【作者】 黄修燕；

【导师】 汤钊猷；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 肝细胞癌（Hepatocellular carcinoma,HCC）（简称肝癌）的研究近年取得了不少进展,治疗多以消灭肿瘤为主要目标（如手术、放疗和化疗）,越来越多的肝癌患者获得长期生存,手术切除仍是最有效的治疗方法之一。然而,近20年小肝癌切除的5年生存率已不再提高,究其原因,主要是术后转移复发率高,而癌转移复发归根到底是由于癌的生物学特性。复旦大学肝癌研究所（简称我所）报道2333例肝癌切除术中,姑息性切除比例占37.2%（868/2333）,其1,3,5,8,10年生存率均低于同期根治组者。姑息性切除疗效较差的原因之一可能是促进了残癌的生长和播散,为此需研究干预的靶点和有效的干预途径。既往有研究表明外科手术促进肿瘤生长、转移和血管生成的作用。迄今,尚无姑息性肝癌切除术对残癌侵袭转移潜能影响的研究报道。本课题通过构建姑息性肝癌切除术模型,研究姑息性肝癌切除术后残癌侵袭转移潜能变化,并检测其侵袭转移潜能相关基因表达的改变,期望发现与残癌侵袭转移潜能改变密切相关的基因表达改变;通过基因表达谱芯片生物信息分析方法,确立用于评估残癌侵袭转移潜能改变的分子诊断模型;应用基于特征性致病基因的基因聚类和多维量表构建高、低转移组基因功能网络,并比较网络结构差异;从mRNA水平加以验证,为肝癌侵袭转移研究提供新的分子标记和治疗靶点。近年,关于中药抗肿瘤的研究引起国内国际重视,肿瘤的常规治疗联合中医（Traditional Chinese Medicine,TCM）治疗有协同作用,可增强抑瘤效应。中药小复方“松友饮”包括丹参、黄芪、枸杞、焦山楂和炙鳖甲5种中药提取物,经中国食品药品监督管理局（Food and Drug Administration,FDA）批准,批准号为国食健字G20070160;由中国科学院（Chinese Academy of Sciences,CAS）上海药物研究所（Shanghai Institute of Materia Medica,SIMM）确定该方剂及各组方的高效液相指纹图谱（fingerprints by high-performance liquid chromatography,HPLC-FP）。本研究通过体内、体外实验研究中药小复方“松友饮”对高转移潜能人肝癌（MHCC97H）细胞的作用及其机制。在我所前期对干扰素α（interferon-α,IFNα）研究的基础上,进一步研究“松友饮”和IFNα联用对姑息术后残癌生长,转移和基因表达的影响,为生物治疗与中药联合应用治疗肝癌患者的临床推广提供一定的实验依据。1.姑息性肝癌切除术对残癌侵袭转移潜能的影响姑息性肝癌切除术后残癌侵袭转移潜能是否发生改变,侵袭转移潜能如何发生改变并无明确的研究报道。我们构建姑息性肝癌切除术模型,肺转移率达100%的高转移潜能人肝癌细胞系MHCC97H用于体内研究。BALB/C-nu/nu雄性裸小鼠经肝左叶原位接种肝癌组织,接种肿瘤后14 d随机分组。姑息组切除大部分病灶和部分肝脏,在瘤体深部保留2 mm大小肝癌组织;对照组做假手术（仅麻醉、开腹）。采用残癌原代培养,侵袭实验,明胶酶谱实验,酶联免疫吸附实验（enzyme-linked immunosorbent assay,ELISA）和免疫组化实验（immunohistochemistryassay,IHA）等方法研究术后不同时段残癌恶性表型变化及其可能的机制。结果显示,姑息性肝癌切除术后残癌侵袭潜能呈增强趋势,术后14 d姑息组与对照组侵袭潜能差异最明显（p=0.015）;术后35 d,姑息组肺转移结节数目多于对照组（14 Vs 9;p=0.041）,上述结果表明姑息性肝癌切除术促进残癌侵袭转移潜能。2.姑息性肝癌切除术对残癌侵袭转移潜能相关基因表达的影响BALB/C-nu/nu雄性裸小鼠经肝左叶原位接种MHCC97H肝癌组织,接种肿瘤后14 d随机分组。姑息组:切除大部分病灶和部分肝脏,在瘤体深部保留2 mm大小肝癌组织;对照组1:做假手术（仅麻醉、开腹）;对照组2:不做任何干预。姑息术后14 d处死裸鼠获取肿瘤标本,利用肿瘤转移相关分类功能基因芯片（Oligo Tumor Metastasis Microarray,Super Array）检测基因表达,使用综合型GEArray表达分析配套软件（GEArray Expression Analysis Suite）进行完整的芯片数据分析。此外,应用差异基因显著性分析,差异基因分组关联度分析及支持向量机（support vector machine,SVM）筛选肿瘤侵袭转移性诊断标志基因;应用基于特征性致病基因的基因聚类和多维量表构建基因功能网络,比较网络密度和凝聚度以及枢纽基因的组间变化特点。结果发现,由12个基因（BAI1,MTA2,MTA1,SMAD2,GNRH1,CDH8,ITGB3,CHD4,GZMA,ITGA7,CXCR4和TSHR）组成的分子诊断模型对肝癌高、低转移分组的阳性率分别为88.9%和93.5%:MTSS1、TGFβ1、SMAD2、IL1β及MMP7基因在高侵袭转移组基因网络中处于核心位置。根据log₂（姑息组/对照组）≥1或log₂（姑息组/对照组）≤—1,且差异有统计学意义（p＜0.05）判断差异基因表达,确定两个与姑息术后残癌侵袭转移潜能变化一致的基因,1个呈负相关（BAI1）,1个呈正相关（MTA2）;并通过实时荧光定量PCR（real-time fluorescence quantitative polymerase chainreaction,Real-time PCR）加以验证。3.中药小复方“松友饮”对肝癌的体内体外作用及可能的机制探索体外研究采用高转移潜能人肝癌MHCC97H细胞,体内研究采用原位接种MHCC97H肿瘤的裸鼠模型。结果发现,“松友饮”可激活caspase-3诱导MHCC97H凋亡,进而抑制肝癌增殖;通过抑制基质金属蛋白酶-2（matrixmetalloproteinase-2,MMP2）抑制肝癌侵袭转移潜能。“松友饮”可减轻荷瘤裸鼠体重下降;抑制增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）和血管生长因子（vascular endothelial growth factor,VEGF）的表达;降低微血管密度（microvessel density,MVD）;肿瘤生长抑制率为53.7%;“松友饮”组肺转移程度下降与对照组相比有统计学意义（p＜0.01）;“松友饮”延长荷瘤裸鼠生存期（与对照组比为了5 Vs 52,d,p＜0.001）。实验过程中没有出现“松友饮”相关性的体重下降超过10%,或者血清转氨酶异常者。4.“松友饮”联合干扰素α（IFNα）对姑息术后残癌的协同抑制作用荷高转移潜能人肝癌（MHCC97H）的裸鼠作姑息性肝切除后,随机分为对照组（皮下注射及口服蒸馏水）,“松友饮”组（口服“松友饮”3.6 g/kg/d,皮下注射蒸馏水）,干扰素α组（皮下注射IFNα7.5×10⁶ U/kg/d,口服蒸馏水）,和“松友饮”合并干扰素α（口服“松友饮”3.6 g/kg/d,皮下注射IFNα7.5×10⁶ U/kg/d）4组。结果,4组肿瘤体积分别为1205.2,724.9,507.6,245.3 mm³;微血管密度（MVD）分别为44,35,23,18个;肺转移结节分别为7,6,4,3个;生存期分别为57,68,77,89 d。与对照组或“松友饮”、IFNα单独应用组相比,联合用药更显著地抑制术后残癌的生长（肿瘤体积,mm³,245.3 Vs 1205.2;p=0.000）,降低微血管密度（MVD,n,18 Vs 44;p=0.000）;抑制肺转移（肺转移结节,n,3 Vs 7;p=0.001）并延长生存期（生存期,d,89 Vs 57;p=0.000）。与对照组相比,BAI1基因mRNA表达上调在“松友饮”组无统计学意义（%,5.7 Vs 5.2;p=0.948）,在IFNα组则表达明显上调（%,30.0 Vs 5.2;p=0.006）,联合用药组其表达上调更为显著（%,39.5 Vs 5.2;p=0.000）。因此,“松友饮”和IFNα联用有协同作用,并显著抑制姑息术后残癌的生长和转移,为中药治疗和生物治疗联合应用的临床推广提供了应用基础。结论1.姑息性肝癌切除术促进残癌侵袭转移潜能。2.与对照组相比,姑息组残癌存在侵袭转移相关基因的差异表达。3.中药小复方“松友饮”可通过激活caspase-3,诱导MHCC97H细胞凋亡,抑制肝癌增殖;并抑制MMP2和VEGF表达,进而抑制肝癌的生长转移。4.“松友饮”联合IFNα协同抑制姑息性肝癌切除术后残癌的生长转移,其分子作用基础可能是调节了某些侵袭转移相关基因的mRNA表达。应用价值1.姑息术后残癌差异表达基因的发现有利于进一步研究肝癌侵袭转移潜能的变化,可能具有一定的预测价值并作为治疗靶点之一。2.中药小复方“松友饮”和IFNα联合应用协同显著抑制姑息性肝切除术后残癌的生长和转移,具有临床应用前景。创新点1.明确了姑息性肝癌切除术促进残癌侵袭转移潜能,筛选出2个与肝癌侵袭转移潜能密切相关的基因;发现BAI1和MTA2可望成为姑息术后肝癌人群复发风险的预测标志和个体化治疗的靶点。2.发现中药小复方“松友饮”通过激活caspase-3诱导凋亡,抑制肝癌生长,通过抑制MMP2和VEGF抑制肝癌的侵袭转移,并延长荷瘤裸鼠生存期;“松友饮”与IFNα二者联用具有协同显著抑制姑息术后残癌生长转移的作用。更多还原

【Abstract】 Primary liver cancer,mostly hepatocellular carcinoma(HCC),is the third cancer killer in the world.More and more HCC patients with long-term survival are benefited from various types of therapy aimed at tumor elimination including hepatic resection,one of the most effective therapies for HCC.Unfortunately,due to the high rate of metastatic recurrence,further improve of 5-year survival after small HCC resection was not found in the recent 20 years.Analysis of 2333 cases of HCC patients following hepatic resection in Liver Cancer Institute of Fudan University indicated that palliative resection amounted to 37.2%,and the 1,3,5,8 and 10-year survival in the palliative resection group were lower than that in the radical resection group.Therefore,it is important to seek for new target and new approach for intervention.Some reports revealed that surgical operation could promote tumor growth and metastasis.Unfortunately,little information is available about the alteration of invasive/metastatic potential of the residual HCC following palliative liver cancer resection.This study was to identify the metastasis-related genes in the residual HCC and provide the intervention regimen for those patients.Recently, Chinese herbs have become a focus of interest in cancer treatment.Experimental studies have shown that extracts from herbal medicines or mixtures have anticancer potential,some herbal extracts have been applied in clinical trials aimed at preventing DNA damage or proliferation;and researchers reported that they have achieved some success with the biotherapy and the therapy of Traditional Chinese Medicine(TCM) combination.The Chinese herbal compound extract "Songyou Yin" contains Salvia miltiorrhiza Bge.(danshen) and other four herbs,which was granted by Chinese Food and Drug Administration(FDA,Grant No.G20070160) with the fingerprints by highperformance liquid chromatography(HPLC-FP) determined by Shanghai Institute of Materia Medica(SIMM),Chinese Academy of Sciences(CAS).This study evaluates the effect of "Songyou Yin" on the tumor growth,invasion,and metastasis of HCC in vitro and in vivo;the further study was applied to investigate the effect of the combined therapy with "Songyou Yin(with the same batch number,#060601)" and interferon alfa(IFNα) on the tumor growth,metastasis and gene expression of the residual HCC following palliative liver cancer resection.Ⅰ.The effect of palliative liver cancer resection on the invasive/metastatic potential of the residual hepatocellular carcinomaLittle is known about the alteration of invasive/metastatic potential of the residual HCC following palliative liver cancer resection.Human HCC tumor models produced by MHCC97H(a human HCC cell line with a lung metastatic rate up to 100%) were established in the BALB/C-nu/nu mice by orthotopic inoculation.Then,the mice were randomized into different groups.The mice were performed a palliative resection with preservation of a small part of tumor(2 mm in diameter),or received a sham operation(control group).The methods of cell primary culture,invasion assay, zymography,immunohistochemistry assay(IHA) and Enzyme-linked immunosorbent assay(ELISA),were used to investigate the alteration of invasive/metastatic potential of the residual HCC.As a result,the invasive/metastatic potential of the residual HCC was increasing slowly.The obvious difference in invasiveness could be found between the palliative group and the control group 14 days after operation(p =0.015),the number of the lung metastatic nodule was 14 in the palliative group, whereas it was 9 in the control 35 days after operation(p=0.041),which indicated that palliative resection enhanced the invasive/metastatic potential of the residual tumor. Ⅱ.The effect of palliative liver cancer resection on the invasive/metastatic -related genes expression in the residual hepatocellular carcinomaWe applied a metastatic human HCC model of palliative resection in this study. Briefly,under anesthesia,the left lobe of the liver was exposed,and a part of the liversurface was mechanically injured with scissors,a piece of MHCC97H tumor tissue(size 2 mm×2 mm×2 mm) was fixed within the left liver tissue.Then,the mice were randomized into palliative resection group(with preservation of 2 mm tumor),control groupl(sham operation),and control group2(without any intervention) 14 days later.All mice were sacrificed by cervical dislocation 14 days following palliative liver cancer resection.Oligo Tumor Metastasis Microarray and GEArray Expression Analysis Suite soft ware were adopted for gene analysis.By the method of support vector machine(SVM),we found the markers composed of 12 genes(BAI1,MTA2,MTA1,SMAD2,GNRH1,CDH8,ITGB3,CHD4,GZMA, ITGA7,CXCR4,and TSHR,) could differentiate the invasive/metastatic potential of HCC with positive predictive rate of 88.9%and 93.5%in the groups with high and low invasive/metastatic potential.In addition,we found MTSS1,TGFβ1,SMAD2, IL1βand MMP7 were situated in the central position of the gene function net with high invasive/metastatic potential,which may be served as therapeutic targets.The analysis of gene expression that was up-or down-regulated more than 2 folds revealed that two differentially expressed genes,in which MTA2 was positively correlated,while BAI1 was negatively correlated with the invasive/metastatic potential,which was confirmed by Real-time PCR.Ⅲ.Inhibition effeets and possible mechanism of Chinese herbal compound extraet "Songyou Yin" on hepatocellular carcinoma in vitro and in vivoIn vitro,Chinese herbal compound extract "Songyou Yin"(containg Salvia miltiorrhiza Bge.-danshen and other four herbs) could cause dramatic attenuation of tumor proliferation by induction of apoptosis that was associated with caspase-3 activation,and inhibit invasiveness of MHCC97H via reducing matrix metalloproteinase-2(MMP2) activity and vascular endothelial growth factor(VEGF) expression.In vivo,"Songyou Yin" minimized cancer-related body weight loss of mice with tumors.Inhibition of tumor growth was observed with stepwise increased dosage of "Songyou Yin" and accorded with the expression of proliferating cell nuclear antigen(PCNA),VEGF and MMP2.The life span of nude mice bearing xenografts was(75.0±3.9) days in "Songyou Yin" group,whereas it was(52.0±2.3) days in the control(p＜0.001).The lung metastatic extent was decreased(p＜0.01, compared with control);and "Songyou Yin" inhibited tumor growth was associated with an increased TUNEL-positive apoptosis as well as a decreased microvessel density(MVD) and VEGF abundance,and inhibited tumor invasion via down-regulation of MMP2.Moreover,there was no animal experienced "Songyou Yin"-related weight loss＞10%,or serum transaminases abnormal after the treatment regimen in this study.Ⅳ.The reinforced antitumor effect of "Songyou Yin" and Interferon alfa combination on the growth and metastasis of residual hepatocellular carcinomaHuman HCC model of palliative resection was established for the study of the intervention strategies.The mice were randomized into 4 groups according to the different therapies:control,"Songyou Yin," interferon alfa(IFNα),and the combination of the "Songyou Yin" and IFNα.The residual tumor volume was 1205.2, 724.9,507.6,and 245.3 mm~3;MVD(n) was 44,35,23,and 18;pulmonary metastatic nodule(n) was 7,6,4,and 3;and life span(d) was 57,68,77,and 89,respectively. The residual tumor growth and invasion was inhibited,accorded with a decreased MVD and VEGF abundance.The combined treatment of "Songyou Yin" and IFNαwas more effective than "Songyou Yin" or IFNαalone with tumor inhibition rate of 79.6%(tumor volume,mm~3,245.3 Vs 1205.2;p=0.000).The combined therapy produced more effective reductions of the number of MVD(n) by 59.1%(MVD,n, 18 Vs 44;p=0.000) and lung metastatic nodules by 57.1%(nodule,n,3 Vs 7;p =0.001),prolonged the life span(d) of nude mice by 56.1%,being 89 days Vs 57 days (p=0.000).Moreover,BAI1 mRNA expression was up-regulated by "Songyou Yin" (%,5.7 Vs 5.2;p=0.948) without significance,but significantly up-regulated by IFNα (%,30.0 Vs 5.2;p=0.006),and more significantly up-regulated by the combined therapy(%,39.5 Vs 5.2;p=0.000) when compared with control group.Conclusions1.Palliative liver cancer resection promotes the invasive/metastatic potential of the residual HCC.2.Palliative liver cancer resection causes differential invasive/metastatic-related gene expression in the residual HCC when compared with the control group.3.Chinese herbal compound extract "Songyou Yin" inhibited HCC growth by inducing apoptosis that was associated with caspase-3 activation,and inhibited tumor invasion via reducing MMP2 activity and VEGF expression.4.Combination of "Songyou Yin" and IFNαreinforced the antitumor effect each other,and significantly improved the survival time of mice with tumor following palliative liver cancer resection.The underlying mechanism may be related to the changes of gene expression in mRNA level in the MHCC97H cells.The potential application of this work1.The differential expressed genes identified by Oligio microarray analysis provide a valuable resource for the next basic and clinical studies of invasive/metastatic potential of HCC,and may be served as potential biomarkers and individualized therapeutic targets for some HCC patients.2.Combination of "Songyou Yin" and IFNαreinforced the antitumor effect each other,which laid a foundation for clinical application. The novelty of this work1.Confirmed the invasive/metastatic potential of the residual tumor was promoted by palliative resection,and analyzed its gene expression profile.The identified gene, BAI1 and MTA2 may be served as potential predictive markers and therapeutic targets for some certain type of HCC patients.2.Discovered Chinese herbal compound extract "Songyou Yin" could inhibit HCC growth and invasive/metastatic potential,and the combined therapy with "Songyou Yin" and IFNαsignificantly reinforced the antitumor effect each other on the residual hepatocellular carcinoma following palliative liver cancer resection.更多还原