【作者】 杨剑丽；

【导师】 王丽；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2009， 博士

【摘要】 宫颈癌是世界女性第二大恶性肿瘤。已经有实验证实血管新生与宫颈癌患者预后不良有关。有多个血管生成因子参与肿瘤血管发生过程,其中主要的有VEGF、bFGF和血管生成素。bFGF是可以与肝素结合的多功能多肽家族的成员之一。它具有促血管生成的作用,它也在多种肿瘤的增殖和分化中发挥重要作用。研究发现多种恶性肿瘤都高表达bFGF。有学者研究显示宫颈癌患者的血清中bFGF含量增高。血管生成素是唯一一个以促血管发生的功能而命名的血管生成因子。血管生成素在内皮细胞可以发生核转移,进而促进内皮细胞的增殖。血管生成素在HeLa细胞中也有核转移现象,并且能够在细胞核内促进rRNA的合成。目前对于bFGF和血管生成素的研究主要集中在其如何诱发肿瘤的血管发生,进而促进肿瘤的进一步生长和恶化,以及它们在肿瘤预后评价中的相关作用。而对于二者的关系的研究比较少。有学者发现,应用血管生成素的反义核苷酸可以抑制体外培养的HeLa细胞和体内的肿瘤细胞的生长,但是细胞和肿瘤内的bFGF表达增加。本实验室前期的研究发现,在转染血管生成素反义核苷酸的黑色素瘤细胞中,bFGF的表达增加,而在转染血管生成素正义核苷酸的黑色素瘤细胞中,bFGF的表达降低。为了进一步的研究bFGF与血管生成素的关系,我们克隆了人bFGF基因,并且构建了含有人bFGF基因的正义和反义的真核表达载体,建立了稳定表达bFGF的细胞株。进而探讨在HeLa细胞中,利用bFGF基因的转染改变细胞内源的bFGF基因的表达水平对血管生成素表达的影响。实验结果显示,在转染反义bFGF基因的bFGF低表达细胞株中,细胞表达的bFGF的mRNA和蛋白都降低,细胞的增殖受到了抑制,但是血管生成素的蛋白表达水平却提高了。相反,在转染正义bFGF基因的bFGF高表达的细胞株中,细胞表达的bFGF的mRNA和蛋白均增加,同时细胞的增殖增加,然而血管生成素的的蛋白表达水平下降了。但是,高表达和低表达bFGF的细胞的血管生成素的mRNA水平无显著性差异。对HeLa细胞及高表达bFGF和低表达bFGF的细胞,施加外源的血管生成素后,HeLa细胞和转染反义bFGF基因的低表达bFGF的细胞株,细胞增殖加速,而转染正义bFGF基因的高表达bFGF的细胞株的细胞增殖无改变。实验结果表明,bFGF和血管生成素均可以直接刺激HeLa细胞的增殖。证明外源bFGF基因的转染导致细胞内源bFGF水平发生改变,这一改变负性地影响细胞内源血管生成素蛋白的表达。更多还原

【Abstract】 Cancer of the uterine cervix is the second most common cancer among women in the world, and the most common gynecological malignancy in P.R. China. Angiogenesis stimulates tumor growth, invasion and metastasis, and therefore is a crucial step in tumorigenesis. Further, evidence of increased neovascularization has been shown to be a negative prognostic indicator in many solid tumors, including cervical cancer. Several growth factors may be involved in the angiogenic process, including angiogenin and basic fibroblast growth factor (bFGF).Basic fibroblast growth factor (bFGF), a member of a group of heparin-binding multifunctional polypeptides, is one of the most potent angiogenic factors. It is also involved in proliferation and differentiation of a variety of normal and malignant cells and tissues. Many malignant tumors showed an increase in bFGF expression. Angiogenin has been shown to play a role in tumor angiogenesis. Its expression is upregulated in many types of cancers. Angiogenin undergoes nuclear translocation in endothelial cells, which has been shown to be necessary for angiogenesis. Tsuji et al have shown that nuclear translocation of angiogenin occurred in HeLa cells regardless of the cell confluence status, and that angiogenin stimulated rRNA synthesis in HeLa nuclei.The role of individual angiogenic growth factors in the formation of new blood vessels and in the progression of tumor growth has been extensively investigated. The expression of angiogenin and bFGF is especially associated with a poor prognosis. Nevertheless, little is known about how these factors coordinately regulate tumor angiogenesis and how they react on the targeted inhibition of a single factor. Kishimoto et al have observed that angiogenin antisense HeLa transfectants and tumors in athymic mice actually expressed a higher amount of bFGF than the controlcells and tumors in athymic mice. Song et al demonstrated that the angiogenin sense transfectants actually expressed a lower amount of bFGF, and the angiogenin antisense transfectants expressed a higher amount of bFGF than A375 cells. To further understand the biological role of angiogenin and bFGF, we investigated the influence of bFGF on angiogenin ggene expression and angiogenin secretion in HeLa cell lines.We cloned bFGF and constructed the expression plasmid of bFGF. We transfected the bFGF gene in the sense and ntisense orientation into HeLa cells, and obtained stable bFGF underexpressing and overexpressing transfectants. In our experiments, we demonstrated that inhibition of bFGF gene and protein expression in the bFGF antisense transfectants induced increased protein expression of angiogenin. In contrast, in the bFGF sense transfectants the expression of angiogenin decreased. Delivery of recombinant angiogenin into transfected and control cells led to increased proliferation in the bFGF antisense transfectants and the control cells. However, the cell proliferation had no change in the bFGF sense transfectants. In conclusion, Furthermore, endogenous bFGF affects the expression of angiogenin in HeLa cells.更多还原