【作者】 李涛；

【导师】 汤钊猷； 曾昭冲； 王鲁； 邱双键； 刘彬彬；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 原发性肝癌(主要是肝细胞肝癌,hepatocellular carcinoma,HCC)是最常见的恶性肿瘤之一,预后极差,目前是世界第三位癌症杀手,而在中国则高居第二位。手术切除仍是治疗肝癌最有效的方法,但由于肝癌的一些特性,如多灶性,早期血管侵犯,合并肝硬化等,只有不到20%的肝癌患者就诊时适合手术治疗。放射治疗(以下简称放疗)用于肝癌的治疗已有半个世纪,近年随着适形放疗技术的出现及放射生物学观念的更新,放疗在肝癌治疗中的地位有所提高。但临床观察到即使接受根治剂量放射治疗后,仍有60%以上的患者有残存肿瘤存在,尽管放疗后肿瘤会缩小甚至消失,病情得到控制或缓解,但缓解期过后不少病人很快出现复发转移,而且进展迅速,预后较差。这一现象从临床上提出了许多问题,如放疗是否会促进残余肿瘤的播散转移,出于何种机制,如何干预以减少放疗后的复发转移并最终提高放疗疗效,等等。本课题即为此而设计,主要应用我所建立的转移性人肝癌MHCC97L裸鼠模型,模拟临床分割放疗,并主要观察放疗后残余肝癌的侵袭转移潜能的变化。通过实验新发现放疗结束后期残余肝癌的侵袭转移潜能增强,其机制主要是通过上调TMPRSS4表达从而诱导上皮一间质转化(Epithelial-mesenchymal transition,EMT)实现的。EMT是指上皮细胞向间质细胞转化的复杂过程,在此过程中上皮细胞的极性消失,迁移和运动能力增强,EMT是上皮来源的恶性肿瘤发生侵袭转移的重要原因。我们还发现放疗后单独使用TMPRSS4的抑制剂AEBSF或合并使用小剂量IFN-α和阿司匹林(Aspirin)可减少放疗后的肝内播散和转移,从而为进一步提高肝癌放疗疗效提供了线索。1.放射线照射可引起肝癌细胞MMP-2、VEGF表达增加及体外侵袭性增强,并与放射线剂量及时间相关分别以不同剂量的6mvX线照射MHCC97L细胞,各照射组吸收剂量分别为4Gy和8Gy,对照组(0Gy)除不接受X线照射外,其他处理与照射组相同。照射后24-48h MHCC97L细胞MMP-2和VEGF的表达或活性增强并与照射剂量及时间相关,组间差异具有统计学意义(P＜0.05)。8Gy照射后48h其表达及活性最强,此后逐渐降低,照射后96h MMP-2活性低于非照射组。放射线照射对MHCC97L细胞体外侵袭能力的影响:0Gy(对照组)、4Gy和8GyX线照射后,MHCC97L细胞穿过Matrigel胶到达Transwell小室膜背面的细胞数分别为7.2±1.9、13.2±2.7和31.2±7.2,组间差异具有统计学意义(P＜0.05)。放射线照射后96h,各组细胞穿膜数无显著差异(P＞0.05)。提示放射线照射可使MHCC97L细胞体外侵袭性短期增强。2.Aspirin能抑制放射线照射后肝癌细胞MMP-2和VEGF的表达、活性及体外侵袭性的增强体外放射线照射前2h细胞培养基内加入Aspirin,使其终浓度为2mM,放疗后继续培养48h。在不抑制细胞增殖的前提下,与对照组相比,Aspirin能在mRNA水平和蛋白水平抑制放疗诱导的VEGF和MMP-2的表达增加和MMP-2活性的增强,以及由此导致的肝癌细胞侵袭性增强。3.放疗结束后残余肝癌体内侵袭转移潜能的变化规律为了在体内进一步验证放疗结束后残余肝癌侵袭转移能力的变化,我们将具有肺转移潜能的人MHCC97L肝癌组织块种植于BALB/c nu/nu雄性裸鼠肝脏,建立转移性人肝癌裸鼠原位模型。待种植后4周瘤体直径1cm左右时开始放疗。周一至周五每天照射2Gy,共计2周,总放疗剂量20Gy。分别于放疗结束后2天和30天处死放疗组及其对照组裸鼠,切除残余肝癌组织再次原位种植于正常裸鼠肝脏,观察6周处死。放疗结束后2d残余肝癌及其对照组肝癌切除再种植肿瘤大小分别为1.61±0.51cm~3和2.25±0.52cm~3(P＜0.05),肺转移率分别为12.5%和66.7%(P＜0.05),而放疗结束后30d残癌及其对照组肝癌切除再种植肿瘤大小分别为和2.60±0.61cm~3和2.15±0.71cm~3(P＞0.05),肺转移率分别为100%和63.6%(P＜0.05)。提示放疗结束后2d残癌生长受到抑制,而放疗结束后30d肿瘤生长速度已恢复到放疗前,甚或过之。放疗结束后30d残癌切除再种植后肿瘤在肝内呈播散性生长,肺转移率增加;而放疗结束后2d残癌切除再种组肿瘤呈外生性生长,无肝内播散,肺转移率降低。提示放疗结束后残余肝癌在体内的侵袭转移能力先减弱后增强。4.放疗结束后不同时段残余肝癌组织肿瘤侵袭转移相关基因表达差异的研究为了寻找放疗结束后残余肝癌在体内侵袭转移能力变化的相关机制,我们采用Super Array肿瘤侵袭转移相关基因芯片,对对照组、放疗结束后2d和放疗结束后30d残余肝癌组织肿瘤侵袭转移相关基因的表达谱进行分析。以上调或下调超过2倍为检测标准。与对照组相比,放疗结束后2d残余肝癌组织抑癌基因Kiss1和TIMP2表达上调。而放疗结束后30d残余肝癌组织癌基因TMPRSS4表达上调,抑癌基因BAI1表达下调。5.实时荧光定量PCR验证基因芯片结果和Western blot分析TMPRSS4在放疗前后不同时段的表达我们采用实时荧光定量PCR法验证基因芯片的结果。结果显示放疗结束后2d残癌组织Kiss1(P＜0.01)和TIMP2(P＜0.05)的mRNA表达显著增强,而放疗结束后30d残癌组织TMPRSS4 mRNA表达显著高于对照组及放疗结束后2d残癌组织(P＜0.01),而BAI1mRNA表达变化无统计学意义(P＞0.05)。由于放疗结束后30d残余肝癌的侵袭转移能力增强是影响放疗疗效的关键,我们用Western blot进一步验证放疗前后不同时段肝癌组织内TMPRSS4的蛋白含量,同样发现放疗结束后30d残癌组织内TMPRSS4蛋白含量显著高于对照组及放疗结束后2d肝癌组织。提示放疗结束后期TMPRSS4表达增加是其侵袭转移增强的重要原因。6.Western blot分析EMT相关分子及COX-2的变化根据文献报道,TMPRSS4表达增强能够抑制E-cadherin从而诱导EMT,我们进一步用Western blot检测上皮细胞的标志E-cadherin和其转录抑制因子SIP1,以及间质细胞的标志Vimentin和N-cadherin,发现放疗结束后30d残癌组织SIP1、Vimentin和N-cadherin的表达明显增加,E-cadherin的表达则显著减少。而TMPRSS4抑制剂AEBSF能抑制放疗诱导的SIP1和TMPRSS4的表达而增强E-cadherin的表达,提示放疗结束后30d残癌侵袭转移能力增强是通过上调TMPRSS4的表达,从而抑制E-cadherin和引起EMT实现的。此外,我们选取部分基因芯片未包括的与肿瘤侵袭转移相关的基因进行分析。Western blot结果显示,放疗结束后30d残癌组织COX-2的表达明显增强,由于COX-2的表达与E-cadherin负相关,因此COX-2表达增强也有助于EMT的发生。7.联合使用小剂量IFN-α和Aspirin或绿脓杆菌制剂能抑制肝癌在裸鼠体内的生长转移据我所前期实验结果,大剂量(1.5×10~7 U/kg·d)长期使用IFN-α能有效抑制转移性人肝癌裸鼠模型肿瘤的生长,并降低手术切除后的复发转移率。但部分患者长期使用大剂量IFN-α会出现白细胞减少等副作用,不能坚持使用影响疗效。鉴于放疗结束后期COX-2表达增加,我们选择联合使用IFN-α和COX-2抑制剂Aspirin观察其对肝癌在裸鼠体内生长转移的抑制作用。结果显示小剂量IFN-α(7.5×10~6 U/kg·d)+Aspirin(15mg/kg·d)可显著抑制肝癌在裸鼠体内的生长转移,其疗效与单独使用大剂量IFN-α(1.5×10~7 U/kg·d)无显著差异。此外,结果还显示绿脓杆菌制剂腹腔注射也可通过促进肿瘤细胞凋亡而显著抑制肿瘤生长转移。为此我们选择小剂量IFN-α(7.5×10~6 U/kg·d)+Aspirin(15mg/kg·d)和绿脓杆菌制剂作为干预放疗结束后期残余肝癌侵袭转移增加的治疗手段。8.联合使用小剂量IFN-α和Aspirin或绿脓杆菌制剂可降低放疗结束后期的肝内播散和转移将放疗结束后30d残癌组织切除再种植于裸鼠肝脏,分为对照组、小剂量IFN-α+Aspirin治疗组和绿脓杆菌制剂治疗组。与对照组相比,小剂量IFN-α+Aspirin治疗组和绿脓杆菌制剂治疗组肝内播散显著减少,而肺转移被完全抑制。Western blot及免疫组化结果显示小剂量IFN-α+Aspirin治疗组可显著减少放疗后残癌组织内的COX-2的表达,而增强E-cadherin的表达,从而逆转放疗结束后期TMPRSS4表达增强所诱导的EMT及侵袭转移的增加,显示出小剂量IFN-α和Aspirin在减少放疗结束后期残癌侵袭转移方面的临床应用价值。结论1.放疗结束后残余肝癌体内侵袭转移潜能随时间变化先降低后增强。2.放疗结束后残余肝癌TMPRSS4表达增加,进而抑制E-cadherin的表达和诱导EMT,是放疗结束后期残余肝癌侵袭转移能力增强的主要原因。3.联合使用小剂量IFN-α和Aspirin或单独使用TMPRSS4抑制剂AEBSF能有效抑制放疗后期COX-2和TMPRSS4表达的上调,增加E-cadherin的表达,抑制残癌的侵袭转移。绿脓杆菌制剂亦可通过促进肝癌细胞的凋亡来抑制放疗后的播散转移。4.联合使用小剂量IFN-α和Aspirin可成为临床肝癌患者放疗后减少复发转移有效实用的干预措施。应用价值1.明确放疗后残余肝癌体内侵袭转移潜能变化规律及其机制,有助于指导放疗后的综合治疗,从而降低放疗后肝癌的复发转移。2.联合应用小剂量IFN-α和Aspirin可显著降低放疗结束后期残癌在体内的侵袭转移,为临床提高肝癌放疗的疗效提供了线索。3.联合应用小剂量IFN-α和Aspirin可显著抑制肝癌的生长转移,有可能成为临床肝癌治疗的新方法。创新点1.首次发现放疗结束后残余肝癌体内侵袭转移潜能的变化规律及其机制,提出放疗后残癌TMPRSS4表达增加,进而抑制E-cadherin的表达和诱导EMT,是放疗结束后期残癌侵袭转移能力增强的主要原因。2.首次提出联合应用小剂量IFN-α和Aspirin可显著抑制肝癌的生长转移并降低放疗结束后期残癌的侵袭转移。更多还原

【Abstract】 Hepatocellular carcinoma(HCC) is the third most common cause of death from cancer in the world and the second cancer killer in China.Surgical resection remains the most effective treatment for HCC.Unfortunately,less than 20%of patients with HCC are candidates for resection at the time of diagnosis because of multinodular pattern,vascular invasion and liver cirrhosis.Radiotherapy has been used to treat HCC for half century.Recently,with the advances of biology and technology,such as 3-dimensional conformal radiotherapy(3D-CRT),radiotherapy has become more important for the treatment of HCC.However,it is reported that even after radiotherapy with curative dosage,residual cancer can be detected in more than 60%of patients.We did observe that even tumor was "eradicated" after radiotherapy,but several months later some of the tumors relapsed or metastasized quickly with poor prognosis.Questions raise based on this observation:does radiotherapy promote the invasiveness and metastatic potential of residual cancer? What is the molecular background in this process? What can be done for intervention to improve the outcome of radiotherapy? Our present study was designed to clarify these issues by treating nude mice bearing human HCC orthotopic xenografts with fractionated radiotherapy.After treatment the invasiveness and metastatic potential of residual HCC was evaluated and possible molecular background was investigated.We found that radiation indeed promotes the invasion and metastasis of residual cancer through upregulation of TMPRSS4,which can induce epithelial-mesenchymal transition(EMT),a major determinant of metastasis. Administration of TMPRSS4 inhibitor AEBSF or combined use of IFN-αand aspirin can inhibit the invasion and metastasis caused by radiation,and may be a novel approach to improve outcome of radiotherapy for HCC.1.Radiation increased the invasiveness of MHCC97L cells in vitro through upregulation of MMP-2 and VEGF Alteration in the invasiveness of MHCC97L cells after radiation was examined using the Matrigel invasion assay.The invasiveness of MHCC97L cells were significantly increased after radiation at a time and dose-dependent manner,and reached the maximum at 48h after 8 Gy radiation.Forty eight hours after radiation at different dose(0、4、8Gy),the number of cells that passed through the transwell inserts was 7.2±1.9,13.2±2.7(P＜0.05) and 31.2±7.2(P＜0.005) respectively,while there was no significant difference 96 hours after radiation.This increase in invasiveness was associated with upregulation of expression or activity of MMP-2 and VEGF.The levels of protein for MMP-2 and VEGF were determined by Western blot analysis and showed a significant increase after exposure to 8 Gy for 48 hours.In order to see if the increase in MMP-2 and VEGF secretion correlates with changes in the expression of the corresponding genes,we tested their mRNA levels by quantitative RT-PCR.There was a dose-dependent increase in MMP-2 and VEGF mRNA expression and the maximal level was observed at dose of 8 Gy;MMP-2 activity was examined by zymographic analysis and was increased in a time and dose dependent manner,attaining a maximum at 48 hours after 8 Gy radiation.2.Aspirin inhibited the activity/expression of MMP-2 and VEGF and thereby suppressed radiation augmented invasiveness of MHCC97L cells.The invasiveness of MHCC97L cell was significantly enhanced after radiation at doses of 4 Gy and 8 Gy.However,when aspirin(2mM) was added before radiation, the invasion rate dropped near to the level of unirradiated cells.Furthermore,aspirin significantly inhibited the radiation induced increase in VEGF and MMP-2 secretion, as well as the MMP-2 activity.Taken together,these findings suggest that the concomitant use of aspirin during radiation could inhibit the radiation enhanced invasion through suppressing the expression or activity of VEGF and MMP-2.3.The invasion and metastasis of residual HCC after radiotherapy in vivoIn order to further investigate the invasion and metastasis potential of residual HCC in vivo,metastatic animal model of human HCC was established by orthotopic implantation of histologically intact MHCC97L HCC tissue into the liver of nude (nu/nu) mice.Four weeks after implanation,the tumors were irradiated at a dose of 2Gy/d,from Monday to Friday for two weeks,and the total radiation dose was 20Gy. The mice were sacrificed 2 or 30 days afer radiation and the residual cancer were replanted into the liver of other nude mice.These mice were sacrificed 6 weeks after replantation.The replanted tumor volume of 2 days- postradiotherapy group and its control group are 1.61±0.51 and 2.25±0.52cm~3(p＜0.05),and the incidence of lung metastasis was 12.5%and 66.7%(P＜0.05);while tumor volume of 30 dayspostradiotherapy group and its control group are 2.60±0.61 and 2.15±0.71cm~3(p＞0.05),and the incidence of lung metastasis was 100%and 63.6%(P＜0.05), respectively.Significant tumor inhibition was only found between control and the 2 days-postradiotherapy group,but not between control and 30 days-postradiotherapy group.The incidence of lung metastasis and inhepatic dissemination were markedly increased in the 30 days-postradiotherapy group.These results suggested that 30 days after radiation the invasiveness and metastatic potential of residual HCC were significantly enhanced.4.Changes in the invasion and metastasis related gene expression profile of residual HCC in nude mice after radiotherapy.In order to investigate the mechanism involved in this process,invasion and metastasis related cDNA microarray was used to analyse changes of gene expression profile after radiotherapy.The results showed that KISS1 and TIMP2,two factors that inbibit invasion and metastasis,were down-regulated in residual cancer 2 days after radiotherapy,while TMPRSS4,which can promote invasion and metastasis,was significantly up-regulated in residual cancer 30 days after radiotherapy.Furthermore, BAI1 was down-regulated in residual cancer 30 days after radiotherapy.5.Validation of cDNA microarray results by quantitative RT-PCR and westen blotIn order to verify the above-mentioned changes in KISS1,TIMP2,TMPRSS4 and BAIl from the cDNA microarray analyses,we tested the effect of radiation on their mRNA levels by quantitative RT-PCR.Compared with control group,the KISS 1(P＜0.01) and TIMP2(P＜0.05) mRNA expression in residual cancer 2 days postradiotherapy were significantly increased.The TMPRSS4 mRNA expression of residual cancer 30 days postradiotherapy was much higher than that of control group and residual cancer 2 days after radiotherapy(P＜0.01),but there was no significant difference in BAI mRNA expression(P＞0.05) between these groups.We further analysed the protein level of TMPRSS4 in these 3 groups and western blot result showed that TMPRSS4 protein was also significantly enhanced in residual cancer 30 days postradiotherapy.These findings indicate that TMPRSS4 is a key oncogene that related to radiation induced invasiveness and metastasis.6.Western blot analysis of EMT related molecules and COX-2According to literature,up-regulation of TMPRSS4 can inhibit the expression of E-cadherin and facilitate EMT,we further detected epithelial marker of E-cadherin and mesenchymal marker of Vimentain and N-cadherin.SIP1/ZEB2,an E-cadherin transcriptional repressor,was also detected by western blot.The results showed that expression of SIP1,Vimentain and N-cadherin was significantly increased while expression of E-cadherin was inhibited.Administration of TMPRSS4 inhibitor AEBSF inhibited the expression of TMPRSS4 and SIP1 and up-regulated the expression of E-cadherin,together with reduced invasion and metastasis in vivo after radiation.It is concluded that radiation promoted the invasiveness and metastasis through TMPRSS4 induced EMT.We also analysed some molecules that were not included in the cDNA microarray by western blot,and found that COX-2 expression was significantly increased in the residual cancer 30 days postradiotherapy.Since there is an inverse relationship between COX-2 and E-cadherin expression,enhanced expression of COX-2 also contributes to EMT.7.Combined administration of IFN-αand aspirin or pseudomonas aeruginosa vaccine(PA) inhibits the growth and metastasis of HCC in vivoWe previously reported that High-dose(1.5×10~7 U/kg·d) and long-term therapy with IFN-αinhibits tumor growth and recurrence in nude mice bearing human HCC xenografts.However,in clinical practice some patients can not stand high-dose therapy with IFN-αbecause of the side effects.Since COX-2 expression is increased after radiation,we choose combined administration of low-dose IFN-α(7.5×10~6 U/kg·d) and aspirin(15mg/kg·d) to inhibit the growth and metastasis of HCC in vivo, which was as effective as high-dose IFN-α(1.5×10~7 U/kg·d).Furthermore, administration of PA intraperitoneally also significantly inhibit the growth and metastasis of HCC through induction of apoptosis.Thereby we choose combined administration of low-dose IFN-αand aspirin or PA to inhibit the radiation induced invasion and metastasis. 8.Combined administration of low-dose IFN-αand aspirin or PA inhibits the dissemination and metastasis of residual cancerThirty days afer radiation,the residual cancer were resected and replanted into the liver of other nude mice.The mice were divided into control group,low-dose IFN-αplus aspirin treatment group and PA treatment group.Six weeks after that,the mice were sacrificed.Low-dose IFN-αplus aspirin or PA treatment significantly decreased the dissemination in liver and completely inhibited the lung metastasis.These findings may provide novel approach to enhance the efficacy of radiotherapy for HCC.Conclusions1.The invasion and metastasis of HCC in vivo was inhibited in early phase and then enhanced in the late phase after radiation.2.Radiation enhanced the invasion and metastasis of residual cancer through upregulation of TMPRSS4,which inhibited the expression of E-cadherin and induced EMT.3.Combined administration of low-dose IFN-αand aspirin or TMPRSS4 inhibitor AEBSF inhibited the dissemination and metastasis of residual cancer through suppression of COX-2 and TMPRSS4 and upregulation of E-cadherin.PA can inhibit the dissemination and metastasis through inducing the apoptosis of HCC.4.Combined administration of low-dose IFN-αand Aspirin could be a potential therapeutic approach to inhibit radiation induced invasion and metastasis and improve the efficacy of radiotherapy for HCC.The potential application of this work1.Clarifying the molecular basis of radiation induced invasion and metastasis of residual HCC helps to instruct the combined therapy postradiotherapy.2.Combined administration of low-dose IFN-αand Aspirin significantly inhibited the dissemination and metastasis of residual HCC postradiotherapy,thereby provides a new clue to improve the outcome of radiotherapy.3.Combined administration of low-dose IFN-αand Aspirin significantly inhibited the growth and metastasis of HCC in vivo,thus provides a new approach for treatment of HCC.Originalities of this work 1.For the first time to clarify the alteration and molecular basis of invasion and metastasis potential of residual HCC after radiation in vivo,and point out that upregulation of TMPRSS4 and induced EMT might play a major role.2.For the first time to point out that combined administration of low-dose IFN-αand aspirin significantly inhibited the growth and metastasis of HCC in vivo, and also suppressed the dissemination and metastasis of residual cancer postradiotherapy.更多还原