【作者】 陈俊霞；

【导师】 崔秀云；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 核糖核酸酶抑制因子(Ribonuclease Inhibitor, RI)是一种胞浆内酸性糖蛋白,分子量约为50KDa。RI是核糖核酸酶A(Ribonuclease A, RNase A)的抑制剂可以有效地抑制其对RNA的水解作用。RNaseA与血管生成因子(Angiogenin,Ang)的氨基酸有着高度保守的同源顺序。Ang是 RNaseA超家族的一员,RI通过与RNaseA(ki=4.0×10-14M)和Ang的紧密结合而抑制他们的活性。Ang是由肿瘤细胞和正常细胞分泌的分子量为14.4kDa的蛋白质,在体内和体外均具有强烈的诱发血管生成的活性。根据cDNA序列分析,Ang与RNase A有35%的同源性;但Ang与RI有更大的亲和力(ki=7.1×10-16M)。X射线分析表明,Ang与RNase A有着极其相似的立体结构,二者都可以与人胎盘RI紧密结合。由于RI可以与Ang形成紧密的非共价结合,从而可以抑制其促血管生成的活性。Ang在结肠癌细胞、肝癌细胞等均高于正常水平,在肿瘤的生长和转移过程中是一种促进新生血管的生成物质。此外RI还可能通过抑制碱性成纤维细胞生长因子(b-FGF)的表达等途径抑制血管生成。抗血管生成将是一种对抑制肿瘤的生长和转移的有效方法。实验证实RI能有效地阻断血管生成因子诱导的血管形成。RI由含有许多亮氨酸的重复顺序的多肽组成,含有这样重复顺序的一百多种蛋白质显示了广泛的功能,包括细胞周期调节、DNA修复、对细胞外基质相互作用以及抑制酶活性等。RI被认为是胚胎发育、创伤愈合及肿瘤发生中新血管形成的一种调节因子。RI定位于染色体的11p15.5,与ras基因邻近,在肿瘤病人中经常存在染色体的11p15.5部位的变异。RI可能与细胞的生长和分化有关。因此,RI 可能还具有尚未知的生物学作用。动物试验显示RI能抑制动物体内某些移植实体瘤的生长,给予荷瘤小鼠RI后可以抑制乳腺癌实体瘤Ca761,肉瘤S180,肝癌实体瘤H22,C6大鼠神经胶质瘤等的生长。我们以前的实验也显示RI在肿瘤病人的血中活性下降,并且RI mRNA在乳腺癌中比其在正常组织中显著降低。。血管生成及新血管的形成,在实<WP=6>体瘤的生长中起重要作用,没有新血管的形成,实体瘤长到1-2mm3 时,肿瘤将会自行退化。这些结果提示RI可能起着候选抑癌基因的作用而可能与某些肿瘤的发生和转移有关。然而,目前除了RI的抗癌作用与其抑制血管生成的活性外,是否还具有其他抑癌功能以及RI抗癌的确切的分子机制还不清楚。 本研究的目的在于进一步了解RI的潜在功能以及探讨RI与肿瘤生长、浸润、转移的关系, 以期阐明RI抗癌的分子机理。本实验研究甲基化抑制剂对肿瘤细胞中核糖核酸酶抑制因子表达的影响;将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317转染到B16小鼠黑色瘤细胞中,观察转染的RI基因在体内外对细胞生长、凋亡、以及浸润、转移特性的影响;以浸润和非浸润的膀胱移行细胞癌细胞系为模型,观察了RI的表达变化及其对肿瘤细胞浸润、转移能力的影响。为此做了以下的一些工作:1.用甲基化抑制剂5-aza-2’-deoxycytidine(5-Aza-CdR)作用人乳腺癌细胞系MCF-7,人胃癌细胞系BGC-823,人前列腺癌细胞系DU-145和人结肠癌细胞系HT-29。通过RT-PCR,蛋白免疫印迹法(western blotting),免疫荧光(immunofluorescence)和免疫细胞化学(immunocytochemistry)技术分析RI基因的表达。结果显示5-Aza-CdR能显著提高RI基因在人乳腺癌细胞系MCF-7,人胃癌细胞系BGC-823,人的前列腺癌细胞系DU-145的表达,但对人结肠癌细胞系HT-29没有明显的影响。结果表明,RI基因可能与胃癌,前列腺癌,和乳腺癌的发生有关。在人类肿瘤中,许多抑癌基因可通过启动子区域异常的甲基化使表达丧失,用甲基化抑制剂5-Aza-CdR处理能恢复基因的表达, RI可能在某些组织中具有抑癌基因的功能。2.将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317转染到B16小鼠黑色素瘤细胞中,用转染空载体和未转染的B16细胞作为对照。通过PCR, RT-PCR, 蛋白免疫印迹, 免疫荧光分析鉴定,获得稳定表达人核糖核酸酶抑制因子的细胞株。结果显示,转染的RI基因在体外能显著地抑制细胞增殖, 调节细胞周期和诱导凋亡以及引起细胞形态的改变,将三种B16细胞接种到C57BL/6小鼠中, 结果, 转染RI基因的实验组显著地抑制的了肿瘤的生长, 与两个对照组相比,荷瘤小鼠有更长的存活时间, 较长的肿瘤生长潜伏期, 更低的肿瘤微血管密度。肿瘤的形成率为63.3%, 肿瘤抑制率分别为90.3% 和 90.6%。结果提示,RI的表达可能与黑色素瘤的生长有关,RI有显著的抗肿瘤效应并部分可能与其抑制血管生成以及降低细胞增殖和诱导细胞凋亡有关。3.将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317转染到B16小鼠黑色瘤细胞中, 用转染空载体和未转染的B16细胞作为对照。通过PCR, RT-PCR, 蛋白免疫印迹, 免疫荧光分析鉴定,获得稳定表达人核糖核酸酶抑制因<WP=7>子的另一细胞株。, 实验表明, 转染的RI基因在体外能显著地抑制细胞增殖和细胞迁移,增加了细胞的粘附以及改善细胞的恶性形态,将三种B16细胞静脉注射到C57BL/6小鼠中, 结果显示,转染RI基因的实验组显著地抑制的了肿瘤的转移, 与两个对照组相比,荷瘤小鼠有更长的存活时间, 更少的转移结节, 更低的肿瘤微血管密度和肺重量。提示RI的表达可能与黑色素瘤的转移更多还原

【Abstract】 Ribonuclease inhibitor (RI) is an acidic cytoplasmic glycoprotein with a 50 kDa estimated molecular weight. RI can inhibit the activity of ribonuclease A (RNase A) by tight combination with it (ki=4.0×10-14M). The amino acid sequence of angiogenin (Ang) is high conservative compared with RNase A. Ang is a member of RNase superfamily. Ang is produced and secreted by tumor cells and normal cells and its molecular mass is 14.4 kDa. It has a robust capacity to induce blood vessel formation in vivo and in vitro. On the basis of analysis of cDNA sequence, Ang is 35% identical with human pancreatic RNase. Ang forms more restricted complex (Ki=7.1×10-16) with RI than that formed by RI and RNase A. X-ray analysis showed that Ang had a very similia space structure. They can both form tight complex with RI. So it can inhibit the activity of Ang. Ang expresses highly and promotes angiogenesis in colon, liver tumor,etc. RI may inhibit angiogenesis through blocking on b-FGF pass way too. So anti-angiogenesis will be an efficient method in the inhibition of the growth and metastasis of cancer. The experiment demonstrated that RI could effectively block the angiogenin induced angiogenesis. RI is constructed almost entirely of leucine-rich repeats. Such repeats have been identified in more than 100 proteins that exhibit a wide range functions, including cell-cycle regulation, DNA repair, extracellular matrix interaction, and enzyme inhibition. RI was recognized as a regulating element on formation of new blood vessels in embryo developing, wound healing, and tumor occurring. RI located on the chromosome 11p15.5, nearly ras gene, 11p15.5 often has change in cancer patients. The study indicated that more than one locus at 11p15 could be involved in tumorigenesis, therefore, RI may has relation with the cell growth and differentiation,which might involve in unclear biological effects. Previous study indicated that RI extracted from human placenta could inhibit the <WP=9>some kinds of transplant tumor growth in the animal body, including Ca761 breast carcinoma, S-180 sarcoma, SHG44 and C6 glioma. Our previous experiments also showed that RI activity reduced in the blood of tumor patients and RI mRNA was significantly lower in the human breast than that in the normal tissue. Angiogenesis, the formation of new blood vessel, plays an important role in the growth of solid tumors. If solid tumor grows to 1-2 mm without new blood vessel formation, the tumor will degenerate. These results suggest RI might be correlated with some tumorigenesis and metastasis as one of candidate tumor suppressor genes (TSGs). However, nowadays whether RI has other tumor-suppressive effects remains unclear, its exact molecular mechanism of antitumor has not been totally ascertained. In order to understand further the function of RI and investigate the relationship between RI and tumor growth, invasion, metastasis for its exact molecular mechanism of antitumor. The study detected the effects on DNA methylation inhibitor on the expression of ribonuclease inhibitor in the cancer cell lines; established a transfection of human RI gene cDNA into murine B16 cells by the retroviral packaging cell line PA317 carrying the pLNCX-RI in vitro to observe the effects of transfected RI on cell proliferation, apoptosis in vitro and tumor growth, metastasis in vivo; analyzed the expression of RI gene using human bladder transitional cell carcinoma with different invasive potential as model to explore effects of RI on invasion and metastasis. The main work is as followings:1. Human breast carcinoma cell line MCF-7, human gastric carcinoma cell line BGC-823, human prostate carcinoma cell line DU-145 and human colon carcinoma cell line HT-29 were treated by methylation inhibitor, 5-aza-2’-deoxycytidine(5-Aza-CdR). The expression of RI gene was analyzed by RT-PCR, Western blotting, immunofluorescence and immunocytochemistry. The data showed that 5-Aza-CdR can significantly elevate the expression of RI at both mRNA and protein leves in MCF-7, BGC-823, DU-145 cell lines, but n更多还原