论文标题:Angiostatin cDNA在人肝癌细胞SMMC-7721中的表达及对裸鼠种植性肿瘤影响的实验研究
Investigation for the Expression of Angiostatin cDNA in Hepatocellular Cancer Cell Line SMMC-7721 and its Effects on Primary Carcinoma of Nude Mouse
论文作者 陶开山
论文导师 窦科峰,论文学位 硕士,论文专业 外科学（肝胆外科）
论文单位 第四军医大学,点击次数 473,论文页数 75页File Size3407k
2001-05-01论文网 http://www.lw23.com/lunwen_2037192/ 血管生成,血管生成抑制因子,血管抑素, 肿瘤发生
Angiogenesis, Anti-angiogenic, angents, Angiostatin, Tumorigenesis
血管生成是指从已存在的血管组织产生新血管的过程，它是哺乳动物个体生长、发育和许多生理及病理过程的重要组成部分。生理状态下，正常成人仅在某些特定时期，如伤口愈合、女性月经期和妊娠期等，血管生成处于活跃状态，经过短暂的增殖后，血管内皮细胞很快回到正常的静止状态。但在一些病理状态下，如慢性炎症、牛皮癣和肿瘤生长转移等，由于正常的血管生成调控机制失控，血管生成变得十分活跃并促使和维持这些疾病状态的延续和发展。 血管生成的启动和调控是诸多因素共同作用的结果，其中血管生成刺激因子和血管生成抑制因子直接作用于血管内皮细胞和（或）周围基质细胞，血管生成是否发生取决于血管生成刺激因子和抑制因子之间的平衡。肿瘤的生长和转移是血管生成依赖性的，在无血供的条件下，原发肿瘤的体积长到1～2mm~3就停止生长，进入休眠期；当刺激因子的力量大于抑制因子时，则血管生成开关开放，出现血管生成，进入肿瘤血管期，肿瘤细胞就可快速增殖，并可发生转移。由于肿瘤的血管依赖性，因此肿瘤血管抑制治疗就成为目前肿瘤治疗的新热点，其具有良好的疗效、不产生耐药 第四军医大学硕士学住论文性、抑瘤谱广泛、无明显毒副作用、对化疗放疗有明显增效作用等优点。随着基因技术的迅速发展，血管治疗与基因治疗的结合在肿瘤治疗中将显示出更广阔的应用前景。 原发性肝癌是我国常见的恶性肿瘤之一，疗效差，以往的实验研究大多针对肿瘤细胞本身，但瘤细胞具有遗传不稳定性、高异型性和高突变率等特点，因此，未能取得满意的疗效。肝癌具有双重血液供应系统，是体内最富于血管的肿瘤之一，抗血管生成治疗将为原发性肝癌的治疗提供一个新的途径。 Angiostatin是近年发现的最有效的血管生成抑制因于之一，它特异性的作用于血管内皮细胞，抑制血管内皮细胞的增殖和迁移，通过抑制血管生成来实现抑制肿瘤的生长和转移，使原发肿瘤和转移瘤的生长呈休眠状态，是肿瘤血管基因治疗中非常有用的目的基因。为了探讨血宫抑素对肝癌细胞的影响及在肝癌治疗中的应用前景，本实验构建了 angiostatin cDNA真核表达载体 pCDNA3．lmAST；利用脂质体基因转染技术将pcDNA3二－mAST导入 SMMC－772，G＊ 筛选抗性克隆；Western blot检测 angiostatin cDNA在 SMMC刁 中的稳定表达；将稳定表达 angiostatinCmA的 SMMCj72瘤细胞种植于裸鼠体内，观察肿瘤细胞致瘤力的变化；通过免疫组化染色，检测瘤体内的微血管密度（MVD）的变化，探讨血管抑素抗肿瘤的机制。 实验结果如下： 1．酶切鉴定和序列分析结果表明己成功地将鼠血管抑素基因克隆入真核表达载体pcDNA3．1卜），即获得了重组质粒pcDNA3．lmAST。 2。利用脂质体法将pcDNA3。lmAST质粒转染到人肝癌细胞SMMC－7721，用G418持续筛选2周，获得了稳定生长的抗G418转染细胞。RI－PCR结果表明已成功地将pcDNA3．lmAST转染入SMMC。7721 5 第四军医大学硕士学位论文细胞。 3．Western七lot 检测结果表明，鼠血管抑素基因在人肝癌细胞SMMC．7721中获得了稳定表达。 4．将H组人肝癌细胞（SMMC－7721、SMMC－7721巾CDNA3．1、SMMCj．1－mAST）用不含G418的培养基常规培养，生长曲线显示：转染细胞和未转染细胞的生长速度无明显差别，表明鼠血管抑素对人肝癌细胞 SMMC－772本身生长无抑制作用。 5．转染血管抑素基因的 SMMC－7721在裸鼠体内的致瘤力明显降低：肿瘤生长缓慢，肿瘤的体积、重量和微血管密度显著低于对照组，说明血管抑素通过抑制肿瘤的血管生成来抗肿瘤生长。 本实验证实：血管抑素angiostatin cDNA在体外对SMMCj 瘤细胞本身的生长无明显影响，但可通过抑制肿瘤血管生成而实现抑制SMMC．7721对裸鼠的致瘤力，为肝癌的治疗提供了一个新的治疗方法。
Angiogenesis, the formation of new capillary blood vessels from pre-existing ones, is a fundamental process required for a variety of physiological and pathological processes. Under physiological conditions, such as wound healing, menstrual cycle in ovaries and endomentrium, angiogenesis is in active situation. Angiognesis occurs over a limited time period and proliferating endothelial cells are rapidly returning to their normal state of quiescence. However, many disease states can arise and are maintained by the persistence of angiogenesis and the loss of normal regulatory mechanisms. These include chronic inflammation, lepra alphos, tumor proliferation and metastasis.The triggering and regulation of angiogenesis is the result of various factors effect, among which angiogenesis stimulators and inhibitors play a direct role on endothelial cells and matrix cells. Angiogenesis is dependent on the balance between angiogenesis stimulators and inhibitors. A tumor is unable to grow above 2mm3 and then towards into the dormancy period without development of a new blood vessel. When angiogenesis stimulators is stronger than angiogenesis inhibitors, the switch of angiogenesis is on and increases angiogenesis, which result in the proliferation and metastasis of tumor. In recent years, a new strategy based on tumor dependence of blood supply, antiangiogenesis for tumor therapy, has been a hot point, which possesses characteristic advantages than normal therapies such as chemotherapy and radiotherapy. With the development of genetic technology, the combination of antiangiogenesis with gene therapy may be is an attractive choice in clinic.Primary hepatocellular carcinoma is one of the most common neoplasmsin China. Most previous experimental researches were against tumor cells which had characters of unable transmissibility, high heterotype and high mutation rate, so satisfied curative effects could not be attained. Hepatocellular carcinoma has double blood supply system, which is one of the tumors with large blood vessels. The treatment of antiangiogenesis will become a new method to the primary hepatocellular carcinoma"s therapy.Angiostatin is one of the most powerful angiogenesis inhibitors which specifically affect endothelial cells, blocking their proliferation and migration, sequentially retarding tumor growth and metastasis and keeping the tumor in the dormancy period. It is a fairly useful target gene in antiangiogenesis gene therapy. In order to investigate the function of angiostatin on hepatocellular carcinoma cells and its possible usage for treatment of hepatocellular carcinoma, we contracted angiostatin cDNA into an eukaryotic expression plasmid pcDNA3.1-mAST and transfected it into SMMC-7721 with lipofectin transfection. After selection by G418, the stable expression on SMMC-7721 of angiostatin cDNA was detected with Western blot and the changes of tumorigenic capacity was also be studied. To observe the changes of microvessel density (MVD) and study the mechanism of retarding tumor of angiostatin with immunohistochemical staining.The results are as follows:1. Enzymatic identification and sequencing results stated that the angiostatin gene clones had been transfected into eukaryotic expression vector of pcDNA3.1(+), i.e. obtained the restructing plasmid.2. pcDNA3.1-mAST plasmid had been transfected into human hepatocellular cancer line SMMC-7721 with lipofectin transfection. The transfected cell clones were selected by G418 after two weeks and the RT-PCR result showed that pcDNA3.1-mAST had been transfected into SMMC-7721 successfully.3. Western-blot detected that the stable expression on SMMC-7721 of angiostatin gene was obtained.4. Cultivated three groups of human hepatocellular carcinoma (SMMC-7721 ,SMMC-7721 /pcDNA3.1 ,SMMC-7721 /pcDNA3.1 -mAST) without G418, the growth curves showed that there were no significant difference between the growth speed of transfected cells and non-transfected cells which stated that angiostatin had no inhibitory effect on the growth of SMMC-7721.
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