【作者】 陈滢；

【导师】 叶笃筠；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 研究背景:在肿瘤的发生、发展和转移过程中,肿瘤血管发生（tumor angiogenesis）、血管网向肿瘤组织内渗透发挥了至关重要的作用。如何调控肿瘤“血管生成开关”一直是癌症防治领域研究的热点,针对肿瘤血管生成这一关键环节研发药物,为癌症的治疗提供了新的行之有效的手段。Rudolf Virchow于1860年首次发现了癌症和炎症的相关性。目前,炎症反应已被证实是肿瘤局部微环境中一个主要特征。脂氧素（lipoxins,LXs）是第一个在活体内被鉴定的内源性炎症反应“刹车信号（braking signal）”家族,被发现在弓形虫感染宿主血清中高表达。此外,弓形虫急性感染的小鼠肿瘤模型中,肿瘤血管生成显著减少,肿瘤生长也得到了明显的抑制。这强烈提示,脂氧素很可能介入到肿瘤的发生过程中。目的:研究LXA⁴对H22荷瘤小鼠肿瘤生长及转移的抑制作用,并初步探讨其可能的作用机制。方法:体外培养小鼠H22肝癌细胞,实验分为四组,分别给予不同浓度LXA4处理,采用免疫酶方法测定常氧下H22细胞培养上清内VEGF表达;用氯化钴（CoCl₂） （200μM）诱导常氧下缺氧诱导因子（HIF）-1α的表达,实验分为空白组、CoCl₂组和CoCl₂+LXA₄组,采用荧光免疫细胞化学方法观察细胞内HIF-1α的表达水平及核转位情况;免疫印迹法用来检测细胞内HIF-1α蛋白含量。整体实验中,采用活体腹水H22肝癌细胞皮下接种法复制成小鼠H22荷瘤模型,实验分为空白组、H22组和H22+BML-111组（1 mg/kg,0.1 ml/次）。实验过程中记录并观察小鼠的一般情况和肿瘤生长状况;于肿瘤细胞接种后第21天取各组小鼠血清,用免疫酶方法测定血清中VEGF的表达;采用颈椎脱臼法处死各组实验小鼠,测定各组小鼠肿瘤体积并计算肿瘤生长抑制率（ IR）;H&E染色后光镜下对各组肿瘤进行病理组织学检查,并观察肿瘤自发性肺转移的情况;免疫组织化学法用来检测肿瘤组织内血管生成及HIF-1α的表达情况。结果:⑴LXA4呈剂量依赖性抑制H22细胞分泌VEGF（*P<0.05）;⑵LXA₄呈剂量依赖性下调H22细胞中HIF-1α的表达（*P<0.05）;⑶整体实验中,BML-111降低小鼠血清中VEGF以及肿瘤组织中HIF-1α的表达水平（*P<0.05）;⑷病理组织学检测结果显示,H22+BML-111组小鼠肿瘤组织内,CD34抗原表达阳性少（*P<0.05）,肿瘤血管生成受到抑制;⑸BML-111抑制小鼠原发性肿瘤的生长,IR=80.78%;⑹BML-111抑制肿瘤自发性肺转移。结论:脂氧素A₄抑制H22荷瘤小鼠肿瘤生长及自发性肺转移,其机制可能是通过抑制肿瘤表达VEGF和HIF-1α从而抑制了肿瘤相关的血管发生。更多还原

【Abstract】 Background: Tumor angiogenesis, proliferation of a network of blood vessels that penetrates into cancerous growths, plays an essential role in carcinogenesis, cancer progression and metastasis. This angiogenic switch of tumor has been a new focus of cancer research over past years, and targeting tumor vasculature represents a new promising tool for cancer therapy. In 1863, Rudolf Virchow first described that cancer and inflammation were connected. Today, the inflammatory response is believed to be a key feature of the environment around and within tumors. As the first family of endogenous“braking signals”in inflammation identified in vivo, LXs was found to reach a high level in the serum of host infected with Toxoplasma gondii. Importantly, it was recently identified that the tumor growth was inhibited concomitant with suppressed angiogenesis during acute T.gondii infection in mice. These observations strongly suggested that LXs might be involved in tumor angiogenesis.Objective: To investigate the effects of Lipoxin A₄ （LXA₄） on inhibiting tumor growth of H22 tumor-bearing mice and the possible mechanisms of LXA₄ in this process. Methods: H22 cells were cultured in vitro and incubated in the absence or presence of LXA4. VEGF protein in supernatant was measured by ELISA kit. Cobalt chloride （CoCl₂） was used to mimic hypoxic condition, and H22 cells were exposed to CoCl₂ （200μM） in the absence or presence of LXA₄, immunofluorescent and western blot assays were used to detect the expression and nuclear accumulation of HIF-1αprotein. In the flowing in vivo experiments, BML-111, a C-7 truncated analog of LXA4, was used to evaluate the effect of LXA₄. Mice were weighted and randomly divided into control group, H22-bearing group and H22-bearing +BML-111 treated group （0.1 ml, final dosage of 1 mg/kg, equals to 1/10 LD50）. The general conditions of the mice including activity, fur growth and weight were observed every day. On day 21 after H22 inoculation mice serum VEGF concentration was determined by ELISA assay. At the time of autopsy on day 21 after inoculation, tumors were dissected and weighted. The tumor volumes and tumor growth inhibition rate （IR） was calculated. For histological detection, tumor and lung sections were stained with hematoxylin and eosin （H&E） routinely for conventional morphological evaluation under light microscope. Immunohistochemical method was used to detect the expression of HIF-1αprotein and microvessel density was determined by the mean number of CD34-positive vessels.Results:⑴LXA₄ inhibited VEGF production in H22 cells in a dose-dependent manner （*P<0.05）.⑵The level of HIF-1αprotein was suppressed by LXA₄ （*P<0.05）.⑶In the in vivo experiments, BML-111 down-regulated both the secretion of VEGF in mice serum and the expression of HIF-1α（*P<0.05）.⑷BML-111 suppressed tumor-related angiogenesis （*P<0.05）.⑸BML-111 inhibited primary tumor growth.⑹BML-111 suppressed spontaneous pulmonary metastasis.Conclusions: LXA4 systemically inhibit the primary growth and spontaneous metastasis of liver tumor, the mechanism is probably through anti-angiogenesis by suppressing the expression of VEGF and HIF-1αof tumor cells.更多还原