【作者】 夏嘉志；

【导师】 陈汉春； 张殿政；

【作者基本信息】 中南大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 GSTpi是谷胱甘肽S-转移酶(GSTs,又称谷胱甘肽转硫酶)家族的重要成员,由一个活跃的多态性基因编码。GSTpi蛋白质的在体功能是清除毒物,避免基因组的损伤,同时可与许多耐药相关基因共表达而成为肿瘤细胞对化疗药物或生物治疗剂耐受的重要原因之一。慢性髓细胞性白血病(chronic myelognous leukemia,CML)是一种起源于骨髓多能造血干细胞的恶性增殖性疾病;干扰素-α(interferon-α,INF-α)是治疗CML的有效制剂之一,但部分CML患者呈现对INF-α治疗效应的耐受。本课题以呈现对INF-α治疗效应不同敏感性的3种CML细胞株:KT-1/A3、KT-1/A3R和K562细胞为研究对象,以抗GSTpi抗体经细胞SP(streptavidin-perosidase)免疫组化法和Western blotting分析这些CML细胞经INF-α处理前、后的GSTpi蛋白质表达水平的差异,结果表明处理前与处理后的KT-1/A3R细胞的GSTpi蛋白质表达水平显著高于KT-1/A3细胞,而K562细胞则完全不表达GSTpi蛋白质;细胞免疫荧光染色后经激光共聚焦显微镜观察发现,KT-1/A3R和KT-1/A3细胞中GSTpi蛋白质主要分布于细胞质,而K562细胞中未见GSTpi蛋白质表达。在上述发现的基础上,将构建的pcDNA3.1-GSTpi重组表达载体分别转染低水平表达和/或不表达GSTpi蛋白质的KT-1/A3和K562细胞,细胞免疫荧光染色后经激光共聚焦显微镜观察证实了经INF-α处理前后其重组载体的细胞表达差异和亚细胞定位;采用流式细胞术检测细胞凋亡率和台盼蓝拒染实验分析细胞成活率的结果表明,GSTpi重组载体的表达明显降低KT-1/A3细胞的INF-α反应性,但并不改变K562细胞的INF-α耐药特征;对所有实验组CML细胞的caspase-3酶活性检测发现,未转染pcDNA3.1-GSTpi重组表达载体的KT-1/A3细胞经INF-α处理后其caspase-3酶活性明显增高,而转染组KT-1/A3细胞经INF-α处理后其caspase-3酶活性没有明显改变,转染组K562细胞经INF-α处理后其caspase-3酶活性略有升高。更多还原

【Abstract】 GSTpi is an important member of GSTs,which is a family of dimeric enzymes encoded by the active and polymorphic genes.The functions in vivo of GSTpi protein are thought to be involved in the elimination of hazardous agents and prevent the genes from injury.The co-expression of GSTpi with a number of drug-resistance associated genes might be an important reason of the tumors resistance to chemotherapeutics and/or biotherapy reagents.Chronic myelognous leukemia(CML) is a malignant chronic myeloproliferative disorder(MPD) of the hematopoietic stem cells.Interferon-a(INF-a) is one of the effective drugs to treat CML,but some of the CML patients exhibit the drug resistance to IFN-a clinically.Three CML cell lines including KT-1/A3,KT-1/A3R and K562 with different sensitivity to IFN-a effect were used in the present study.By using the technologies of immunocytochemistry with streptavidin peroxidase and Western blotting with anti-GSTpi antibody,the different expression levels of GSTpi in KT-1/A3,KT-1/A3R and K562 cells were comparatively detected before and after IFN-a induction.The results showed that the expression level of GSTpi protein in KT-1/A3R cells was significantly higher than that in KT-1/A3 cells in the conditions of both before and after IFN-a treatment.However,there was no GSTpi protein expression in K562 cells with or without IFN-a induction.The laser immunofluorescence-scanning confocal microscopy showed that GSTpi proteins were mainly localized in cytoplasm of KT-1/A3R and KT-1/A3 cells,but also no GSTpi expression was seen in K562 cells.Based on the results mentioned above,pcDNA3.1-GSTpi recombinant expression vector was constructed and transfected into KT-1/A3 cells with little GSTpi expression and K562 cells without GSTpi expression.The expression level difference and the subcellular localization of GSTpi protein in the pcDNA3.1-GSTpi recombinant expression vector transfected cells before and after IFN-a treatment were analyzed.The results of Flow Cytometry(FCM) for the apoptosis ratio detection and Typan Blue dye exclusion test for cell viability analysis showed that the IFN-a response of KT-1/A3 cells was significantly decreased by pcDNA3.1-GSTpi recombinant expression vector transfection.However,the resistant features of K562 cells to IFN-a were not affected by pcDNA3.1-GSTpi recombinant expression vector transfection.The results of caspase-3 activity detection showed that the caspase-3 enzymatic activity of KT-1/A3 cells without pcDNA3.1-GSTpi recombinant expression vector transfection and then induced by INF-a was significantly increased,while the caspase-3 enzymatic activity of KT-1/A3 cells transfected with pcDNA3.1-GSTpi recombinant expression vector and then induced by INF-a was almost not changed, meanwhile,the caspase-3 enzymatic activity of K562 cells transfected with pcDNA3.1-GSTpi recombinant expression vector and then induced by INF-a was slightly increased.更多还原