肿瘤低氧微环境与氧载体化疗辅助效应研究

【作者】 韩建群；

【导师】 修瑞娟； 刘谦；

【作者基本信息】 中国协和医科大学 ， 生物医学工程， 2008， 博士

【摘要】 目的:探讨氧载体在辅助肿瘤化疗中的潜在价值,建立两种荷瘤小鼠模型,动态活体显微观测氧载体对接种肿瘤新生血管微循环结构和功能的影响:研究氧载体对肿瘤低氧微环境的影响及其机制。方法:(1)体外培养人宫颈癌Hela细胞,将2×10~6个HeLa细胞接种于雌性金黄地鼠右侧颊囊上,构建颊囊部位的粘膜下肿瘤模型。(2)将动物模型随机分为4组(n=10),第一组:对照组,颈静脉插管,注射生理盐水,1周两次;第二组:单纯化疗组,腹腔注射顺铂注射液(5毫克/公斤体重),1周两次;第三组:化疗联合低剂量氧载体(PEG-Hb)组,腹腔注射顺铂注射液(5毫克/公斤体重)+颈静脉插管注射PEG-Hb(0.3克/公斤体重),1周两次;第四组:化疗联合高剂量氧载体(PEG-Hb)组,腹腔注射顺铂注射液(5毫克/公斤体重)+颈静脉插管注射PEG-Hb(0.6克/公斤体重),1周两次。(3)接种肿瘤细胞前及接种后第3、5和7天利用计算机辅助的活体显微观测系统动态连续观测和记录瘤体及其新生血管的形态变化,并通过图像分析软件计算瘤体近、远端微血管面积密度和迂曲度的动态变化;观察并定量分析接种肿瘤细胞后第七天血管中白细胞运动状态的变化,选择直径在40μm左右的小静脉,定量计算滚动及黏附白细胞的数量,测量方法分别采用1分钟内通过观测区段的非黏附白细胞占全部白细胞的百分比和观测区段每平方毫米血管内皮表面的黏附白细胞数。(4)1周后处死动物,处死前1小时腹腔注射外源性生物还原低氧标记物哌莫硝唑(60毫克/公斤体重),处死后免疫组化检测外源性低氧标记物hypoxyprobe-1,以及内源性低氧标记物HIF-1α的表达,评价肿瘤组织hypoxyprobe-1和HIF-1α的变化。(5)ELISA法检测血清中可溶性内皮细胞黏附分子-1(sICAM-1)以及致炎细胞因子TNF-α和IL-6的含量。(6)体外培养人宫颈癌HeLa细胞,将2×10~6个HeLa细胞接种于3～4周雌性BALB/c裸鼠腋下,构建荷瘤裸鼠模型。(7)接种肿瘤细胞两周后,将裸鼠随机分为5组(n=10),第一至四组分组和处理方法与金黄地鼠实验类似,相应处理1周两次。第五组:单纯氧载体PEG-Hb组,尾静脉注射PEG-Hb(0.45克/公斤体重),1周两次。(8)药物处理1月后处死动物。处死前大约1小时腹腔注射外源性生物还原低氧标记物哌莫硝唑(60毫克/公斤体重),处死后免疫组化检测外源性和内源性低氧标记物(HIF-1α)的表达水平,检测肿瘤组织Hypoxyprobe和HIF-1α的变化。免疫组化测定EPO/EPOR、MDR1和CD31的表达水平;(9)提取肿瘤组织总蛋白,免疫印迹检测氧载体辅助化疗后,瘤体组织中EPO/EPOR、MDR1和HIF-1α的表达。(10)ELISA法检测血清中EPO的表达水平。结果:(1)实体瘤的生长过程中伴随有明显的血管新生,新生血管表现为迂曲,扩张,囊化和血管周围血液渗出增加等形态变化,尤以瘤体近端明显。模型鼠一般特性的变化:各药物处理组动物体重在第3、5和7天均较对照组明显降低(P＜0.05),瘤体体积在第3、5和7天均较对照组明显降低(P＜0.01),各处理组间无显著差异。(2)图像分析显示各药物干预组在第3、5和7天较对照组瘤体近端的微血管面积密度和迂曲度均降低(P＜0.05)。在瘤体远端的微血管迂曲度各组间无明显差异。瘤体远端微血管面积密度在第5和7天第四组较第二组明显降低(P＜0.05)。瘤体近端微血管迂曲度仅在第5天第四组较第二组明显降低,瘤体近端微血管面积密度在第5、7天第四组较第二组明显降低,应用顺铂后引起白细胞在小静脉内明显黏附和滚动,加用PEG-Hb后对白细胞的黏附和滚动没有影响。(3)免疫组化染色显示金黄地鼠瘤组织Hypoxyprobe-1和HIF-1α表达在第4组均较第2组明显降低(P＜0.05)。(4)金黄地鼠各组间血清sICAM-1、TNF-α和IL-6表达水平在第二、三和四组均较对照组明显增加,加用PEG-Hb并未降低炎性因子浓度。(6)在裸鼠实验中,联合应用PEG-Hb可以增强顺铂的抑瘤效应,瘤体体积在第4组下降最明显,较单纯化疗组显著(P＜0.05)。单纯PEG-Hb组与对照组没有明显差异。(7)免疫组化测定低氧标记物的结果显示Hypoxyprobe-1和HIF-1α表达在第四组较第二组下调明显(P＜0.05)。EPO/EPOR、MDR1和CD31在大剂量PEG-Hb联合化疗组较单纯化疗组表达明显降低。而小剂量PEG-Hb联合化疗组较单纯化疗组无明显下降。EPO与HIF-1α表达水平呈正相关(P=0.021 r=0.76)。(8)Western blot检测瘤体HIF-1α、EPO/EPOR和MDR1表达在第四组较第二组明显降低(P＜0.05)。(9)ELISA测定裸鼠血清中EPO浓度在第四组较第二组明显下降(P＜0.05)。结论:(1)金黄地鼠颊囊荷瘤模型适合于观测肿瘤新生血管微循环的变化,为综合评价和分析肿瘤、抗癌药物和肿瘤微环境之间相互作用提供了一个良好的平台。(2)肿瘤生长过程中存在血管新生和组织低氧。大剂量PEG-Hb联合化疗可明显改善金黄地鼠肿瘤模型肿瘤低氧区域的氧供,抑制因低氧而导致的血管新生,降低瘤体近端的微血管面积密度和迂曲度,使不规则和紊乱的微血管网趋于正常化。(3)在较短药物干预和观测期内,加用氧载体与否对动物体重和瘤体大小没有明显影响。(4)化疗药物顺铂可以引起内皮损伤和炎症反应,小静脉中黏附和滚动白细胞数量增加,血清炎性因子TNF-α、IL-6以及内皮损伤标志物sICAM-1表达增加,联合不同剂量的PEG-Hb未能减轻顺铂诱发的炎症反应。(5)裸鼠肿瘤模型可用于客观的观察PEG-Hb联合化疗对瘤体生长和动物一般特性的影响,化疗联合大剂量PEG-Hb较单纯化疗增加抑瘤率,瘤体明显缩小,PEG-Hb的化疗增敏效应与剂量有关。(6)大剂量PEG-Hb的辅助化疗效应可能是通过改善低氧微环境,内源性及外源性低氧标志物下调;HIF-1α发挥转录因子的中枢作用,作用相关调控基因,减少低氧适应性反应;抑制EPO/EPOR信号的传递,降低MDR1的表达逆转耐药和继发的抗血管新生效应。目的:观察不同浓度和作用时间的氯化钴对体外培养的肿瘤细胞活力、增殖和凋亡的影响,探讨建立合理实用的体外肿瘤化学低氧模型的模式。方法:(1)体外培养肺癌细胞株A549和人宫颈癌HeLa细胞株,MTT方法和流式细胞仪(FCM)检测不同浓度的氯化钴(0、10、50、100、200、400、800和2000μmol/L),在相应的时间(4、8、12、24、48小时)内对肿瘤细胞活力以及增殖和凋亡的影响。(3)氯化钴处理肿瘤细胞后提取总蛋白,免疫印迹测定内源性低氧标记物HIF-1α和相关凋亡蛋白的表达。结果:(1)终浓度≤200μmol/L氯化钴处理肿瘤细胞24小时,MTT检测细胞活力几乎未受影响,而加大氯化钴浓度或延长处理时间明显降低细胞活力。(2)流式细胞仪测定低浓度氯化钴(200μmol/L)处理细胞后受损细胞多为早期凋亡细胞。高浓度氯化钴(800μmol/L)作用后受损细胞多为晚期凋亡和坏死细胞,而且A549细胞的凋亡率明显高于HeLa细胞,分别为39%和29%。(3)Western blot结果显示氯化钴(200μM)处理A549相应时间(4、8、12、24、48hr)后,HIF-1α表达上调,24小时达高峰(P＜0.05),延长处理时间则下调,Bax和P53表达上调,而Bcl-2表达下调;氯化钴(200μmol/L)处理HeLa细胞相应时间后HIF-1α表达上调,24小时达高峰,延长处理时间则下调,Bax表达上调,而Bcl-2表达下调。P53基础表达水平低,且细胞经过氯化钴处理后表达无变化。氯化钴处理A549不同浓度(0、50、100、200和400μmol/L)相同时间(24小时)HIF-1α表达与作用浓度呈正相关,200μmol/L时达高峰(P＜0.05),增加浓度则下调,Bax和P53表达上调,而Bcl-2表达下调。相同方式处理HeLa细胞,结果HIF-1α表达与氯化钴浓度平行变化,200μmol/L时达高峰,增加浓度则下调。Bax表达上调,而Bcl-2表达下调,P53表达水平低且无显著变化。结论:(1)氯化钴依作用浓度和时间影响体外培养的肿瘤细胞增殖和凋亡,中低浓度氯化钴在较短时间内处理细胞,受损细胞多为早期凋亡。高浓度(800μmol/L)处理后受损细胞为晚期凋亡和坏死成分。(2)HeLa细胞P53表达水平低不参与细胞凋亡过程,不同细胞株间凋亡率有差异,对氯化钴的反应性不同。(3)氯化钴诱导内源性低氧标志物HIF-1α的表达在一定范围内(～200μmol/L,24小时)是正相关的,与抗凋亡蛋白的表达同向平行。浓度增加和处理时间延长导致HIF-1α表达下调和细胞凋亡率增加,背离模拟低氧的初衷。(4)在体外进行化学性模拟低氧时,要慎重考虑氯化钴的作用浓度和时间,本试验提示氯化钴在浓度200μmol/L左右处理24小时是一个较佳的模拟低氧模式。更多还原

【Abstract】 Objective:The aim of current study was to investigate the potential benefit of oxygen carrier in tumor chemotherapy.The effect of oxygen carrier on the microcirculation of tumor tissues based on two established tumor models was observed and evaluated.The changes of tumor oxygenation and neovascularization after oxygen carrier administration and the possible underline mechanism were explored.Methods:（1） Cultured HeLa cells were injected into submucosa of 3-4-week-old female golden hamster cheek pouch to build the tumor model.（2） Fourty tumor burden hamsters were randomly assigned to four groups（n=10） and treated respectively:group 1: control group,hamsters were administrated with saline twice a week through a right jugular vein catheter;group 2:cisplatin group,the hamsters were administrated with cisplatin（5mg/kg b.w.） twice a week;group 3:cisplatin group plus low concentration of PEG-Hb,the hamsters were administrated with cisplatin（5mg/kg b.w.） plus PEG-Hb （0.3g/kg b.w.） twice a week.Group 4:cisplatin plus high concentration of PEG-Hb group, the hamsters were administrated with cisplatin（5mg/kg b.w.） combined with PEG-Hb （0.6g/kg b.w.） twice a week.（3） Computor assisted intravital microscopy system was used in observing and recording tumor neovascularization on 0、3、5、7 day post innoculation, then off-line analyze images to detect the changes of the microvessel tortuosity and area functional capillary density.Leukocytes movement pattern in venule was also measured on the 7th day after innoculation.（4） Immunohistochemistry of endogenous hypoxia maker （HIF-1α） and exogenous hypoxia maker（Hypoxyprobe-1） were tested in tumor tissues.（5） Serum level of soluble Intercellular adhesion molecule-1（sICAM-1）,TNF-αand IL-6 were detected by enzyme-linked immunosorbent assay（ELISA）.（6） Cultured HeLa cells were subcutaneously injected to the armpits of 3-4-week-old female BALB/c nude mice to built another tumor model.（7） One month post-innoculation,the nude mice were randomly assigned to five groups（n=10） and treated respectively,group 1-4 were similar to golden hamsters’ arrangement;group 5,PEG-Hb group,the nude mice were administrated with PEG-Hb（0.45g/kg b.w.） twice a week.（8） Animals were sacrificed after a month of respective treatment,tumor tissue hypoxia situation was also evaluated by endogenous hypoxia maker（HIF-1α） and exogenous hypoxia maker（Hypoxyprobe-1）. Immunohistochemical staining method was used to detected the expression level of EPO,EPOR,MDR1 and CD31 in tumor tissue.（9） Western blot method was used to detected the expression of EPO/EPOR,MDR1 and HIF-1αin tumor tissue.（10） Serum level of EPO was detected by ELISA.Results:（1） In the concomitance with solid tumor development the angiogenesis is notable,tortuosity,dilation,sacculation and permeability morphological changes are the specific features of newly formed tumor vessels.In golden hamster experiment,the body weight and tumor volume on 3,5,7 day post-innoculation both were significantly decreased in group 2,3 and 4 compared with control group.（2） Microvessel tortuosity was increased post-innoculation,which was more intensive in the inner ring than the outer ring of the tumor.On the fifth day the tortuosity in group 4 was markedly reduced compared with group 2 in peritumoral area.Area capillary density was increased parallel with the tumor development,chemotherapy treatment significantly decreaed it in the inner ring surrounding tumor mass.On the fifth and seventh day the area capillary density in group 4 was markedly reduced compared with group 2.Cisplatin can induce leukocyte rolling and sticking to venule,which was not effected with PEG-Hb administration.（3） The expression of Hypoxyprobe-1 and HIF-1αwere significantly decreased in group 4 compare with group 2（P＜0.05）.（4） Cisplatin can up-regulate pro-inflammatory factors（sICAM-1, TNF-α,IL-6）,which was not influenced with PEG-Hb treatment.（6） In BALB/c nude mice study,chemotherapy combined with PEG-Hb administration can enhance its tumor inhibition effect,after treatment the tumor volume was decreased sharply in group 4.（7） The lowest hypoxia marker expression was detected in group 4.EPO/EPOR,MDR1 and CD31 were decreased in group 4 compare with group2（P＜0.05）.EPO level was positive correlation with HIF-1αexpression（P=0.021 r=0.76）.（8） Serum level of EPO was decreased in group 4 compare with group2（P＜0.05）.Conclusion:（1）The tumor model established in golden hamster cheek pouch is a ideal model for microvascular observation.It is favorable in tumor newly formed vessels observation and evaluation of drugs influence on tumor microcirculation.（2）Tissue hypoxia and angiogenesis are concomitant with neoplasma,high concentration of PEG-Hb combined with cisplatin can be benefit to tumor tissue oxygenation,subsequently contribute to anti-angiogenesis and tumor microvessels normalization.（3） There are no significant difference in body weight and tumor volume changes when cisplatin combined with or without blood substitute in a short time period.（4） Cisplatin treatment involves in endothelium injury and inflammatory reaction,adding blood substitute could not depress inflammation.（5）After one month treatment nude mice tumor volume inhibited obviously in group 4,blood substitue concentration is an important factor in assistant tumor chemotherapy.（6） EPO/EPOR and MDR1 which are modulated by HIF-1αmight participate in the process of blood substitue chemotherapy sensitizasion. Objective:To investigate the effect of cobalt chloride on tumor cells proliferation and apoptosis in vitro,to explore the reasonable strategy of cobalt chemical mimic hypoxia.Method:Two tumor cell lines（A549 and HeLa） were cultured in vitro,After the cells were exposed to CoCl₂（0.05～2mmol/L） for different time period（4～48h）,cells viability、proliferation and apoptosis were maesured by MTT and FCM methods.HIF-1αand related apoptosis proteins expression were detected by Western blot.Result:At concentration of 2001μmol/L within 24hr cell viability was weakly changed. However,higher dose or prolonged challenge of CoCl₂ significantly decreased cell survival rate（p＜0.05）.It is clearly showed that most of the damaged cells were early apoptosis population after CoCl₂（200μmol/L） incubation within 24hr,contrary to a higher concentration of CoCl₂（800umol/L） most of the damaged cells were late apoptosis and necrosis parts.Moreover,A549 cell apoptosis rate was significantly higher than HeLa cell （39%vs29%）.Western blot analysis revealed that CoCl₂（200μmol/L） exposure implicated in up-regulating the expression of HIF-1α,Bax,p53 and down-regulating the expression of Bcl-2 in A549 cell within 24hr,Similar results were observed in HeLa cell,except p53.A low level of p53 protein expression was detected with or without CoCl₂ in HeLa cell.But higher concentration（～400μM） or prolonged CoCl₂ challenge changed the tendency of HIF-1αexpression in the two lines as well as Bcl-2,Bax.Therefore,this feature illustrated that prolonged or higher dose of cobalt exposure was not positive to expected chemical hypoxia effect（HIF-1αexpression）.Conclusion:Dose and time-dependent effect of cobalt chloride on the tumor cells proliferation and apoptosis,most of the damaged cells were early apoptosis population after cobalt（200μM） incubation within 24hr,higher dose of CoCl₂ caught late apoptosis and necrosis.P53 did not involved in the process of HeLa cell’s apoptosis.The apoptosis effect of CoCl₂ is tumor cell specific.In a limited range of cobalt concentration HIF-1αexpression posseses positive relation with cobalt.But prolonged or higher dose of cobalt exposure was not positive to expected chemical hypoxia effect（HIF-1αexpression）.The concentration around 200μM within 24hr period time may be a reasonable choice in chemical hypoxia mimicry.The concentration and duration of cobalt chloride should be taken into consideration in chemical mimic hypoxia.更多还原