【作者】 邓锒梅；

【导师】 张桂英；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 研究背景与目的：胃癌是最常见的恶性肿瘤之一,晚期胃癌患者化疗的有效率约20%-40%。胃癌的多药耐药(multidrug resistance,MDR)是造成化疗失败的极为重要的原因,如何逆转胃癌MDR,提高化疗效果是当前亟需解决的关键问题。近年来虽然胃癌MDR机制被广泛研究,一些新的耐药相关分子如核糖体S13和锌带基因ZNRD1等不断被发现,但仍未能全面阐明其产生机制和找到有效的靶点完全逆转胃癌MDR,提示可能存在不为我们所知的胃癌MDR机制,有必要进一步寻找新的耐药机制及耐药相关基因。在我们的早期研究中,首次发现可溶性耐药相关钙结合蛋白Sorcin在胃癌耐药细胞株SGC 7901/VCR中表达明显增强,用Sorcin寡核苷酸下调其在SGC7901/VCR中的表达,可以部分逆转SGC7901/VCR细胞的多药耐药性,从而证明Sorcin与胃癌多药耐药密切相关。然而,目前国际上关于Sorcin在多药耐药细胞中的具体机制仍不十分清楚。目前研究认为细胞中绝大多数酶的功能和调控过程是以蛋白质复合体或蛋白质网络协同作用实现的,蛋白质的相互作用是目前研究的热点。因此,本课题旨在通过分离、鉴定Sorcin相互作用蛋白质,探讨其在胃癌多药耐药中的作用机制,为逆转胃癌多药耐药提供新靶点。方法：(1)通过脂质体介导将pcDNA3.1-Sorcin-FLAG融合表达载体瞬时转染至HEK293T细胞,RT-PCR和Western blotting分别检测转染细胞中Sorcin mRNA和蛋白表达水平；利用FLAG标签的亲和层析技术提纯Sorcin蛋白复合体；1D-SDS-PAGE技术预分离蛋白复合体；ESI-Q-TOF串联质谱分析鉴定Sorcin相互作用蛋白质；(2)采用免疫共沉淀结合Western blotting在SGC7901/VCR细胞中验证3个可能与Sorcin相关的蛋白质(GSTP1、HSP70、Tubulin);采用RT-PCR和Western blotting分别检测SGC7901/VCR和SGC7901两种细胞中HSP70 mRNA和蛋白表达水平的差异；HSP70siRNA下调HSP70在SGC7901/VCR中的表达,并给予不同浓度的长春新碱干预,采用MTT法分析细胞存活率,Hoechest 33258及流式细胞学检测细胞凋亡；采用RT-PCR检测长春新碱处理后凋亡相关基因bcl-2及bax mRNA的表达,初步探讨HSP70在胃癌多药耐药细胞中可能的作用机制。结果：(1)共鉴定出72个Sorcin相关蛋白质,功能分类包括蛋白质代谢酶类、分子伴侣、生物氧化相关酶类,细胞骨架蛋白、信号转导相关蛋白,酶解相关蛋白和一些功能未知蛋白等；(2)在Sorcin免疫复合物中检测到HSP70、Tubulin、GSTP1蛋白,与质谱分析结果一致；与SGC7901细胞相比,HSP70在胃癌多药耐药细胞SGC7901/VCR中表达明显增强；HSP70 siRNA转染SGC7901/VCR细胞后,RT-PCR和Western blotting结果显示HSP70表达下调；经过长春新碱处理后,与阴性对照组及空白对照组相比,HSP70 siRNA转染组对长春新碱敏感性增加,细胞凋亡明显增加；经长春新碱处理后,SGC7901细胞中bcl-2 mRNA表达较长春新碱处理前下调约42.7%,HSP70 siRNA转染的SGC7901/VCR细胞较阴性siRNA转染的SGC7901/VCR细胞bcl-2 mRNA表达下调约28.6%；而长春新碱处理前后各组细胞bax mRNA表达均无明显变化。结论：(1)成功鉴定出72个Sorcin相互作用蛋白质；(2)免疫共沉淀结合Western blotting的结果证实Sorcin与HSP70、Tubulin、GSTP1存在相互作用；(3)HSP70在SGC7901/VCR细胞株中呈高表达；HSP70 siRNA抑制HSP70的表达可促进长春新碱诱导的bcl-2基因表达的下调,从而上调bax/bcl-2比值,促进长春新碱诱导的细胞凋亡,增强胃癌多药耐药株SGC7901/VCR对长春新碱的敏感性,HSP70有望成为逆转胃癌多药耐药的新靶点。更多还原

【Abstract】 Background and objective:Gastric cancer (GC) is one of the most common cancer worldwide.The effect of chemotherapy in advanced GC is only 20%-40%. Multidrug resistance (MDR) is one of the major problems preventing the chemotherapeutic efficacy of GC. To over-come MDR is a critical task in cancer chemotherapy. Even though lots of mechanisms of MDR have been found recent years, some new moleculars related to MDR have been found, such as ribosome protein S13, ZNRD1 and so on, the mechanisms of MDR have not been fully elucidated yet; treatment of MDR cells with inhibitors of the known molecules did not restore full sensitivity of gastric cancer cells to chemotherapeutic drugs, which indicates that there is some new mechanisms of MDR in gastric cancer. So it is necessary to detect new mechanisms and proteins related to chemoresistance.We had confirmed that the expression of Sorcin, a Calcium-binding protein, in SGC7901/VCR cells was significantly higher than that in its parent SGC7901 cells for the first time. Supression of Sorcin expression by Sorcin antisense oligonuceotides could partially reverse MDR. However, the exact mechanisms of Sorcin in MDR is still unknown. With the development of Proteomics, more and more research indicated that cellular and molecular functions and vital movements are almost carried out through complex or even network referred a great deal of proteins. Protein-protein interactions have been researched extensively recent years. Based on our previous studies, this study aim to identify and validate proteins interacting with Sorcin, elucidate the mechanisms of Sorcin encoded gene in MDR of gastric cancer, and to search some new targets related to chemoresistance meanwhile.Methods:(1) Firstly, pcDNA3.1-Sorcin-FLAG plasmid was transfected into HEK293T cells mediated by Lipofectamine. The mRNA and protein expression levels of Sorcin were detected by RT-PCR and Western blotting. Then, the proteins extracted from these cells were purified by anti-FLAG M2 affinity gel, prefractionated by one dimensional SDS-polyacrylamide gel electrophoresis (1D-SDS-PAGE). The fractionated proteins were trypsined and then analyzed by ESI-Q-TOF/MS; (2) Coimmunoprecipitation coupled with Western blotting were done to confirm the partial Sorcin-interacting proteins, such as HSP70、GSTP1、Tubulin, in the SGC7901/VCR cells; The mRNA and protein expression levels of HSP70 in SGC7901/VCR and SGC7901 cells were detected by RT-PCR and Western blotting; Suppressing HSP70 expression in SGC7901/VCR by HSP70 small interfering RNA(siRNA), then deal with VCR of different concentration, Proliferation of cells was detected by MTT assay; cell apoptosis was detected by Hoechest 33258 staining and flow cytometry; The mRNA expression levels of bcl-2 and bax were detected by RT-PCR.Results:(1) A total of 72 Sorcin-interacting proteins were identified, which could be classified into seven categories based on their functions: metabolic enzymes, molecular chaperone, enzyme related to biological oxidation, cytoskeleton organization, proteins relative to signal transduction, proteins related to enzymolysis and proteins of unknown functions; (2) HSP70、Tubulin、GSTP1 were detected in the immune complex of Sorcin, which were consistent with the results of mass spectrum analysis; The mRNA and protein levels of HSP70 were up-regulated in SGC7901/VCR cells compared with SGC7901; The mRNA and protein levels of HSP70 were obviously down-regulated in SGC7901/VCR cells indicated that HSP70-suppressing SGC7901/VCR cell line was established; Compared with control groups, after treated by Vincristine (VCR), cell apoptosis of HSP70 siRNA group was increased detected by MTT assay, Hoechest 33258 staining and flow cytometry; In SGC7901 and HSP70-suppressing SGC7901/VCR cell line groups, bcl-2 mRNA was down-regulated by 42.7% and 28.6% respectively, while the expression of bax mRNA did not change significantly after VCR treatment detected by RT-PCR.Conclusions:(1) Successfully separate and identified 72 proteins interacting with Sorcin; (2) The result of coimmunoprecipitation coupled with Western blotting confirmed that HSP70, GSTP1 and Tubulin were interacted with Sorcin, which were consistent with MS data; (3) HSP70 was up-regulated in SGC7901/VCR cells compared with SGC7901; The ratio of the expression of bax/Bcl-2 was up-regulated accordingly with suppression of HSP70 in SGC7901/VCR by HSP70 siRNA, which increased the VCR-induced apoptosis and the sensibility to anti-cancer drug of SGC7901/VCR cells. HSP70 is hopefully to be a new target protein for reversing MDR.更多还原