【作者】 郅克谦；

【导师】 温玉明；

【作者基本信息】 四川大学 ， 口腔临床医学， 2004， 博士

【摘要】 Steeg首先从鼠黑色素瘤细胞株中分离出nm23基因,认为它与肿瘤转移呈负相关,随后的实验也证实nm23基因在抑制肿瘤转移中的作用。最近研究表明,nm23-H1与抗肿瘤药的敏感性有一定关系,在乳腺癌研究中发现,nm23-H1与顺铂化疗敏感性密切相关,本研究旨在对nm23-H1提高顺铂化疗敏感性的可能机制作一初步探讨。 首先采用基因转化技术,制备高纯度的nm23-H1真核表达质粒并对其进行鉴定。通过阳离子脂质体介导,将nm23-H1导入Tca8113细胞,建立稳定表达细胞株,采用免疫组化、western-blot和流式细胞仪技术对表达产物进行鉴定。我们成功制备大量nm23-H1真核表达质粒,采用限制性内切酶酶切证实nm23-H1插入片断长度为986bp。转染后稳定表达的Tca8113细胞,nm23-H1蛋白表达明显增高。 然后通过体外实验观察转染前后Tca8113细胞对顺铂的化疗敏感性,分别用MTT法、检测罗丹明的荧光强度、流式细胞仪和等离子质谱仪检测杀伤率、线粒体膜电位、细胞凋亡和细胞内铂离子浓度的变化。体外实验中发现,顺铂对转染后Tca8113细胞的杀伤率,细胞凋亡比例和细胞内铂离子浓度均有明显提高,这种作用能被Na(+)/K(+)-ATP酶的抑制剂哇巴因明显抑制;顺铂可明显降低转染后Tca8113细胞的线粒体膜电位。更多还原

【Abstract】 Since the initial description of the nm23 gene as an antimetastatic factor whose expression is correlated inversely with tumor metastatice potential in murine melanoma cell lines. Numerous studies have supported its suppressive effects on tumor metastatasis. Recently, the role of nm23-Hl in sensitivity to anticancer agents has attracted a great deal of attention. In addition to the antimetastastic property, nm23-Hl has been showen to be associated with sensitivity to cisplatin in breast carcinoma.Firstly, to transfect nm23-Hl into Tca8113 cell line using Lepfect liposomal transfection reagent and get stable Tca8113 cell line. Detect the protein expression of nm23-Hl in stable cell line using immunohistochemistry, western-blot and flow cytometer. we get pCMV-Bam- neo-nm23-Hl successfully and establish the stable expression of nm23-Hl. It is showed that 986bp of nm23-Hl is inserted into pCMV-neo-Bam vector by electrophoresis on a 1% agarose gel and stained with ethidium bromide. nm23-Hl expression was increased in the stable expression cell line by immunohistochemistry, western-blot and flow cytometer.Secondly, Using MTT, flow cytometer and VG PQ Excell, we detectthe changes of cell mortality rate, mitochondrial membrane potential, apoptosis and intracelleular platinum (Pt) by nm23-H13. we found that, in vitro, the cell mortality rate, apoptosis and intracellular platinum are increased significantly in stable expression cell line of nm23-Hl, comparing with Tca8113 cell line of non-transfection; this effect can be inhibited by oubain which is an inhibitor of Na(+)/K(+)-ATPase. mitochondrial membrane potential is decreased significantly in stable expression cell line of nm23-Hl.Thirdly, to establishing nude mice xenotransplanted model of Tca8113 cell, we detect the changes of tumor volume and weight by nm23-Hl and cisplatin. In vivo, the therapy of nm23-Hl plasmid and cisplatin can inhibit significantly the growth of tumor which include the volume and weight in nude mice xenotransplanted model, comparing with non-therapy group and therapy of cisplatin group.we establish the stable expression cell line of nm23-Hl. nm23-Hl can increase the chemosensitivity of cisplatin significantly; the mechanism is correlated with the activity of Na(+)/K(+)-ATPase. The therapy of nm23-Hl plasmid and cisplatin can inhibit the growth of tumor in nude mice. All thesis results are important for the combination of gene and chemotherapy to use in clinics.更多还原