TGF-β在HepG2细胞中对VEGF表达的影响及机制研究

发布时间：2018-03-10 22:57

本文选题：转化生长因子-β1　切入点：血管内皮生长因子　出处：《石河子大学》2005年硕士论文　论文类型：学位论文

【摘要】：原发性肝癌是我国最常见的恶性肿瘤之一,其中肝细胞肝癌(HCC)占原发性肝癌的90%。HCC转移和复发是影响患者术后长期存活的主要障碍之一。而肿瘤新生血管的形成已经被认为是肿瘤发生复发转移的重要机制之一,探讨肿瘤微血管的形成,将极大的有助于解决肿瘤的进一步发展、以及术后的存活率等问题。血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)是被公认的作用最强的促内皮细胞生长因子,在肿瘤的新生血管形成中成为最关注的因素。然而肝癌细胞中VEGF基因和蛋白表达的具体信号通路以及在新生血管形成中的具体作用尚不明确。此外转化生长因子-β(Transfoming Growth Factor β,TGF-β)由于其在肿瘤的发生发展中的双重作用而受到关注,因此其在肿瘤的发生、发展过程中的作用不可忽视。在鼠平滑肌细胞、或其他类型肿瘤细胞中的研究发现TGF-β能够通过Smads或者MAPK信号通路刺激VEGF的表达。然而在肝癌细胞内它是通过什么信号通路引起VEGF表达增加的,目前尚不清楚。并且肿瘤生长过程中必定伴有不同程度的缺氧,那么它能否协同缺氧促进VEGF表达,它们之间存在什么样的联系,仍需进一步的探索。本研究在体外培养肝癌细胞的基础上分6组:1)空白对照组;2)TGF-β1组(不同浓度);3)氯化钴组(不同浓度);4)TGF-β1+PD98059组;5)TGF-β1+氯化钴组;6)TGF-β1+氯化钴组+PD98059组;免疫细胞化学分别检测TGF-β1和氯化钴两组VEGF的蛋白表达,一步法RT-PCR半定量技术检测全部分组的VEGFmRNA的表达。结果:免疫细胞化学检测TGF-β1组或氯化钴组都有明显的VEGF蛋白阳性表达。RT-PCR结果也显示:分别使用氯化钴和TGF-β1处理的细胞VEGFmRNA的表达显著高于对照组(p0.01);并且不同浓度之间VEGFmRNA表达的差异也有显著性。TGF-β1+PD98059组与单纯TGF-β1组相比有显著性差异(p0.01);TGF-β1+氯化钴组VEGFmRNA表达与氯化钴组有差异(p0.05),TGF-β1+氯化钴+PD98059组与TGF-β1+氯化钴组比较VEGFmRNA表达也有差异(p0.05); 研究结果表明:肝细胞肝癌的新生血管形成中,TGF-β1或缺氧都起到了促进VEGF表达的作用,并且TGF-β1能够协同缺氧上调VEGF的表达;而ERK/MAPK信号通路对TGF-β1引起的VEGF表达以及TGF-β1协同缺氧诱导VEGF的表达产生了抑制作用。
[Abstract]:Hepatocellular carcinoma is one of the most common malignant tumors in China, including hepatocellular carcinoma (HCC) for primary hepatocellular carcinoma 90%.HCC metastasis and recurrence is one of the major obstacles to the long-term survival of patients after surgery. The tumor angiogenesis has been considered as an important mechanism of occurrence of metastasis of tumor recurrence. The formation of tumor angiogenesis, will greatly contribute to the further development of the problem and solve the tumor, postoperative survival rate.
Vascular endothelial growth factor (Vascular Endothelial, Growth Factor, VEGF) is promoting endothelial cell growth factor most recognized, in tumor neovascularization in become the most attention. However the specific signal expression of VEGF gene in hepatocellular carcinoma cells and protein pathway and the specific role in neovascularization is not clear in addition, transforming growth factor beta (Transfoming Growth Factor beta, beta TGF-) because of its dual role in the development of tumor and attention, therefore in the tumorigenesis, development process can not be ignored. In rat smooth muscle cells, or of other types of tumor cells in TGF- beta can be found the expression of Smads or MAPK stimulate VEGF signaling pathways in hepatoma cells. However, it is through what signal pathway caused by increased expression of VEGF, it is unclear and the tumor. There must be different degrees of hypoxia during the long term, so whether it can cooperate with hypoxia to promote the expression of VEGF and what relations exist between them, we still need further exploration.
In this study, based on the in vitro cultured hepatoma cells were divided into 6 groups: 1) blank control group; 2) TGF- beta 1 groups (different concentrations); 3) cobalt chloride group (different concentrations); 4) TGF- beta 1+PD98059 group; 5) TGF- beta 1+ cobalt chloride group; 6) TGF- beta 1+ cobalt chloride group +PD98059; immunocytochemistry were used to detect the expression of TGF- 1 and cobalt chloride two group VEGF protein expression, expression of one-step RT-PCR semi quantitative technique all packets of VEGFmRNA.
Results: immunocytochemical detection of TGF- beta 1 or cobalt chloride group had the positive expression of VEGF protein.RT-PCR results also show that the expression of using cobalt chloride and beta TGF- 1 cells treated with VEGFmRNA was significantly higher than the control group (P0.01); and different concentration between VEGFmRNA expression significantly.TGF- beta 1+PD98059 group and simple TGF- beta 1 groups have significant difference (P0.01); TGF- beta 1+ cobalt chloride group VEGFmRNA differentially expressed with cobalt chloride group (P0.05), TGF- beta 1+ cobalt chloride +PD98059 group and 1+ group TGF- beta cobalt chloride VEGFmRNA expression also has difference (P0.05);
The results showed that the angiogenesis of hepatocellular carcinoma in the formation of TGF- beta 1 or hypoxia plays a role in promoting the expression of VEGF and TGF-, the expression of beta 1 synergistically upregulated VEGF and hypoxia; inhibited ERK/MAPK signaling pathway on TGF- induced expression of VEGF beta 1 and beta 1 TGF- co induced by hypoxia VEGF the expression.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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