胞上清液诱导的EPCs细胞周期的影响不同浓度(5、10、20μg/ml)β-榄香烯作用48h及20μg/mlβ-榄香烯作用不同时间(12、24、48h)后,与对照组相比,G1期细胞所占比例明显上升(P<0.05),S期细胞所占比例明显下降(P<0.05),即p-榄香烯作用后EPCs细胞周期停滞于G1期。六、β-榄香烯对肿瘤细胞上清液诱导的EPCsVEGF及VEGFR.2mRNA表达的影响不同浓度(5.10.20μg/ml)β-榄香烯作用48h及20μg/mlβ-榄香烯作用不同时间(12、24、48h)后,与对照组相比,EPCsVEGF及VEGFR-2mRNA表达明显减少(P<0.05)。结论1、β-榄香烯可抑制肿瘤细胞上清液诱导的EPCs的增殖率,并呈时间和剂量依赖性；2、β-榄香烯可减少肿瘤细胞上清液诱导的EPCs的管腔形成数量,并呈时间和剂量依赖性；3、β-榄香烯可促进肿瘤细胞上清液诱导的EPCs的凋亡,并将EPCs阻滞于G1期,呈时间和剂量依赖性；4、β-榄香烯可使肿瘤细胞上清液诱导的EPCs的VEGF及VEGFR-2mRNA表达下降,呈时间和剂量依赖性。更多还原

【Abstract】 Endothelial progenitor cells (EPCs) are the precursor of endothelial cells in the embryonic development period, which can differentiate into endothelial cells (ECs). To be born, EPCs mainly in the morrow.In some cases EPCs can be mobilized to peripheral blood and homed to the target tissue participating the physiological or pathological angiogenesis. Endogenous factors like ischemia、hypoxia、damage、stress and extrinsic factors as cell factors VEGF、GM-CSF、G-CSF、drugs can promote the mobilization of EPCs.After VEGF injeceted in the mice,the EPCs of circulating blood increase generously,the ability of generation and migration also increased.The researchers discover that EPCs play an important role in many ways as ischemia disease、wound healing、tumor angiogenesis.The elementary process is EPCs mobilized and migrated to peripheral blood,homing and recruitment to ischemia tissues,differented to endothelial cells.The growth of entity tumor depended on tumor neovascularity. Inhibiting neovascularization to restrain the development of tumou will become a new important target and method.In the past,the researchers consider that the migration and generation of tumor local endothelial cells to form the tumor vessel.But the therapy of anti-tumor angiogenesis to aim at endothelial cells of tumor vessel is not very good. It also can not explan tumor angiogenesis mimicry and so on very well.Now the reseachers discover that EPCs play a great role in the tumor angiogenesis. EPCs may become another antineoplaston target.Elemene extracted in the traditional Chinese medicine curcuma aromatica salisb (our country’s unique new kind names radix curcumae) aetherolea is the active component of anticancer.β-elemene is the main active component of elemene.The specific study about the main mechanism ofβ-elemene is in the primary phase.The in vitro study demonstrate thatβ-elemene can arrest many tumor cells from S to G2/M phase,inhibit their proliferation,down regulate proteinum expression of VEGF-C、VEGFR-3、β-EGF of adenocainoma of lung and throat squamous cell carcinoma,depress the microvessel density.This research purpose is studying the effect ofβ-elemene on the EPCs induced by tumor cells supernatant and whether can inhibit EPCs in the tumor vasculogenesis.Method一.The separation, culture, purifies and identification of EPCsUse the Ficoll density gradient centrifuge combined with difference-speed adherence screening method to separate EPCs from rat bone marrow. Collect EPCs cultured 4 day, identify with laser confocal microscopy and the cells that AC133 and vWF are both positive are the differentiating EPCs.二.MTT assay EPCs proliferationDigest and collect EPCs; Put cells into the 96 well cell culture cluster by 5×103 and 200μl per well,37℃,5%CO2 incubation 1-2h; After the cells fusion, non-blood serum DMEM to affect 12h; Add 40% volume fraction tumor cells supernatant,after 12h add different densityβ-elemene Oμg/ml,5μg/ml, 10μg/ml,20μg/ml;after 48h add 20μl MTT (5mg/ml) per well,incubation 4h, then DMSO 150μl per well; Shake 10min in the micro oscillator; Measure the OD value in wave length 490nm.三.Matrigel assay the vasculogenesis activity of EPCsDigest and collect EPCs; Put cells into the 24 well cell culture cluster by 5×103 per well; Add 40% volume fraction tumor cells supernatant,after 12h add different densityβ-elemene 0μg/ml,5μg/ml, 10μg/ml,20μg/ml,to continue culture 24h.Observe the number of the tube formation,choosing 5 field of vision per well random and getting the average value.四.Flow cytommetry detect EPCs cell cycleSelect 5d EPCs; Add 40% volume fraction tumor cells supernatant,after 12h add different densityβ-elemene 0μg/ml,5μg/ml, 10μg/ml,20μg/ml to continue culture 48h.Trypsinizate EPCs and make it into ventrifuge tube,1000s/min,centrifuge 10min,PBS washing two times. Abscising supernatant, fix it with 4℃precooling 75% alcohol,4℃overnight.1000s/min,10min,PBS wash one time,abscise supernatant,and add 200μg/ml RNase A,37℃incubating one hour; Add 20μg/ml PI,protected from light 30min 4℃.Flow cytomertry analysis EPCs cell cycle.五. Annexin-V/FITC analysis EPCs apoptosisSelect 5d EPCs;Add 40% volume fraction tumor cells supernatant;After 12h add different density (3-elemene Oμg/ml,5μg/ml,10μg/ml,20μg/ml to continue culture 48h.Trypsinizate EPCs and make it into ventrifuge tube,1000s/min,centrifuge 10min,PBS washing two times, abscising supernatant.Differently add FITC and Annexin-V according to kit ditection,incubation 10min and flow cytomertry analysis.六.RT-PCR analysis mRNA expression of EPCs VEGF and VEGFR-2Extract every group EPCs totol RNA with Trizol,according to kit ditection reverse transcription reaction;PCR.七. Statisticsl AnalysisUse SPSS13.0 software to carry out statistical analysis.Experimental result use ANOVA analysis.P<0.05 means the difference to be significant.Result八.Cultivation and identification of EPCsAfter 24h original generation cultured, the cells are small and circular majority; after Id, a few cells adhere; after 3d, some cells extend to spindle-shaped; at 5th day, there are cell colonies; after 7d, the spindle-shaped cells continue to adhere and grow well; at 14d, the cells fuse to the monolayer like endothelial cell as the paving-stone shape. The cells that AC 133 and vWF are both positive are the differentiating of EPCs.二.The effect ofβ-elemene on the proliferation of EPCs induced by tumor cells supernatantAffter different density(5、10、20μg/ml)β-elemene affect EPCs 48h and 20μg/mlβ-elemene affect EPCs different time(12、24、48h). Compare to the control, the proliferation of EPCs is depressed obviously (P＜0.01)三. The effect of p-elemene on the activity of vasculogenesis of EPCs induced by tumor cells supernatantAffter different density(5、10、20μg/ml)β-elemene affect EPCs 48h and 20μg/mlβ-elemene affect EPCs different time(12、24、48h). Compare to the control,the tube formation of EPCs is depressed obviously (P＜0.01)四. The effect of P-elemene on the apoptosis of EPCs induced by tumor cells supernatantWhen the density of P-elemene is 5μg/ml, the apoptosis of EPCs changes not obviously;When the density set up to lOμg/ml, the apoptosis of EPCs advances manifest;When 20μg/mlβ-elemene affect EPCs 24h even 48h, the apoptosis of EPCs advances obviously too.五. The effect ofβ-elemene on the cell cycle of EPCs induced by tumor cells supernatantAffter different density(5、10、20μg/ml)β-elemene affect EPCs 48h and 20μg/ml P-elemene affect EPCs different time(12、24、48h). Compare to the control, the ratio of cells in Gl phase rises greatly (P＜0.05) but in S phase decrease obviously (P＜0.05).In a word, the EPCs cell cycle was arrested in G1 phase.六. The effect ofβ-elemene on the VEGF and VEGFR-2 mRNA expression of EPCs induced by tumor cells supernatantAffter different density(5、10、20μg/ml)β-elemene affect EPCs 48h and 20μg/ml P-elemene affect EPCs different time(12、24、48h). Compare to the control,the VEGF and VEGFR-2 mRNA expression of EPCs decreased obviously (P＜0.05)Conclusions1.β-elemene can restrain the proliferation of EPCs induced by tumor cells supernatant in the time and dose dependented manner;2.β-elemene can decrease tube number formation of EPCs induced by tumor cells supernatant in the time and dose dependented manner;3.β-elemene can promote the apoptosis and arrest cells entering S phase from G1 phase of EPCs induced by tumor cells supernatant in the time and dose dependented manner;4.β-elemene can decrease VEGF and VEGFR-2 mRNA expression of EPCs induced by tumor cells supernatant in the time and dose dependented manner.更多还原