【作者】 胡珊珊；

【导师】 钱家鸣；

【作者基本信息】 北京协和医学院 ， 内科学， 2014， 博士

【摘要】 胰腺癌是恶性程度最高的消化系统肿瘤之一,是西方国家第四大癌症死亡原因；在我国,胰腺癌的发病率逐年上升,在所有肿瘤中居第7位。由于早期诊断困难,胰腺癌患者确诊时约50%出现全身转移；由于缺少有效治疗手段,5年生存率仅6%,中位生存时间仅为4-6个月。肿瘤的生长和转移与肿瘤血管生成密切相关,当肿瘤大小超过1～2mm3时,其继续生长依赖于新生血管生成。研究发现多种信号分子参与肿瘤血管生成,其中CXCL8和VEGF是最重要的促血管生成因子,同时多种趋化因子和受体及相关信号通路的参与亦影响肿瘤血管生成。针对胰腺癌血管生成的研究将有利于发现胰腺癌治疗的新靶点。ARHI基因是一个母源性印迹基因,是Ras超家族成员之一；与大部分Ras家族成员不同,ARHI基因是一个抑癌基因,其编码的蛋白在人类多种组织中如卵巢、乳腺中表达,但在乳腺癌、卵巢癌中表达下调。北京协和医院消化内科实验室的前期研究发现,ARHI基因是胰腺癌抑癌基因,可以抑制胰腺癌细胞的增殖、促进细胞周期停滞、诱导细胞凋亡及自噬发生和抑制细胞迁移。ARHI基因是胰腺癌发生发展重要抑癌基因,然而肿瘤细胞的生长和转移受到微环境的调控,而在这种调控中血管生成是重要的关键环节,并且是目前和未来治疗的重要靶点。因此探讨ARHI基因对胰腺癌肿瘤血管生成的调控作用及其机制有重要意义,目前尚未见报道。本研究目的：通过人胰腺癌组织、胰腺癌细胞株、裸鼠胰腺癌荷瘤,探讨ARHI基因对胰腺癌血管生成的影响及相关机制,探讨ARHI基因对肿瘤微环境中趋化因子和受体的影响及意义。为进一步研究胰腺癌发病机制和抗肿瘤血管生成治疗在胰腺癌中的应用,提供重要的理论依据。第一部分ARHI基因对人胰腺癌组织血管生成的影响及其相关临床与病理意义目的：探讨ARHI基因与胰腺癌组织血管密度、血管生成相关蛋白和临床与病理的相关性方法：1.收集23例胰腺癌组织和27例正常胰腺组织标本,登记临床病理资料。2.应用免疫组化方法检测ARHI、CD34、VEGF和CXCL8蛋白在胰腺癌组织和正常胰腺组织中的表达。3.分析各蛋白表达和胰腺癌临床与病理因素的关系。4.分析ARHI与MVD、VEGF、CXCL8四者之间的相互关系。结果：1.ARHI蛋白在正常胰腺组织和胰腺癌组织中表达的阳性率分别为88.9%和21.7%,两者有显著差异；正常胰腺组织MVD值显著低于胰腺癌组织,分别为27.84±2.01和43.47±6.44；正常胰腺组织VEGF蛋白表达阳性率(44.4%)显著低于胰腺癌组织中VEGF (100%),两者比较有显著统计学差异；CXCL8蛋白在正常胰腺组织和胰腺癌组织中表达的阳性率分别为44.4%和100%,两者比较有显著统计学差异。2. ARHI的表达与肿瘤分化程度呈相关性,肿瘤分化程度越低,ARHI表达缺失率越高；CXCL8的表达与CA199水平呈相关性,CA199水平越高,CXCL8阳性表达率越高,与肿瘤分化程度呈相关性,肿瘤分化程度越低,CXCL8阳性表达率越高；VEGF的表达与年龄、性别、吸烟史、糖尿病史、血清CA199水平、血清CA242水平、临床分期、分化程度、肿瘤大小、肿瘤位置、淋巴结转移、血管浸润均无相关性。3.胰腺癌中MVD与CXCL8的表达呈正相关,MVD与VEGF、 CXCL8与VEGF、ARHI与MVD、ARHI与VEGF、ARHI与CXCL8的表达均无显著相关性。4.在所有统计病例中,CXCL8与VEGF的表达呈正相关,ARHI与MVD、VEGF及CXCL8的表达呈负相关。小结：1)在胰腺癌组织中,ARHI表达缺失或显著下调,MVD值显著增多,VEGF和CXCL8表达显著上调。2) ARHI、VEGF和CXCL8与肿瘤分化程度相关；MVD与血清CA199水平、临床分期、分化程度、肿瘤大小、淋巴结转移均呈正相关。3)在所有统计病例中,CXCL8与VEGF的表达呈正相关,ARHI与MVD、 VEGF及CXCL8的表达呈负相关。第二部分胰腺癌细胞株和裸鼠胰腺癌荷瘤验证ARHI基因对肿瘤血管生成的影响及相关机制的初探目的：探讨ARHI基因对胰腺癌血管生成的影响及相关机制。方法：1.应用携带ARHI基因的质粒pIRES2-ARHI-EGFP和pLNCX2-ARHI-EGFP,采用脂质体瞬时转染胰腺癌PANC-1细胞系,并用G418筛选建立稳定转染的多克隆细胞株。2.验证ARHI基因表达情况,用MTT法检测ARHI基因对胰腺癌细胞增殖的影响。3.通过Real-time PCR和Western blot检测胰腺癌细胞株中ARHI基因转染对mTOR、SOSC3、Stat3、NF-κB、VEGF、CXCL8mRNA和(或)蛋白表达的影响。4.将ARHI稳定转染PANC-1细胞株、空载体稳定转染PANC-1细胞株和未转染PANC-1细胞株接种于裸鼠腋窝皮下,比较三组裸鼠体重变化、肿瘤重量和体积。5.荷瘤标本一部分进行病理学检测,通过免疫组化检测ARHI、 Ki-67、CD34、VEGF、CXCL8蛋白的表达；一部分标本通过Real-time验证ARHI基因对mTOR、SOSC3、Stat3、NF-κB、VEGF、CXCL8mRNA表达的影响。结果：1.G418筛选4周收集稳定转染胰腺癌细胞,通过RT-PCR和Western blot方法检测,在ARHI基因稳定转染组可见ARHI mRNA和蛋白表达,而空载体和对照组无表达。2.慢病毒质粒pLNCX2-ARHI-EGFP转染效率高,荧光更强,用于后续实验。3.MTT结果显示,pLNCX2-ARHI-EGFP组稳定转染多克隆细胞株比pLNCX2-EGFP组稳定转染细胞株增殖缓慢。4. pLNCX2-ARHI-EGFP组mTOR、 Stat3、NF-κB、VEGF、CXCL8mRNA表达比pLNCX2-EGFP组显著降低,SOCS3mRNA表达显著升高；p-mTOR、p-Stat3、NF-κB、CXCL8蛋白表达显著降低。5.接种后所有裸鼠均有一定程度的消瘦,pLNCX2-ARHI-EGFP组消瘦显著,肿瘤质量和体积显著偏小。6. pLNCX2-ARHI-EGFP组裸鼠荷瘤中ARHI mRNA和蛋白表达阳性,pLNCX2-EGFP组和对照组无表达,证明胰腺癌荷瘤成功建立。7.pLNCX2-ARHI-EGFP组裸鼠荷瘤中Ki-67蛋白表达显著下降,MVD显著减少,VEGF和CXCL8蛋白表达显著下降。8. pLNCX2-ARHI-EGFP组裸鼠荷瘤中mTOR、 Stat3、NF-κB、VEGF、CXCL8mRNA表达显著下降,SOCS3mRNA表达显著升高,与细胞结果一致。小结：1)成功构建稳定表达ARHI基因的多克隆PANC-1细胞株。2) ARHI基因再表达可以抑制胰腺癌细胞的增殖。3)通过裸鼠胰腺癌荷瘤的建立,在动物水平验证了ARHI基因对胰腺癌生长及肿瘤血管生成的抑制作用。4) ARHI基因抑制胰腺癌细胞和裸鼠荷瘤中VEGF、CXCL8的表达,ARHI基因可能通过mTOR/NF-KB和STAT3信号通路途径抑制CXCL8和VEGF的表达,进而抑制胰腺癌血管生成。第三部分ARHI基因对胰腺癌细胞中血管生成相关趋化因子和受体表达的影响目的：探讨ARHI基因通过对肿瘤微环境中趋化因子和受体的作用,对胰腺癌生长、侵袭转移、免疫和血管生成的影响。方法：1.通过Real-time PCR Array检测稳定转染ARHI基因对84个趋化因子和受体mRNA表达水平的影响。2.挑选与肿瘤血管生成相关的趋化因子和受体,设计引物在ARHI基因稳定转染细胞株和胰腺癌裸鼠荷瘤中进行验证。结果：1. PCR Array结果：1) pLNCX2-ARHI-EGFP组中有36个基因mRNA表达下调,9个基因mRNA表达上调；2)与肿瘤血管生成相关基因：促肿瘤血管生成基因中,CCL2、CXCL1、CXCL2、CXCL8、CXCL12和CXCR4表达显著下调,CXCL3、CCR1和CCR2表达轻度下调；抗肿瘤血管生成基因中,CXCL9、 CXCL10、CXCL11和CXCR3表达显著上调,以CXCL9最明显,达27.8倍；3)与肿瘤转移和侵袭相关基因：CXCL12和CXCR4表达显著下调,MMP-2表达轻度下调；与对远端器官定位浸润和潜伏相关基因：CXCL12、CXCR4和CCR7表达显著下调,CXCR5表达轻度下调；与肿瘤免疫调节相关基因：CXCL8、CXCR1和CCR7表达显著下调,其中以CXCR1尤为明显,达-41.3倍。2.验证结果显示,pLNCX2-ARHI-EGFP组较pLNCX2-EGFP组CXCL8、CXCL1和CXCR4mRNA表达显著下降,CXCR3mRNA表达显著上调,与PCR Array结果一致；CXCR2表达显著下降。3. pLNCX2-ARHI-EGFP组裸鼠荷瘤较pLNCX2-EGFP组裸鼠荷瘤中CXCL8、CXCL1、CXCR4和CXCR2mRNA表达显著下降,CXCR3mRNA表达显著上调,与细胞结果一致。小结：ARHI基因影响胰腺癌细胞趋化因子和受体的表达,提示ARHI基因抑制胰腺癌生长作用部分通过趋化因子及其受体调节肿瘤微环境的血管生成和免疫而实现的。结论：1. ARHI在78.3%胰腺癌组织中表达缺失或下调,再次验证ARHI基因是胰腺癌抑癌基因。而ARHI蛋白表达与MVD、VEGF及CXCL8蛋白表达呈负相关。2. ARHI基因抑制胰腺癌细胞株增殖,抑制胰腺癌裸鼠荷瘤的生长,抑制胰腺癌裸鼠荷瘤中血管生成和相关蛋白VEGF和CXCL8的表达。3. ARHI基因抑制肿瘤中血管生成和相关蛋白VEGF和CXCL8可能通过mTOR/NF-KB和STAT3信号传导通路,进而抑制胰腺癌血管生成。4. ARHI基因促进或抑制肿瘤微环境中多种趋化因子和受体,进而影响胰腺癌生长和转移。更多还原

【Abstract】 Pancreatic cancer is one of the most common gastrointestinal malignant tumors, which has become the fourth cause of cancer death in western countries. Recently, the incidence of pancreatic cancer is increasing in China, which are ranked the7th among all of the cancers. For the low early diagnosis rate, half of the patients have developed systemic metastasis. Overall, pancreatic cancer carries an unfavorable prognosis with short survival rate and poor quality life due to lacking of effective treatment. The5-year overall survival rate is less than6%and the median survival time is4-6months.It is known that tumor angiogenesis is critical to tumor growth and metastasis. When the tumor is growing with a size larger than1-2mm3, its growth is angiogenesis-dependent. Many studies showed that a number of signaling molecules were involved in tumor angiogenesis, including CXCL8and VEGF as pro-angiogenesis factors. Furthermore, some chemokines and their receptors could regulate tumor angiogenesis.ARHI, a maternally imprinted gene belongs to the Ras superfamily is a tumor suppressor gene, which appeared high expression in ovarian and breast normal tissues, but down-regulated expression in ovarian and breast carcinomas. A series of studies in our group showed that ARHI was a tumor suppressor gene in pancreatic cancer. Re-expression of ARHI in pancreatic cancer cells can inhibit cell proliferation and invasion, induce apoptosis, autophagy and cell cycle arrest, and inhibit cell mobility. ARHI is an importment tumor suprressor gene in the occurrence and development of pancreatic cancer. Actually, the growth and metastasis of tumor cells is regulated by the tumor microenvironment, in which angiogenesis play a key role. Angiogenesis has become the important target for the current and future treatment strategy. Therefore, the study for ARHI, angiogenesis and related mechanisms has the important significance. Until now, there is no report to study ARHI-induced angiogenesis in pancreatic cancer.The aim of this study was to disclose the effect of ARHI on angiogenesis and related mechanism in pancreatic cancer and to analyze the impact of ARHI on chemokines and receptors in tumor microenvironment by human pancreatic cancer tissues, cell lines, and pancreatic cancer xenografted in Nude Mice. According to the above researches, we hope to find more theoretical basis of pathogenesis and feasibility of antiangiogenic therapy in pancreatic cancer for the further study.PART I:ARHI and angiogenesis-related protein in pancreatic cancer and its clinicopathological significance.Aim:To explore the relationship between pancreatic cancer and angiogenesis, and illustrate the role of ARHI in the pancreatic angiogenesis.Methods:1.23cases of pancreatic cancer and27cases of normal pancreas with clinical data were collected and recorded.2. Immuohistochemical technique was performed to detect the expression of ARHI, CD34, VEGF and CXCL8in the specimens.3. The relationship between the expression of above proteins and clinicopathological factors of pancreatic cancer was analyzed.4. The relationship among ARHI, MVD, VEGF and CXCL8analyzed.Results:1. The positive protein expression rate of ARHI in normal pancreas and pancreatic cancer tissue was88.9%and21.7%, there was a significant difference between the two groups. MVD in normal pancreas and pancreatic cancer are27.84±2.01and43.47±6.44, there was a significant difference. The positive expression rate of VEGF in normal pancreas and pancreatic cancer was44.4%and100%, there was a significant difference. The positive expression rate of CXCL8in normal pancreas and pancreatic cancer was44.4%and100%, there was a significant difference.2. ARHI expression showed significant negative correlation with tumor differentiation. CXCL8expression showed significant positive correlation with CA199and tumor differentiation. The expression of VEGF protein showed a significant correlation with age, gender, smoking history, drinking history, history of diabetes, serum CA199level, serum CA242level, clinical stage, tumor differentiation, tumor size, tumor location, lymphatic metastasis and vessel infiltration.3. In pancreatic cancer cases, MVD showed significant positive correlation with CXCL8, and there were no significant correlation on MVD with VEGF, CXCL8with VEGF, ARHI with MVD, ARHI with VEGF, ARHI with CXCL8.4. In all cases, CXCL8showed significant positive correlation with VEGF, ARHI showed significant negative correlation with MVD, VEGF and CXCL8.Summary:1) ARHI expression in pancreatic cancer was loss or significantly down-regulated, however, MVD, VEGF and CXCL8showed a significant high expression in pancreatic cancer.2) ARHI expression had significant correlation with tumor differentiation. MVD showed a significant correlation with serum CA199level, clinical stage, tumor differentiation, tumor size and lymphatic metastasis.3) In all cases, CXCL8showed significant positive correlation with VEGF, and ARHI showed significant negative correlation with MVD, VEGF and CXCL8. PART II:The effect of ARHI on angiogenesis of pancreatic cancer cell lines and xenografted in nude mice and the related mechanisms Aim:To investigate the effect of ARHI on angiogenesis of stably transfected PANC-1cell line and pancreatic cancer xenografted in Nude Mice, and to explore the related mechanisms.Methods:1. Both plasmids of pIRES2-ARHI-EGFP and pLNCX2-ARHI-EGFP were transfected into human pancreatic cancer cell line PANC-1by lipofectamine2000, the polyclonal stable PANC-1cell line with G418was selected.2. The expressions of ARHI mRNA and protein in pLNCX2-ARHI-EGFP group were analyzed by RT-PCR and Western blot methods respectively. MTT assay were used to determine the effect of ARHI on the cell proliferation.3. The expressions of mTOR, SOSC3, Stat3, NF-κB, VEGF and CXCL8were detected at the mRNA level by Real-time PCR.4. The cells with pLNCX2-ARHI-EGFP, pLNCX2-EGFP and control were injected subcutaneously into nude mice to establish the pancreatic cancer xenografted model. The tumor growth, size, and body weight change of nude mice were recorded and compared.5. The protein expression of ARHI, Ki-67, CD34, VEGF and CXCL8protein in xenografted tumor tissues were studied by immunohistochemistry. The expressions of mTOR, SOSC3, Stat3, NF-κB, VEGF and CXCL8were detected at the mRNA level in xenografted tumor tissues by Real-time PCR.Results:1. After4weeks of G418selection, the polyclonal stable transfected PANC-1cell line was identified with ARHI mRNA and protein expression by RT-PCR and western blot.2. The stable cell lines transfected with lentivirus plasmid pLNCX2-ARHI-EGFP were chose for further studies due to the stronger fluorescence density.3. The MTT assay showed that ARHI could inhibit cell proliferation in pLNCX2-ARHI-EGFP group with a significant difference compared with that in vector group at the third day.4. The expression of mTOR, Stat3, NF-κB, VEGF and CXCL8at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group compared with the polyclonal vector stable cell line, while the SOSC3gene was significantly up-regulated. Western blot analysis showed that the expression of p-mTOR, p-Stat3, NF-κB and CXCL8proteins were significantly down-regulated in pLNCX2-ARHI-EGFP group.5. The growth of tumor in nude mice was much slower in pLNCX2-ARHI-EGFP group than the vector group and control group, meanwhile, the size of tumors of ARHI group were also smaller than the other two groups, and the body weight change of vector group and control group were more obvious than pLNCX2-ARHI-EGFP group.6. Pancreatic cancer xenografted models was successfully established with ARHI positive expression.7. In pLNCX2-ARHI-EGFP group xenografted model, the ARHI protein expressed in the higher level than that in vector group and control group, while the Ki-67index, MVD (characterized with CD34), VEGF and CXCL8protein expressed in lower level inversely.8. The expression of mTOR, Stat3, NF-κB, VEGF and CXCL8at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group xenografted model than the vector group, while while the SOSC3gene was significantly up-regulated, and the result was in accordance with that in the cell level.Summary:1) The polyclonal stable transfected PANC-1cell line which could express ARHI was successfully established.2) Re-expression of ARHI in stable transfected PANC-1cell line could inhibit proliferation of pancreatic cancer cells.3) The pancreatic cancer xenografted nude mice model was successfully established, and the data in the animal levels showed that ARHI gene had an inhibition effect on tumor growth and angiogenesis in pancreatic cancer.4) ARHI gene may inhibit the expression of CXCL8and VEGF by means of mTOR/NF-KB and STAT3signaling pathway, eventually inhibit angiogenesis of pancreatic cancer.Part Ⅲ:The effect of ARHI transfection on chemokines and receptors gene expression profileAim:To explore the effect of ARHI transfection on the chemokines and receptors gene expression profile of PANC-1cells.Methods:1. Expression profile, including84chemokines and receptors related genes was detected by using quantitative real-time PCR based RT2Profiler(?) PCR Array.2. Real-time PCR was performed to detect mRNA expression of CXCL8, CXCL1, CXCR4, CXCR3and CXCR2in stable transfected cell lines and xenografted cancer tissues.Results:1. PCR Array:1) In PANC-1cells transfected with pLNCX2-ARHI-EGFP, the expression levels of mRNA of36genes were down-regulated, and9genes were up-regulated.2) Among these genes related with tumor angiogenesis, CCL2,CXCL1,CXCL2,CXCL8,CXCL12,CXCR4were significantly down-regulated, CXCL3,CCR1,CCR2were slightly down-regulated. CXCL9,CXCL10,CXCL11,CXCR3were significantly up-regulated, and the increase of CXCL9was the most significant (-27.8fold).3) Among these genes related with tumor metastasis and invasion, CXCL12and CXCR4were significantly down-regulated, MMP-2was slightly down-regulated. Among these genes related with location of organ infiltration and latency, CXCL12,CXCR4,CCR7were significantly down-regulated, CXCR5was slightly down-regulated. Among these gene with tumor immunity, CXCL8,CXCR1,CCR7were significantly down-regulated, and decrease of CXCR1was the most significant (-41.3fold).2. The expression of CXCL8, CXCL1and CXCR4at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group compared with the vector group, while the CXCR3gene was significantly up-regulated, the result was the same as PCR Array. We also found the expression of CXCR2was down-regulated in pLNCX2-ARHI-EGFP group compared with the vector group.3. In xenografted cancer tissues, the expression of CXCL8, CXCL1, CXCR4and CXCR2at the mRNA level were significantly down-regulated in pLNCX2-ARHI-EGFP group compared with the vector group, while the CXCR3gene was significantly up-regulated, and the result was in accordance with that in the cell level.Summary:ARHI gene could influence the expression of many chemokines and receptors, which suggests that the mechanisms of ARHI gene inhibiting tumor growth partly correlate with chemokine and receptors to regulating tumor angiogenesis and immunity in microenvironment in pancreatic cancer.Conclusions:1) The expression of ARHI gene in pancreatic cancer was loss or significantly down-regulated, which verified the inhibition effect of ARHI gene. ARHI showed significant negative correlation with MVD, VEGF and CXCL8.2) Re-expression of ARHI gene could inhibit proliferation of pancreatic cancer cells. ARHI gene could inhibit the growth and angiogenesis of xenografted cancer tissues, as well as the expression of VEGF and CXCL8.3) ARHI gene may inhibit the expression of CXCL8and VEGF by means of mTOR/NF-KB and STAT3signaling pathway, eventually inhibit angiogenesis of pancreatic cancer.4) ARHI gene could influence expression of many chemokines and receptors, and then inhibit tumor growth and metastasis.更多还原