【作者】 钟大平；

【导师】 梁后杰；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2007， 博士

【摘要】 背景与目的侵袭转移是恶性肿瘤的基本特征,也是肿瘤患者死亡的主要原因之一。淋巴道是大部分肿瘤最常见的转移途径,有无淋巴结转移也是判断患者预后的重要指标。近年来随着淋巴管标记物的发现,在某些恶性肿瘤中已经证实有淋巴管生成,被称为淋巴管生成因子的VEGF-C、VEGF-D通过其受体VEGFR-3能够诱导淋巴管的生成。恶性肿瘤中的淋巴管生成因子不但能够促进肿瘤淋巴管的生成,而且增加淋巴道转移的几率。结肠癌(colon cancer)是临床最常见的恶性肿瘤之一,淋巴结转移是其发生侵袭转移的重要途径,同时也是影响患者预后的重要因素。因此了解结肠癌淋巴管生成的情况及其存在的内在机制对于结肠癌的临床治疗有相当重要的意义。T细胞淋巴瘤侵袭转移诱导因子1(Tiam1)是从小鼠T淋巴瘤细胞中分离出来的一个基因,是Ras相关的C3肉毒素底物1(Rac1)的特异性鸟苷酸转换因子(GEF),在细胞外信号刺激下可以激活Rac1,影响细胞的骨架重建、粘附运动、增殖分化和凋亡等。Tiam1与肿瘤的侵袭转移密切相关。但促进肿瘤侵袭转移的基因Tiam1是否与肿瘤淋巴管生成有关尚不清楚。因此,我们拟通过Tiam1基因沉默对结肠癌组织中淋巴管生成因子VEGF-C/D表达影响的研究,了解其是否对结肠癌的淋巴管生成、淋巴结转移产生影响,从而初步探讨结肠癌发生淋巴道转移的机制。研究方法首先,通过免疫组化的方法,检测结肠癌组织中Tiam1、Rac1和VEGF-C/D的表达情况,同时利用统计学方法,分析Tiam1表达同Rac1、VEGF-C/D、微淋巴管密度、淋巴结转移情况以及其他一些临床病理特征的相关性。其次,构建Tiam1 RNA干扰载体,体外培养结肠癌细胞,用RT-PCR和Western blot筛选高表达Tiam1的细胞并检测Tiam1基因沉默对结肠癌细胞Rac1、VEGF-C/D表达的影响。最后,将Tiam1 RNA干扰载体转染结肠癌细胞HCT116,筛选转染稳定表达的细胞。采用背部皮下种植的的方法造成裸鼠荷瘤模型。比较干扰组与对照组种植瘤在淋巴管生成、淋巴结转移方面的差异。结果在结肠癌组织切片免疫组化检测中,可观察到Tiam1和Rac1蛋白在癌细胞中呈高表达,而周围正常组织表达阴性。在50例标本中,Tiam1和Rac1的阳性率均为84%,二者表达具有一致性;有淋巴结转移的患者与无转移的患者之间,Tiam1/Rac1的阳性率差异显著(P<0.05)。VEGF-C/D也表达于癌细胞的细胞质,在肿瘤周围正常组织中则基本没有表达,二者中以VEGF-C表达多见。淋巴结转移患者的VEGF-C/D阳性率高于无淋巴结转移的患者的阳性率,差异有统计学意义。结肠癌组织中Tiam1也与VEGF-C和淋巴管密度有关。结肠癌组织中Tiam1、Rac1和VEGF-C/D的表达在患者的年龄、性别、分化程度等方面差异不显著(P>0.05)。在体外实验中,成功构建了Tiam1 RNA干扰载体,转染并筛选稳定表达的结肠癌细胞,经RT-PCR和Western blot方法证实Tiam1 RNAi质粒可明显抑制Tiam1的表达。当Tiam1基因沉默后,VEGF-C/D表达减弱,而且Rac1表达也减弱。由此推测Tiam1可以促进VEGF-C/D表达,而且可能通过Tiam1/Rac1信号途径起作用。在动物体内实验中,将Tiam1 RNA干扰载体转染HCT116细胞。干扰组裸鼠种植瘤与对照组种植瘤相比较,生长相同天数后,干扰组的淋巴管密度比对照组明显减少,并有统计学意义。结论本课题从临床观察、体外实验及动物体内实验三个层面探讨了Tiam1/Rac1信号通路在结肠癌淋巴管生成中的作用,认为(1)人结肠癌组织中不同程度表达Tiam1、Rac1及VEGF-C/D蛋白;Tiam1与Rac1表达密切相关,并且Tiam1和VEGF-C表达、淋巴管密度、淋巴结转移显著相关。(2)成功构建Tiam1 RNAi质粒,经酶切鉴定和测序证实干扰质粒序列完全正确。(3)成功将Tiam1 RNAi质粒转染结肠癌细胞,经绿色荧光、G418抗性筛选后获得稳定表达干扰质粒的细胞。经RT-PCR和Western blot方法证实Tiam1 RNAi质粒可明显抑制Tiam1的表达。(4)体外实验证实Tiam1基因沉默可抑制结肠癌细胞VEGF-C/D和Rac1的表达,推测Tiam1可能通过Rac1影响VEGF-C/D的表达。(5)动物实验证实Tiam1可通过肿瘤局部微环境中VEGF-C/D的表达,进而影响肿瘤淋巴管的生成。更多还原

【Abstract】 Background and ObjectiveInvasion and metastasis are the basic characteristic of malignant tumor and the main lethal cause. Lymphatic is the most common metastasis way of the majority cancers. Having lymphonode metastasis or not is an important index of prognostic. With the finding of the specific markers of lymphatic and lymphangiogenesis factors, tumor cells had been reported that they could induce lymphangiogenesis by autocrine or paracrine VEGF-C/D.Tiam1 was separated from the T lymphoma of mice in 1994. It was only in testicle and brain. Recently more and more studies reported that it was related to migration and metastasis in breast cancer, rhinopharynx cancer and other cancers. The mechanism is regarded that Tiam1 promote tumor cells migration and adherence through activation of Rac1.Colon cancer, a common malignant tumor in digestive system, its metastasis is mainly through lymphatic. Several researches have shown that Rac1, the backward position of Tiam1, could affect the angiogenesis in tumor, but Tiam1’s role in lymphangiogenesis is still unknown. In this study we detected Tiam1’s effect on VEGF-C/D in order to unveil its contribution in colon cancer lymphangiogenesis and lymphonode metastasis and its potential mechanism, which may laid a foundation for further understanding the mechanism of lymphatic metastasis route in colon cancer.Methods1. Colon cancer tissue immunohistochemistry assayHuman colon cancer samples, obtained from southwest hospital surgery department, were sliced and detected Tiam1, Rac1, VEGF-C/D and lymphantic with immunohistochemical analysis. The results were analyzed statistically to investigate the correlations of Tiam1, VEGF-C/D, lymphonode metastasis, lymphatic vessel density and some other clinical pathology characteristics. 2. Tiam1’s effect assayAfter Tiam1 silencing, the changes in mRNA and protein of VEGF-C/D was detected in conlon cancer cell line HCT116 by RT-PCR and Western blot. RNAi vector, carrying siRNA targeting Tiam1, was constructed, and transfected into HCT116 cell line. And the expression of VEGF-C/D in transfected cells and control group cells was compared to get some information about the mechanism that Tiam1 affects VEGF-C/D.3. Tiam’s effect on tumor lymphangiogenesisThe plasmid of pGenesil-1.neo.EGFP/Tiam1-RNAi was constructed and transfected into HCT116 cell line, then transplanted into nude mouses. After harvested the transplanted neoplasm, the expression of Rac1, VEGF-C/D, LMVD and lymphonode metastasis in the transplantation tumor were dectected and compared with the control group.Results1. Colon cancer tissue immunohistochemistry assayTiam1/Rac1 and VEGF-C/D were detected in cancer cells . Tiam1/Rac1 and VEGF- C/D were much higher in lymphonode metastasis group than that in non-metastasis group (P<0.05). Furthermore, the data statistics suggested that the expression of VEGF-C was consistent with the expression of Tiam1. Tiam1 and VEGF-C are closely correlated with lymphatic vessel density, but not with patient’s age, sexual, cellular differentiation.2. Tiam1’s effect assayBased on the RT-PCR and Western blot results, data demonstrated that the expression of VEGF-C/D had a positive relationship wiht Tiam1. After transfected the siRNA vector targeting Tiam1, the expression of VEGF-C/D was suppressed.3. Tiam’s effect on tumor lymphangiogenesisIn the transplanted tumor, the lymphatic microvessel density in transfected group was significantly lower than that in the control group (P<0.05).ConclusionAfter studying the Tiam1’s effect on VEGF-C/D expression and its mechanism in colon cancer, we hypothesized (1) Tiam1/Rac1 and VEGF-C/D were expressed in conlon cancer tissue, and Tiam1 was significantly correlated with Rac1, VEGF-C, LMVD and metastasis. (2) The plasmid of Tiam1-RNAi was constructed successfully, and verified by enzyme incision and sequencing. (3) Based on the RT-PCR and Western blot results, the expression of Tiam1 was suppressed after transfected the plasmid of Tiam1-RNAi. (4) In vitro Tiam1 gene’s Silencing can reduce the expression of VEGF-C/D in colon cancer cells. Tiam1 maybe affect VEGF-C/D through the Rac1 signaling pathway. (5) Tiam1 can induce lymphangiogenesis in colon cancer and further affect the lymphatic metastasis by its effect on VEGF-C/D in local microenvironment.更多还原