【作者】 刘安安；

【导师】 胡先贵；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 胰腺癌是一种凶险的恶性肿瘤,在我国年发病率为5.1/10万,居全身恶性肿瘤的第8位,并呈逐年上升的趋势。胰腺癌的显著特点是恶性程度高,淋巴结转移发生早,即使接受根治性手术切除及放射、化学药物治疗,局部复发及转移的发生率仍很高,胰腺癌的大多数胰腺癌患者切除术后预后差。美国国立卫生研究院报告,胰腺癌1年生存率为8%,5年生存率为3%,中位生存期仅2～3月。我国的统计资料显示,5年生存率在5%左右。大量研究表明,胰腺导管腺癌细胞主要向周围淋巴结、胰内神经、胰周结缔组织及肠系膜上动脉周围神经丛进行侵袭和转移,淋巴结转移与神经侵犯是影响患者预后最重要的因素之一,因此在探索胰腺癌的有效治疗方法方面,如能有效的预防和控制胰腺癌的淋巴转移,将有望大大提高为胰腺癌的综合治疗效果。长期以来淋巴管被视为肿瘤转移最重要的路径。2000年以后,淋巴管上皮特异性单抗的出现,大大推进了肿瘤组织中微淋巴管的研究。大部分的研究结果都显示肿瘤诱导的新生淋巴管与肿瘤侵袭、转移及预后有密切的相关性。近年来的研究发现,血管内皮生长因子(vascular endothelial growth factor,VEGF)C、D能够通过激活血管内皮生长因子受体3(vascular endothelial growth factor receptor,VEGFR-3)刺激淋巴管新生,与包括胰腺癌在内的多种实体瘤淋巴结转移和局部侵犯有密切关系。在促进肿瘤转移的机制上,淋巴管新生相关因子可能不仅仅增加肿瘤相关新生淋巴管的数量,而且可能激活淋巴上皮分泌化学性因子、粘附分子以及受体来参与肿瘤细胞与淋巴内皮之间的相互作用,从而促进肿瘤的扩散,目前淋巴管新生的分子介导,无论是临床还是实验上都认为VEGF-C/D-VEGFR-3信号传导通路是新生淋巴管的主要调节环节,有迹象研究表明VEGF-C与肿瘤的淋巴结转移呈正相关而通过阻断VEGF-C/D-VEGFR-3信号传导通路,能够抑制淋巴管生成及淋巴结转移。RNA干扰(RNA interference,RNAi)是广泛存在于生物体内的一种通过双链RNA,(double-stranded RNA,dsRNA)来抵抗病毒入侵和调节基因表达的自然机制,RNAi自1998年首先提出以来立即成为了分子生物学领域的研究热点,近几年的研究基本阐明了RNAi的机制,RNAi可分为3个阶段,即较长dsRNA经RnaseⅢ酶Dicer的切割,RNA诱导沉默复合物(RNA-induced silencing complex,RISC)的组装和目的mRNA的降解。目前在RNAi作用特征和应用的研究上也取得了较大进展,小分子干涉siRNA(small interfering,RNA)在哺乳动物细胞中能够介导转录后基因沉默(post-transcriptional gene silencing,PTCG),因其具有高效性和序列特异性,已经成为功能基因组学研究的有力工具之一,尤其在肿瘤相关基因的研究和肿瘤基因治疗研究领域取得了突破性进展。本实验利用RT-PCR的方法对胰腺癌组织以及癌旁组织和正常胰腺组织中淋巴管新生相关因子VEGF-C,VEGFR-3的表达进行研究,比较与淋巴管密度的相关性,最后进行VEGF-C基因靶向RNAi抑制VEGF-C表达的实验研究,为探寻基因治疗抑制胰腺癌淋巴管生成及淋巴结转移,提高胰腺癌治疗效果及远期生存率的新方法进行一定的实验基础。第一部分:胰腺癌组织淋巴管新生因子VECF-C、VEGFR-3的表达的测定目的:分析胰腺癌组织、癌旁组织以及正常胰腺组织中淋巴管新生因子VEGF-C、VEGFR-3表达的差异及与临床病理特点的关系。方法:选至长海医院胰腺外科2007.3-2007.6切除胰腺肿瘤组织标本共20例,手术切除后30s内迅速以液氮保存肿瘤组织、肿瘤边缘3mm内的癌周组织,以及远离肿瘤的阴性切缘的正常组织,运用RT-PCR方法测定这三种不同组织中淋巴管新生相关因子表达的差异以及与临床病理特点的关系。结果:VEGF-C、VEGFR-3在胰腺癌组织、癌旁组织、正常组织中表达量差异显著(p＜0.05),其中VEGF-C在肿瘤组织组织中表达明显高于癌旁组织即正常胰腺组织,VEGFR-3在癌旁组织明显高于癌组织及正常胰腺组织,肿瘤组织中VEGF-C、癌旁组织中VEGFR-3表达在不同年龄、性别、肿瘤部位、分化程度的患者间差异不显著(p＜0.05);而在淋巴结转移阴性和阳性患者中表达量差异显著(p＜0.05),相关分析显示肿瘤组织中VEGF-C、癌旁组织中VEGFR-3表达与淋巴结转移情况呈正相关;肿瘤组织中VEGF-C、癌旁组织中VEGFR-3表达在TNM分期为Ⅰ期、Ⅱ期的患者与Ⅲ期、Ⅳ期患者间差异显著(p＜0.05)。第二部分:胰腺癌中淋巴管密度与淋巴管新生因子VECF-C、VEGFR-3相关性目的:比较胰腺癌组织、癌旁组织、正常胰腺组织中微淋巴管密度的差异,以及研究与VEGF-C、VEGFR-3表达的相关性。方法:应用Envision法免疫组化技术对第一部分收集的20例胰腺癌组织、癌旁组织、正常胰腺组织进行LYVE-1染色,按Weider法进行微淋巴管密度的判定,分析在这三种组织中微淋巴管分布的差异,同时与第一部分所得的VEGF-C及VEGFR-3的结果进行比较,研究VEGF-C、VEGFR-3的表达与微淋巴管密度的相关性。结果:在胰腺癌组织、癌旁组织、正常组织中微淋巴管密度差异非常显著(p＜0.05),其中癌旁组织明显高于癌组织,统计分析显示VEGF-C、VEGFR-3表达与微淋巴管密度呈正相关。第三部分:siRNA抑制胰腺癌细胞PANC-1的VEGF-C表达的实验研究目的:在体外观察VEGF-C基因靶向siRNA转染胰腺癌细胞株PANC-1,对VEGF-C表达的影响。方法:根据VEGF-C基因序列以及siRNA的设计原则,利用在线siRNA设计软件设计并合成VEGF-C基因靶向的siRNA,以阳离子脂质体Lipofectamine 2000稀释后与合成的siRNA混合后形成脂质体-siRNA复合物,将该复合物加入培养细胞中,在转染24小时、48小时及72小时后收集细胞。以RT-PCR方法测定在RNA干扰后VEGF-C的mRNA的表达。选择干扰试剂盒附带的阳性对照GAPDH-siRNA的正反义寡核苷酸模板,与合成的干扰序列的正反义模板一起在体外进行转录,将得到的GAPDH-siRNA以同样方法导入肿瘤细胞株PANC-1作为阳性对照。另外同时随机设计引物序列,以同样脂质体法导入培养的细胞株PANC-1作为siRNA的阴性对照组,阴性和阳性对照组均同样在24小时、48小时、72小时后收集细胞,RT-PCR检测阴性对照组VEGF-C和阳性对照组GAPDH的表达。同时分别以siRNA、脂质体、Opti-MEMI(作为对照)处理培养的胰腺癌PANC-1细胞,分别计算存活率,进行细胞毒性检测。结果:分别以siRNA、脂质体、Opti-MEMI(作为对照)处理培养的胰腺癌PANC-1细胞,各组细胞存活率无明显统计学差异,证实单独siRNA或脂质体对培养的胰腺癌PANC-1细胞均没有明显毒性。阳性对照组RT-PCR法测试证实GAPDH基因表达明显受到抑制,证实脂质体法将体外合成的siRNA导入胰腺癌细胞的可行性。以自行设计的两列VEGF-C基因靶向siRNA(40nM)和VEGF-C靶向siRNA的错义序列,分别以脂质体法分别导胰腺癌细胞PANC-1,RT-PCR法测试VEGF-C基因表达,证实导入VEGF-C基因靶向siRNA的胰腺癌细胞VEGF-C表达与阴性对照组相比明显受到抑制。在转染24小时后VEGF-C表达明显受到抑制,并在转染72小时后始表达开始恢复。更多还原

【Abstract】 The morbility of pancreatic carcinoma in P.R.China is 5.1/100,000,It is increasing year by year.This disease has very poor prognosis because of early metastasis and invasion through lymphatic system.In a report of U.S.National Institutes of Health,Survival rate in 1 year is 8%and in 5 years is 3%.Metastasis of this neoplasm through lymphatic system is one of the most important factors which affect the prognosis.Preventing the metastasis through lymphatic system may make great progress in treament of pancreatic carcinoma.Discovering of monoclonal antibody of epithelium of lympgatic vessel has improved the study in microlympgatic vessel in tumour tissue since 2000.It is shows that malignancy cell’s metastasis and invasion correlates with lymphangiogenesis induced by the neoplasm.Recently studies show that vascular endothelial growth factor receptor 3 （VEGFR-3） is activated by its specific ligand vascular endothelial growth factor C（VEGF-C） and then promotes Lymphangiogenesis.lt may cause metastasis and invasion of the neoplasms.These facts which correlated with Lymphangiogenesis not only promote lymphangiogenesis but also active several chemical factor adhesion、molecule and receptor secreted by epithelium of lympgatic vessel which infact the interaction between the tumor cell and endothelial cell of lympgatic vessel.So these facts can promote cancer progression. The VEGF-C/VEGFR-3 asix affects tumour progress by regulation lymphagiogensis.And it may be the most important regulators of lymphagiogensis.Some studies show inhibiting it can depress lymphagiogensis and metastasis of tumour through lympgatic vessel.RNA interference（RNAi） by double stranded RNA（dsRNAs） molecules of approx--imately 20-25 nucleotides termed short interfering（siRNAs） is a powerful method for pr--eventing the expression of a particular gene.The Mechanism of RNAi can be divided in three steps:the dsRNAs processed into small interfering RNAs（siRNAs） by an RNaseⅢ--like enzyme,RNA-induced silencing complexes（RISCs） packed and RISCs cleave and destroy the cognate RNA.In mammalian cells,introduction of siRNA initiates post-tran--scriptional gene silencing（PTCG）.It is going to be a powerful method in study of fun--ctional genomics especially as tumor related gene and gene therapy because of its highly efficacy and specificity.We study expression of VEGF-C and VEGFR-3 in tissues of pancreatic carcinoma, adjacent tissues of pancreatic carcinoma and tissue of normal pancreas by realtime PCR and the relationship With the lympgatic vessel density in these tissues.Then we use VEGF--C gene targeted siRNA to depress the expression of VEGF-C gene in vitro in order to find a new method of gene therpy for pancreatic carcinoma by inhabiting lymphagiogensis and metastasis of neoplasmsPartⅠ:Expression of VEGF-C and VEGFR-3 in pancreatic adenocareinoma, adjacent tissues of pancreatic carcinoma and normal pancreasObject:Analysis the expression of VEGF-C and VEGFR-3 in pancreatic carcinoma, adjacent tissues of pancreatic carcinoma and normal pancreas to find the relationship bet--ween these expressions and the features of clinical pathology.Method:To preserve 20 tissues of pancreatic carcinoma、their adjacent tissues and tissues of their negative margin in liquid nitrogen（LN） at the pancreectomy in Changhai Hospital.Studing the expression of VEGF-C and VEGFR-3 in these tissues by RT-PCR and studying the relationship with the features of clinical pathology by statistical method.Result:The expression of VEGF-C in tissue of pancreatic carcinoma and VEGFR-3 in adjacent tissues of pancreatic carcinoma is higher than it in pancreatic carcinoma and the normal pancreas（p＜0.05）.And these expressions has no relationship with the the features of clinical pathology such as age、gender、position of the neoplasm、degree of differentiation（p＞0.05）.But it is higher in tissue with positive lymph node than with negative lymph node（p＜0.05）.PartⅡ:Relationship of the expression of VEGF-C and VEGFR-3 in tissues of pancreatic carcinoma,adjacent tissues of pancreatic carcinoma and normal pancreas with lympgatic vessel density（LVD）in these tissuesObject:Studing the difference of lympgatic vessel density（LVD） in pancreatic carcinoma, adjacent tissues of pancreatic carcinoma and normal pancreas and the relationship with expression of VEGF-C and VEGFR-3 in these tissues.Method:Immunohistochemistry were used to detect the expression of LYVE-1 to detect LVD in 20 cases of pancreatic carcinoma,adjacent tissues of pancreatic carcinoma and normal pancreas collected in partⅠand studying the relationship with expression of VEGF-C and VEGFR-3 in these tissues by statistical method.Result:The LVD in adjacent tissues of pancreatic carcinoma is higher than it in pancreatic carcinoma and normal pancreas（p＜0.05） And it correlated with the expression of VEGF-C and VEGFR-3（p＜0.05）PartⅢDepression of the expression of VEGF-C by VEGF-C gene targeted siRNA in vitroObject:To observe change in the expression of VEGF-C of human pancreatic cell （PANC-1 ） after introduction of VEGF-C gene targeted siRNA.Method:VEGF-C gene targeted siRNA is designed and composed according to siRNA design software online.VEGF-C gene targeted siRNA is brought by cationic liposome(Lipofectamine^TM 2000 Reagent) to transfect PANC-1 cell.GAPDH-siRNA is introducted as positive control,Missense sequence of these siRNA is also introducted as negative control.24h, 48h、72h after these transfection.The transfected cell is collected.RT-PCR is performed to analysis the expression of VEGF-C in transfected cell and negative control.Result: PANC-1 cell’s survival rate has no changed after dealed with siRNA and gationic liposome respectively.The expression of VEGF-C in transfected cell is highly depressed（p＜0.01） in 24h,and has recovered in 72h.更多还原