【作者】 成薇婷；

【导师】 田德英；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 【研究背景】肝细胞癌(Hepatocellular carcinoma，HCC)是我国高发的恶性肿瘤，其发病率、复发率、死亡率都有增加趋势。手术切除仍然是其最有效的治疗，但是术后由于容易复发和转移，预后仍较差，积极探讨新的治疗靶点具有重要的意义。Hedgehog(Hh)信号通路是高度保守的调控胚胎组织分化发育的通路，在脊柱动物体内，Hh蛋白与跨膜受体Ptch结合后，解除了Ptch对Smo的抑制，随后Smo从共受体上解离下来进入胞浆，将信号下传并激活锌指转录因子Gli，后者启动Ptch-1、Hip、Gli1、Wnt等目的基因的表达，进而调控细胞的增殖、分化。最近的研究发现，在前列腺癌、基底细胞癌、胰腺癌、结肠癌以及胃癌等众多恶性肿瘤中Hh信号通路异常持续激活，通过阻断该信号通路后，可以抑制肿瘤的发生和进展。但是该信号在HCC发病机制中的作用尚不清楚。【目的】探讨Hh信号通路在原发性肝细胞癌形成过程中的作用及其可能机制。为进一步深入了解肝细胞癌的发生、发展过程并为其诊断和治疗提供新的科学依据。【方法】1．构建含有肝癌组织和相应癌旁组织的组织芯片；2．用免疫组织化学染色法检测Shh、Ihh、Ptch-1和Gli-2的表达，并对表达情况与临床病理组织学参数之间的关系进行分析；3．采用RT-PCR方法检测Hh信号通路各分子在5株肝细胞系(L02、HepG2、Hep3B、SMMC-7721、Huh7)中的表达情况；4．选用Hh信号活性最强的人肝癌细胞株SMMC-7721作为研究模型，使用不同浓度的Hh信号通路特异性抑制剂KAAD-cyclopamine处理细胞，采用MTT法检测细胞增殖，BrdU掺入法结合流式细胞仪检测DNA合成情况和细胞周期；5．采用倒置显微镜观察细胞的形态学变化，AnnexinV／PI流式细胞术和TUNEL染色检测细胞凋亡率，Caspase-3活性检测试剂盒检测Caspase-3活性；6．采用细胞划痕愈合实验、细胞体外运动和侵袭实验研究细胞粘附、运动和侵袭；7．Westernblot检测CyclinD1、p21、caspase-3、bcl-2、E-cad等调节基因的表达。【结果】1．成功构建含有88个位点的组织芯片，44对肝癌和癌旁组织以一对一点阵方式排列，显微镜阅片证实取材正确，与原组织诊断吻合，结构保存良好。2．HCC组织中Hh信号通路活性较癌旁组织显著增强。Shh、Ptch-1在HCC中的阳性表达率均高于癌旁肝组织(P＜0．05)；但二者的表达与各项临床病理指标，包括年龄、组织学分型、肿瘤大小、转移以及HBV感染之间均无明显关联(P＞0．05)。Ihh在两种组织中的表达无明显差异(P＞0．05)，其表达与各种临床病理参数无明显关联。Gli-2在HCC中的阳性表达率显著高于癌旁组织(P＜0．01)；且与组织学分型组以及肿瘤是否发上转移相关。低分化和伴有门静脉转移的病例中的表达率高于高分化组和无门静脉转移的肿瘤，差异具有统计学意义(P＜0．05)。3．Hh信号通路在5株肝细胞系中均有不同程度表达。其中Shh、Ptch-1、Smo、Gli-2的mRNA表达水平在肝癌细胞系中明显增强，尤以SMMC-7721细胞为甚，而正常肝细胞系L02细胞中上述各分子的表达较弱。特别是Gli2在这两种细胞中的差别更为明显。Gli1在Hep3B和SMMC-7721细胞中表达水平也显著升高。而Ihh和Gli3在各种细胞系中表达均较弱。4．应用Hh信号通路特异性抑制剂KAAD-cyclopamine处理人肝癌细胞系SMMC-7721后，细胞生长增殖能力显著减弱，而且随着KAAD-cyclopamine浓度的增加以及作用时间的延长，其抑制效果也相应提高，呈现一定的剂量和时间依赖性。BrdU掺入法结合流式细胞仪检测结果显示KAAD-cyclopamine能显著减少SMMC-7721细胞DNA合成，提高G0／G1期细胞比例并降低S期细胞比例(P＜0．05)。同时，Western blot检测结果显示p21的蛋白表达水平明显升高而CyclinD1的表达明显降低。5．Hh信号通路阻断后，SMMC7721细胞的生存能力下降，凋亡率明显升高。干预后细胞由饱满舒展、贴壁生长状态转变为圆钝缩小、脱落悬浮状态。AnnexinV／PI检测结果显示细胞早期凋亡显著增加；TUNEL法检测发现干预组细胞凋亡明显增多，荧光显微镜下反光增强(P＜0．01)。促凋亡蛋白Caspase-3活性明显上调而抗凋亡基因bcl-2的表达下调。6．KAAD-cyclopamine使SMMC-7721细胞的运动侵袭能力显著下降。划痕愈合试验结果显示，干预组细胞的愈合程度较对照降低了65％，细胞的运动速度明显下降(P＜0．01)。细胞体外运动能力和侵袭能力均受到明显的抑制，与对照组相分别下降了54．4±7％和48．3±11．9％(P＜0．01)。Western blot检测显示E-钙粘蛋白的表达量随着Hh信号阻断而上调。【结论】HCC组织中，Hh信号转导通路呈现高表达现象，并且与肝癌的分化和转移之间存在明显关联，与此一致的是，在5株肝细胞系细胞中，恶性程度较高且具有较强侵袭能力的细胞株SMMC-7721中Hh信号通路的活性最强。利用KAAD-cyclopamine特异性阻断SMMC-7721细胞中Hh通路后，可明显抑制细胞DNA合成，进而抑制肝癌细胞生长增殖能力，其机制可能与促进p21的表达，抑制CyclinD1的表达有关。阻断Hh通路后，可以降低细胞生存能力，诱导肝癌细胞凋亡，原因可能与上调了凋亡促进因子Caspase-3、下调了凋亡抑制因子bcl-2的表达有关。此外，Hh信号通路被阻断后，肝癌细胞的运动迁徙能力和侵袭力显著降低，其机制可能与促进E-钙粘蛋白的表达有关。因此，Hh信号通路在肝细胞癌的发生发展过程中具有重要作用。参与了肝癌细胞的增殖、凋亡以及侵袭转移的多种生物学过程的调节。对Hh信号通路更加渗入、全面的探讨有助于我们进一步了解原发性肝癌的发生发展机制，为其诊断和治疗提供新的科学依据。更多还原

【Abstract】 [Background and Objective]Hepatocellular carcinoma(HCC)is one of the most common malignancies in ourcountry,and has the increased uptrend in incidence,recurring rate and death rate.Thehepatic resection remains the most effective treatment,but the prognosis of HCC isgenerally poor,due to the high post-operative recurrence and invasiveness of primary tumor.It is vital to explore new molecular markers for treatment strategies.Hedgehog(Hh)signaling pathway is a highly conserved system,which plays a crucial role in embryonictissue patterning,cell differentiation and proliferation.In vertebrate organisms,thesignaling pathway is initiated by the binding of ligands(Shh,Ihh,Dhh)to the membranousreceptor patched(Ptch)which in turn alleviates the suppression on smoothened(Smo),subsequently Smo triggers a series of intracellular events with resultant activation of thezinc-finger transcription effectors,glioma-associated oncogenes(Glil,Gli2,Gli3)transcription factor,which induces the expression of numerous target genes,such as Ptch-1,Hip,Gli1 and Wnt,that regulate proliferation,differentiation.Recent studies revealed thataberrantly persistent activation of Hh signaling pathway and overexpression of target geneslead to several malignant tumorigenesis,such as cancer of prostate,basal cell,pancreas,colon and stomach.However,the role of the pathway in pathogenesis of HCC is stillindistinct.[Aim]To investigate the effects of Hh signaling pathway on HCC and its possible mechanism,which not only furthered to detect the mechanisms of onset and developmentof HCC but provided the new scientific evidence of its molecular diagnosis and treatment.[Methods]1.Tissue microarray(TMA)contained HCC and corresponding tumor-adjacent livertissues were constructed.2.The expressions of Shh,Ihh,Ptch-1 and Gli-2 in HCC tissues and correspongdingadjacent-tumor liver tissues were detected by immunohistochemistry and the correlationbetween their expressions and clinicopathologic parameter was analyzed.3.The mRNA expressions of Hh signaling components in 5 hepatoma cell lines(L02,Hep3B,HepG2,SMMC-7721 and Huh-7)were detected by RT-PCR.4.After treated with different concentrations of KAAD-cyclopamine,a specificinhibitor of Hh signaling pathway,Methyl thiazolyl tetrazolium(MTT)assay was used toexamine the changes in the proliferation of SMMC-7721 cells,BrdU incorporation assaywas applied to measure DNA synthesis rates and flow cytometry(FCM)was employed toanalyze the cells cycle.5.The morphological changes of cells were observed by inverted microscope,theAnnexin V/PI and TUNEL(terminal deoxynucleotidyl transferase-mediated nick endlabeling of DNA fragmentation sites)were applied to detecte the apoptosis rate induced byKAAD-cyclopamine.The change of Caspase-3 relative activity was analyzed bycolorimetric assay.6.Wound healing assay,Transwell assay and Modified Boyden chamber techniquewere performed to determine the cells adhesion,motility and invasiveness.7.The expressions of the regulative genes,such as Cyclin D1,p21,Caspase-3,bcl-2,E-cadherin were detected by Western blot.8.Statistical analysis:Date were expressed as mean±SD.Statistical correlation ofdata was cheched for significance by the ANOVA and paired Student’s t test.P＜0.05 wasconsidered significant. [Results]1.The TMA was constructed involving 88 spots,totally 44 HCC tissues andcorresponding tumor-adjacent tissues ranked lattice one-by-one.Those spots wereconfirmed that based on correct diagnosis in line with the original organization bymicroscope,and the structure of lesions were well-preserved.2.Hh signaling pathway activity enhanced significantly in HCC compared withcorresponding tumor-adjacent liver tissues.The expressions of Shh and Ptch-1 in HCCtissues were higher than the corresponding tumor-adjacent liver tissues(P＜0.05),but nocorrelation between expressions and clinicopathologic factors was found,including age,histological typing,tumor size,metastasize and HBV infection(P＞0.05).Neithersignificance different expression of Ihh between HCC and tumor-adjacent tissues,norrelationship between its expression and clinicopathologic factors was found.(P＞0.05).Inaddition,positive expression of Gli2 was remarkably stronger in HCC than tumor-adjacentliver tissues(P＜0.01)and overexpression of Gli2 was significantly associated with HCChistologic differentiation and portal venous invasion(P＜0.05).3.All Hh pathway components were expressed in 5 HCC cell lines to different extent.Expression of Shh,Ptch,Smo and Gli2 mRNAs was robustly observed in hepatoma celllines,with predominance in SMMC-7721,but weaker expression in normal liver cell lines(L02)was noted,especially Gli2 was significantly over expressed in SMMC-7721 butalmost undetectable in L02.The enhanced expression of Gli1 was only observed in Hep3Band SMMC-7721.However,Ihh and Gli3 were expressed at similarly low levels in all celllines.4.Blockade of Hh signaling pathway in SMMC-7721 cells,which pathway wassignificantly activated,by using KAAD-cyclopamine,a specific antagonist of signaling.The antagonist inhibited the cell proliferation markedly.The inhibitory effect enhancedwith increasing concentration and action time of KAAD-cyclopamine,in a dose-andtime-dependent pattern.The FCM analysis showed that KAAD-cyclopamine deduced the synthesis of DNA in SMMC-7721 cells,and induced cell cycle G1/G0 phase arrest(P＜0.05).Additionally,up-regulation of p21 and down-regulation of cyclin D1 protein expressionswere detected by Western blot.5.Blockade of the Hh pathway reduced SMMC-7721 cells survival ability andincreased cells apoptosis.The treatment induced dramatic morphologic changes of cells,thecells transformed from a flat,elongate and adherent growth status into a rounded sharps,subsequently lost contact with neighboring cells and finally floated into medium.thetreated cells apoptosis rates increased significantly measureb by Annexin v/PI and TUNELassay(P＜0.01).Proapoptotic protein Caspase-3 expression down-regulated andanti-apoptotic gene bcl-2 expression up-regulated.6.The motility and invasive capacity of SMMC-7721 cells were greatly suppressed byKAAD-cyclopamine.Wound assay revealed significant reductions in wound closure by＞65% for treated cells and significantly descend in mean velocities compared with controlcells(P＜0.01).Transwell Invasion assay displayed that SMMC-7721 exhibited the stronginvasive potential to penetrate the basement membrane components Matrigel,but to asignificantly lesser extent after application of KAAD-cyclopamine(P＜0.01).Accordantly,the expressions of E-cadherin was up-regulated by KAAD-cyclopamine.[Conclusion]Overexpression of Hh signaling pathway in HCC compared with correspondingadjacent-tumor liver tissues,and the ectopic activation of Hh pathway was associated withhistologic differentiation and portal venous invasion of HCC.All of HCC cell linesexpressed Hh pathway to different extent,with predominance in SMMC-7721,which wereregarded as poorly differentiated.Blockade Hh pathway by KAAD-cyclopaminesignificantly inhibited the DNA synthesis and resultantly inhibited proliferation inSMMC-7721 cells.Moreover,blackade Hh signaling pathway induced apoptosis of andKAAD-cyclopamine attenuated invasiveness and motility of SMMC-7721 cells remarkably.The possibely machenism are Hh signaling pathway regulate the expression of genes, including CyclinD 1,p21,Caspase3,bcl-2 and E-cadherin.Taken together,Hh signaling pathway plays an important role in onset anddevelopment of human hepatocellular carcinama.The blockade of singaling inducedbroadly biological effects including cell growth and invasion inhibition,apoptosisenhancement in HCC.A deeper and full understanding of the function of Hh signalingpathway may provide signifieant insights into the pathogenesis,development andprognostic of hepatocellular carcinoma and a novel applicable strategy for diagnosis andtherapy.更多还原