【作者】 陈孝平；

【导师】 陈正望；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 硫氧还蛋白(Thioredoxin,Trx)是一类分布广泛的小分子多功能蛋白,它们在进化上相当保守,都具有一个相同的有氧化还原活性的Cys-Gly-Pro-Cys的氨基酸序列。哺乳动物的Trx家族包括Trx-1和Trx-2。它们在其保守的活化区域内含有具备氧化还原活性的二硫巯基和二硫醇,因此硫氧还蛋白在细胞内行使着各种不同的功能。硫氧还蛋白系统是由硫氧还蛋白、硫氧还蛋白还原酶、NADPH三部分组成的。硫氧还蛋白系统可催化还原氢过氧化物、有机过氧化物以及一些被氧化的蛋白,是一个在自然界中普遍存在的系统。癌组织中Trx-1的过量表达会促进细胞的增殖和抑制细胞的凋亡,其机制是为DNA的合成提供平衡的氧化还原环境和活化转录因子等。关于它抑制细胞凋亡的机制仍在不断的研究中。由于Trx-1对肿瘤细胞的促进生长和抑制凋亡作用,它正成为治疗癌症和阻止癌症发生的分子靶点。某些癌症病人的血清中Trx-1的浓度是较高的,但乳腺癌病人的血清和正常人的血清中的Trx-1含量并不清楚。Trx-1对细胞周期的作用也未见报道。Trx-1对细胞凋亡中的作用有一不致的报道。为此我们进行了以下的研究:1)利用RT-PCR技术从MCF-7细胞中扩增获得了Trx-1基因,构建了该基因的原核表达载体pET32a-Trx,在E.coli中获得了高效表达的融合蛋白,并利用亲和层析的方法成功地对其进行了纯化,纯化的蛋白大小与预期一致。2)利用纯化的Trx-1蛋白对新西兰大白兔进行免疫,制备出相应的多克隆抗体,经间接ELISA法检测其效价为1:10000,并对Trx-1具有特异性,可以用于ELISA或western blot实验。3)利用间接ELISA法对正常人和乳腺癌病人的血清中的Trx-1的浓度进行测定,发现乳腺癌病人的外周血中Trx-1的含量低于正常人。4)构建了真核表达载体pcDNA-Trx,并将其转染到乳腺癌细胞系MCF-7中。5)研究了Trx-1对细胞周期的影响。用维生素C刺激乳腺癌细胞MCF-7,或者用pcDNA-Trx转染MCF-7使细胞中的Trx-1表达升高,另外在细胞培养基中加入不同浓度的Trx-1,再用流式细胞仪检测细胞中进入S期细胞的比例。结果发现维生素C的刺激使细胞体内Trx-1表达上升,更多的细胞进入到S期。转染pcDNA-Trx的细胞Trx-1的表达上升,进入S期的细胞比例明显增加。将Trx-1蛋白加入到培养基中后,进入S期的细胞比例明显地随Trx-1的浓度的提高而升高。这些结果都说明无论在细胞体外还是体内,Trx-1水平的上升均会促使细胞进入S期。6)细胞中的总Trx-1水平升高而核内Trx-1水平无变化时,不能影响MCF-7细胞对顺铂的敏感性。但核内Trx-1水平增加而总TRx-1水平不变时MCF-7明显的表现出对顺铂的抗性增加了。MCF-7抗性株中的核内Trx-1水平高于敏感株,而且MCF-7细胞用顺铂处理时,顺铂敏感细胞株中核内Trx-1会下降,而顺铂抗性株中核内Trx-1的水平没有明显的变化。同时研究发现,抗癌基因p53的mRNA水平明显和核内Trx-1水平正相关。这些结果说明,核内Trx-1是MCF-7抵抗顺铂诱导的凋亡中所必需的,而可能的机制是核内Trx-1促使抗癌基因p53的表达。从以上的实验可以得出如下结论:乳腺癌病人的血清中Trx-1的浓度和正常人相比略有下降但并不明显;Trx-1无论在MCF-7细胞的体外还是体内均可以细胞滞留于S期;用不同的方法诱导MCF-7细胞凋亡时,Trx-1的表达量变化并没有固定的趋势,而核内Trx-1则均出现增强的表达;核内Trx-1对MCF-7抵抗顺铂诱导的凋亡是必需的。更多还原

【Abstract】 The thioredoxins are ubiquitous proteins containing conserved Cys-Gly-Pro-Cys redox catalytic site. Mammalian thioredoxin family members include thioredoxin-1 (Trx-1), and mitochondrial thioredoxin-2 (Trx-2). Thioredoxin is reduced by NADPH and thioredoxin reductase and, in turn reduces oxidized cysteine groups on proteins. When thioredoxin levels are elevated there is increased cell growth and resistance to the normal mechanism of programmed cell death. An increase in thioredoxin levels seen in many human primary cancers compared to normal tissue appears to contribute to increased cancer cell growth and resistance to chemotherapy. Mechanisms by which thioredoxin increases cell growth include an increased supply of reducing equivalents for DNA synthesis, activation of transcription factors that regulate cell growth, and an increase in the sensitivity of cells to other cytokines and growth factors. The mechanisms for the inhibition of apoptosis by thioredoxin are just now being elucidated. Because of its role in stimulating cancer cell growth and as an inhibitor of apoptosis, thioredoxin offers a target for the development of drugs to treat and prevent cancer.Our studies are as following:1) Trx-1 gene was obtained by RT-PCR. Prokaryotic fusion gene expression vector, pET32a-Trx, was constructed. A high level of expression of fusion protein in E.coli was detected after IPTG induction and purified proteins were obtained by affinity chromatography.2) The purified Trx-1 proteins were mixed with Freund’s complete or incomplete adjuvant as antigen to immune rabbits. ELISA assay revealed that the titer of the prepared antiserum against Trx-1 protein was as high as 1:10000. IgG was purified with saturated ammonium sulfate. These polyclonal antibodies are special to Trx-1 and can be used to detect Trx-1. 3) The contrations of Trx-1 in the serum of breast cancer patients were detected with ELISA, and we found that the contration of Trx-1 is a little lower than control.4) Trx-1 gene was cloned into eukaryotic expression vector, pcDNA3.1+ and transferred into breast cancer cells, MCF-7.5) Trx-1 overexpressed in MCF-7 cells after dealed with Vc or transferred with Trx-1 cDNA. At the same time, more cells were in S phase. Meanwhile, higher Trx-1 in vitro can also bring more cells into S phage. So Trx-1 contributes to prolonging S phase of MCF-7 cells, both in vivo and in vitro.6) We explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced by cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53.Based our data, we can include that i) the contrations of Trx-1 in the serum of breast cancer patients is a little lower than control, ii) Trx-1 contributes to prolonging S phase of MCF-7 cells, both in vivo and in vitro, and iii) the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53.更多还原