【作者】 罗俊辉；

【导师】 邹良建； 黄盛东；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 食管鳞状细胞癌(Esophageal squamous cell carcinoma,ESCC)是我国常见的恶性肿瘤,严重威胁人民群众的健康。经过多年研究,虽然其诊断与治疗状况有了很大的发展,但其发病率仍居高不下,5年生存率仍在15%-24%左右,并未得到根本性的改善。导致食管癌治疗失败的主要原因是肿瘤的复发,而造成其复发主要原因就是体内仍残存有癌细胞。两种主要因素导致体内癌细胞残留,即癌细胞的转移和肿瘤细胞对化疗药物的耐药性,前者受癌细胞的自身生物学特性影响,后者则受癌细胞耐药基因的调控。乳腺癌耐药蛋白(BCRP)作为一种肿瘤多药耐药(MDR)蛋白,同时也被认为是肿瘤干细胞的标志,因此其不仅仅影响到肿瘤的耐药性,而且还对肿瘤的很多其他生物学特征有影响,包括侵袭性、增殖能力、致瘤性等。通过研究BCRP对肿瘤的生物学特性的影响以及对转移、复发、化疗药物耐药性的关系,对指导临床上对肿瘤的早期诊断与治疗、判断患者的预后均有重大的意义,这些已在一些肿瘤中有了初步的研究,但目前尚无BCRP在食管癌生物学特性中的作用以及和食管癌转移、复发、耐药性的关系。因此,有必要对食管癌中BCRP的表达以及其作用做一个研究。鉴于以上原因,本实验首先研究BCRP在食管鳞状细胞癌组织中的表达及与患者病理分型、转移、复发、预后等的关系,然后通过建立表达BCRP的食管鳞状细胞癌细胞系来研究BCRP在食管癌耐药性中的作用及对其生物学特性的影响,并探讨其可能的机理,为食管癌的早期珍断和治疗提供新的思路和理论依据。第一部分BCRP在食管鳞癌中的表达及其临床病理意义目的:研究BCRP在人食管癌组织中的表达及与临床病理的相关性,以初步探讨BCRP在人食管癌中表达的生物学意义。方法:50例新鲜食管癌及对应癌旁正常食管组织标本,采用免疫组化法和Western-blot检测BCRP蛋白的表达,30例2年前手术的食管癌石蜡标本,采用免疫组化法检测BCRP蛋白的表达,结合临床资料对实验结果统计分析,探寻BCRP基因在人食管癌中表达的临床意义。结果:1、Western-blot结果显示食管癌组织中BCRP的蛋白表达水平高于癌旁组织,经图像分析系统分析表明,癌与癌旁组织BCRP和β-actin产物条带积分光密度值之比分别为0.37±0.19和0.30±0.15,BCRP蛋白在食管癌组织中的表达水平为正常组织的1.22倍,统计分析显示差异有显著统计学意义(P＜0.01)。2、免疫组化结果显示在食管癌组织中有50%为BCRP阳性,癌旁组织为33.75%,两者有显著统计学意义(P＜0.01);3、BCRP表达程度与患者中期生存率及早期复发相关(P＜0.05),与其他临床因素无关。(4)、术前化疗可引起癌组织BCRP的上调结论:BCRP上调表达于食管鳞癌组织,其表达与患者中期生存率、患者的早期复发有关,可以作为判断肿瘤预后的标记;化疗药物可引起BCRP的上调表达。第二部分表达BCRP的食管鳞癌可传代细胞系的建立目的:通过建立表达BCRP的食管癌可传代细胞系,为下一步研究BCRP在食管癌中的作用奠定基础。方法:通过化疗药物cDDP和羟喜树碱作用于食管癌可传代细胞株,筛选出食管癌耐药细胞株。流式细胞仪检测细胞株中BCRP阳性细胞的比例,Western-blot和细胞免疫荧光检测该细胞株中的BCRP的蛋白表达情况。检测细胞中已知对顺铂耐药起关键性作用的谷胱甘肽—S转移酶(GST)、肺耐药蛋白(LRP)的表达,检测P-gp和MRP1在细胞株中的表达,将其与BCRP表达的升高进行相关性分析,以对化疗药物引起食管鳞癌中BCRP升高的机制做初步的探讨。结果:建立了对顺铂耐药的细胞株SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0,流式细胞仪检测SHEC-4、SHEC-4-D-0.3、SHEC-4-D-0.75、和SHEC-4-D-1.0中BCRP阳性细胞的比率依次上升,SHEC-4与其他三种细胞的BCRP阳性细胞比率有统计学差异(p＜0.01),其他三种细胞系之间的差异无统计学意义(p＞0.05),Western-blot结果显示SHEC-4-D-0.3、SHEC-4-D-0.75和SHEC-4-D-1.0中BCRP的蛋白表达水平高于SHEC-4,细胞免疫荧光显示SHEC-4无荧光细胞,而SHEC-4-D-0.3、SHEC-4-D-0.75和SHEC-4-D-1.0中均可见大量的荧光细胞。LRP在SHEC-4与SHEC-4-D-1.0中的表达与其他细胞系有统计学差异(p＜0.05),GST在四种细胞系中的表达依次升高,有统计学意义(p＜0.01)。结论:通过化疗药物筛选建立了高表达BCRP的食管癌可传代细胞株。顺铂引起BCRP升高的机制可能和顺铂筛选的耐药细胞同时高表达BCRP及其他耐药蛋白有关第三部分BCRP对食管鳞癌耐药性的作用目的:研究食管癌可传代细胞系的耐药性与BCRP的关系,探讨BCRP在食管癌耐药中的作用。方法:1、BCRP的表达对食管癌耐药性的作用:SHEC-4、SHEC-4-D-0.3、SHEC-4-D-0.75和SHEC-4-D-1.0分别用不同浓度的cDDP、5-FU、卡铂、羟喜树碱、柔红霉素进行处理48小时,绘制细胞抑制率曲线,计算细胞耐药指数(RF),评价其对化疗药物耐受性的差异。2、抑制BCRP和GST活性后食管癌耐药细胞系的耐药性的变化:将BCRP的特异性抑制剂双嘧达莫和GST的特异性抑制剂利尿酸与细胞系共培养,分别用不同浓度的cDDP、5-FU、卡铂、羟喜树碱、柔红霉素进行处理,绘制细胞抑制率曲线,计算细胞耐药指数(RF)及抑制剂的逆转指数(RI)。并与未加抑制剂的细胞系进行比较。结果:SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0对cDDP、卡铂、柔红霉素、羟喜树碱的耐药指数均升高,而对5-FU的耐药指数降低。在加入双嘧达莫后,SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0对柔红霉素、羟喜树碱的RI降低,而对其他药物的RF无明显影响;加入利尿酸后,SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0对cDDP、卡铂的RF下降,对其他药物的RF无明显影响。结论:BCRP的高表达是引起食管癌耐药的一个重要原因。第四部分BCRP对食管鳞癌细胞系生物学特性的影响目的:研究BCRP对食管鳞癌细胞系生物学特性具有何种作用方法:检测食管癌可传代细胞系SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0与SHEC-4的生物学性状的异同。包括:一、BCRP对食管癌可传代细胞系体外生物学性状的作用:1、细胞形态观察;2、免疫组织化学染色检测细胞爬片的CK及Ki67表达3、细胞生长方面的检测:生长曲线、平板克隆形成率、细胞贴壁率、细胞周期;4、细胞运动实验;5、Transwell小室细胞侵袭性实验;二、BCRP对食管癌可传代细胞系体内生物学性状的作用;1、裸鼠接种成瘤实验:将不同浓度的ECCs接种于Balb/C裸鼠背部皮下,观察裸鼠皮下出现移植瘤的时间及所需细胞量;2、成瘤组织的常规病理及成瘤细胞的再培养3、流式细胞仪及western-blot检测成瘤组织中BCRP的表达;结果:一、BCRP对食管癌可传代细胞系体外生物学性状的作用:SHEC-4较另三种细胞体积略大,四种细胞形态差别不大;四种ECCs均呈CK及Ki67阳性,生长曲线均呈“S”型,群体倍增时间分别为28.79h、34.98h、35.69h、35.8h,后三组ECCs的群体倍增时间与SHEC-4相比明显延长,有显著的统计学意义(p＜0.01)。后三组细胞系在平板形成细胞数大于50个的克隆形成率较SHEC-4明显上升,但无统计学意义(p＞0.05);ECCs冻存复苏后细胞的接种率分别为70%～81%;细胞周期显示SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0的S期比例增加,SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0的细胞迁徙能力和细胞侵袭能力均较SHEC-4明显上升。二、BCRP对食管鳞癌可传代细胞株体内致瘤性的影响接种1×10~5个瘤细胞的SHEC-4、SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0在4周后各分别有75%、75%、100%、75%成瘤,接种1×10~4个瘤细胞SHEC-4未见成瘤,而SHEC-4-D-0.3、SHEC-4-D-0.75、SHEC-4-D-1.0的成瘤率均为25%,接种1×10~3个瘤细胞的成瘤率分别为0%、25%、50%和25%,接种1×10~2个瘤细胞组均未成瘤。成瘤的体积无明显差别。体内的肿瘤细胞的形态和BCRP含量与亲代无明显差别。结论:BCRP在食管鳞状细胞癌中的表达明显提高了肿瘤细胞的增殖能力、致瘤性、侵袭性,可能与食管癌的转移、复发相关。全文总结1、BCRP上调表达于食管鳞癌组织,其表达与患者中期生存率、患者的早期复发有关,化疗后患者BCRP表达升高,说明某些食管鳞癌天生具有耐药性,化疗药物可以调高BCRP在癌组织中的表达,BCRP可以作为预测患者的预后的一个指标。2、利用顺铂对ECCs进行筛选可建立高表达BCRP的食管鳞癌可传代细胞株,BCRP的表达升高的可能机制是BCRP与其他耐药蛋白如LRP共表达于同一细胞亚群并被优势克隆,因此,在临床上使用化疗药物会可能导致BCRP的高表达。3、BCRP的上调使食管癌对某些化疗药物(喜树碱类、柔红霉素等)具有耐药性,这些耐药性可为BCRP的抑制剂所逆转,为临床上解决食管癌对化疗药物的耐药提供了思路。4、BCRP的高表达在体内和体外均与食管鳞状细胞癌ECCs的增殖能力、侵袭性、致瘤性有关,可能与食管癌的转移、复发相关。更多还原

【Abstract】 Esophageal squamous cell carcinoma(ESCC) is a common cancer in China with high incidence,which threaten the health of people seriously.Although great progress in the diagnosis and therapy of EC have been made in the last decades,the cure rate of EC has not been improved yet and 5 year survival rate still remained around 20%-30%. Recurrences occur frepuently in EC patients,which are the leading cause of death,and the main cause of recurrences is the remnant of cancer cell.Metastasis and drug resistance is the main cause of remnant of cancer cell.Metastasis was adjusted by bionomics of cancer,and drug resistance was adjusted by drug resistance gene.As a multi-drug resistance(MDR) protein,breast cancer resistance protein(BCRP) is a marker of tumor stem cell.So BCRP not only effect the drug resistance of cancer,but also effect the bionomics of cancer,including invasion activity,proliferation activity and oncogenicity.To study the effect of BCRP in cancer biological characteristics, metastasis,recurrences and drug resistance is owned important significance on caner early diagnosis,therapy and Prognostic in clinically,which indicated in some cancer.To our knowledge,there is no report about the effect of BCRP in ESCC’s biological characteristics,neither report about the relationship between BCRP and ESCC’s metastasis,recurrences and drug resistance now.So it is necessary to study the expression and effect of BCRP in ESCC.According to the mentioned above,we performed the following experiments.Fistly, we investigated the expression of BCRP in esophageal cancer samples and explored its correlation with clinical and pathological characters.Then we established ESCC cell line. Based on above work,we investigated the effect of BCRP in ESCC’s drug resistance and the relationship between BCRP and ESCC’s biological characteristics.We also explored the possible mechanism of this effect.Purpose a new theoretical basis of early diagnosis and therapy for esophageal cancer.Part1:Expression of BCRP in ESCC and its possible implicationsObjective:To determined the expression of BCRP in ESCC,and to investigate the association of BCRP with ESCC.Methods:Western-blot and immunohistochemical technique were used to detect the expression level of protein of BCRP in 50 pairs of fresh esophageal cancer tissues and according para-cancer normal tissues.Paraffin-embedded sections were obtained from 30 patients with ESCC for immunohistochemical studies to detect the expression level of protein of BCRP.Implications between the expression of CBP and clinical and pathological characters was Analyzed.Results:1.The expression levels of BCRP by Western-blot in cancer tissues were 1.22-fold(P＜0.05) higher than those in para-cancer normal tissues respectively.The decreased expression of CBP gene in lung cancer tissues was significantly associated with TNM stage(P＜0.05)and lymph node metastasis(P＜0.05);2.The positive rate of BCRP in cancer and normal tissue was 50%and33.75%respectively and the difference was very significant(P＜0.01) by immunohistochemical;3.The increased expression of BCRP gene in esophageal cancer tissues was significantly associated with histology differentiated stage (P＜0.05),survival time and recurrences(P＜0.05).Conclusions:Increased expression of BCRP in esophageal cancer is tumor specific, contributed to survival time and recurrences in ESCC,may be considered as a supplementary predictor prognostic.Part 2:Establishment of ESCC continuous cell line which expressed BCRPObjective:To establish ESCC continuous cell line which expressed BCRP,in order to study its effects of BCRP on the biological behavior of esophageal cancer.Methods:To establish a multi drug-resistant cell line,ESCC continuous cell line cells were cultured with intermittent gradual-increasing-concentration of cisplatin(cDDP) and hydroxycamptotbecine(HCT).Detect proportion of BCRP positive cells of ESCC by FCM,and detect BCRP protein by Western-blot and cell immunofluorescence.Detection known to cisplatin resistance from the key role of glutathione-S-transferase(GST), pulmonary resistance protein(LRP),P-gp and MRP1 expression to identify whether resistant to cDDP was originated by BCRP.Results:Established SHEC-4-D-0.3,SHEC-4-D-0.75 and SHEC-4-D-1.0 which resistant to cDDP.The proportion of BCRP positive cells increased one by one(SHEC-4 vs SHEC-4-D-0.3(0.75,1.0),p＜0.01),but not statistically significant between SHEC-4-D-0.3,SHEC-4-D-0.75and SHEC-4-D-1.0.Western-blot showed that protein expression level of SHEC-4-D-0.3 and SHEC-4-D-0.75,SHEC-4-D-1.0 increased to SHEC-4.Immunofluorescence showed SHEC-4 has no fluorescence cell,however,a great deal of fluorescence cells was found in SHEC-4-D-0.3/0.75/1.0.Expression of LRP in SHEC-4 and the three resistance cells with a significant difference(p＜0.05),and GST in the four cell lines were increased in statistical significance(p＜0.01).Conclusions:Established ESCC continuous cell line which expressed BCRP by chemotherapy drugs.The up regulation mechanism of BCRP by Cisplatin may be cisplatin resistant cells at the same time high expression of BCRP and other resistance proteinPart 3:Effect of BCRP to drug resistant of ESCCObjective:To study the relationship between ESCC continuous cells’ drug resistant with BCRP,to explore the effect of BCRP to drug resistant of ESCC.Methods:1.Effect of expression of BCRP in ESCCs’ drug resistant:SHEC-4, SHEC-4-D-0.3,SHEC-4-D-0.75 and SHEC-4-D-1.0 was cultured with different concentration of cDDP,5-Fu,Carboplatin,HCT and Daunorubicin(DNR) for 48 hours,then draw curve of cell inhibition ratio and calculate cell resistant index(RF) in order to evaluate their resistance to chemotherapy drugs.3.Drug resistant of ESCCs changed by use inhibitor of BCRP and GST:ESCCs were cultured with dipyridamole (specific inhibitor of BCRP) and Ethacrynic acid(specific inhibitor of GST),then cultured with different concentration of cDDP,5-Fu,Carboplatin,HCT and Daunorubicin(DNR) for 24 hours,draw curve of cell inhibition ratio and calculate cell resistant index(RF) and reverse index(RI) in order to evaluate their resistance to chemotherapy drugs,compared with ESCCs which not cultured with inhibitor.Result:SHEC-4-0.3 cDDP,SHEC-4-D-0.75,SHEC-4-1.0 pairs cDDP,carboplatin,DNR, HCT resistance index increased,while the 5-FU resistance index lower.After joining the dipyridamole,the RI of SHEC-4- cDDP-0.3,SHEC-4-D-0.75,SHEC-4-D-1.0 to daunorubicin and I hydroxycamptothecin lower,and on other drugs RF no significant impact.In SHEC-4—0.3 cDDP,SHEC-4-D-0.75,SHEC-4—1.0,RF dropped by join Ethacrynic acid,but no significant effect on the other drugs.Conclusion:The expression of BCRP in esophageal cancer is an important reason for drug resistance.PartⅣ:The Effect of BCRP on biological characteristics of esophageal squamous cell carcinomaObjective:To study the effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line. Methods:Detect the similarities and differences of esophageal cancer cell lines SHEC-4—0.3 cDDP,SHEC-4-D-0.75,SHEC-4—1.0 cDDP SHEC-4’s biological characteristics.Include:A:The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vitro:1.Cell morphology;2.Immunohistochemical staining of CK and Ki67 expression3.Detection of cell growth:Growth curve,flat colony formation rate,the rate of adherent cells,the cell cycle;4.Experimental of cell migration5.Transwell cave invasive experimentalB:The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vivo:1.Tumorigenicity in nude mice inoculated experiment:Different concentrations of ECCs will be inoculated in Balb/C mice back subcutaneous,observed in nude mice transplanted tumor cells and the necessary amount of time to observe the size of a tumor;2.The routine pathological of tumor tissue and cultured tumor cells again.3.FCM to tumor tissue in the expression of BCRPResult:A.The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vitro:SHEC-4 cell volume than the other three slightly,four cell morphology little difference;CK and Ki67 showed positive in four ECCs.Growth curve showed the "S" curve,population doubling time was 28.79 h,34.98h,35.69h,35.8h,the three ECCs’ population doubling time compared with SHEC-4 was significantly prolonged,and have statistical significance(p＜0.01).The number of colony formation rate of the three groups of cells increased than SHEC-4,but has no significance(p＞0.05);ECCs frozen cells after vaccination recovery rate of 70%to 81%.Cell cycle show SHEC-4—0.3 cDDP, SHEC-4-D-0.75,SHEC-4—1.0 cDDP proportional to the S phase,SHEC-4—0.3 cDDP, SHEC-4-D-0.75.SHEC-4—1.0 cDDP cell migration and cell invasion capacity increased significantly than SHEC-4.B The effect of BCRP in biological characteristics of esophageal squamous cell carcinoma cell line in vivo: Four weeks after Vaccination of 1×10~5 cells SHEC-4,SHEC-4-D-0.3, SHEC-4-D-0.75,SHEC-4-D-1.0 the tumor occurred 25%,62.5%,50%,75%respective. For 1×10~4 cells:SHEC-4 no tumor,and SHEC-4—0.3 cDDP,SHEC-4-D-0.75,cDDP SHEC-4-DDP-1.0 rate of 12.5%,1×10~3 inoculated tumor cells into the rate was 0%,0%, 25%and 12.5%,1×10~2 inoculation of tumor cells did not found,no significant difference between volume,no significant difference of the shape of the tumor cells and BCRP% between pro-generation content and filial generation(p＞0.05).Conclusion:Expression of BCRP in esophageal squamous cell carcinoma was significantly increased tumor cell proliferation,tumor,invasive,and may related with esophageal cancer metastasis and recurrence.Full summary:1.BCRP up-regulated expression in esophageal squamous cell carcinoma,correlated with differentiation,survival of patients with life,the recurrence rate.BCRP can be used for predicting the prognosis of patients.2.The use of cisplatin on ECCs screening can establish high expression in BCRP of esophageal squamous cell carcinoma.The possible mechanisms of the BCRP increased is the co-expression of LRP/BCRP expression in the same cell subsets and advantages cloning,therefore,clinical use of cisplatin also may lead high expression of BCRP.3.Up regulation of BCRP to esophageal cancer can lead resistant to some chemotherapeutic drugs(camptothecin,daunorubicin,etc.),these resistance can be reversed by the BCRP inhibitors.Clinical solution of esophageal cancer chemotherapy could use BCRP inhibitors too.4.Expression of BCRP in vivo and in vitro can be markedly increase esophageal squamous cell carcinoma’s proliferation,invasiveness,tumorigenicity,and may be related to esophageal cancer metastasis and recurrence.更多还原