兰州大学机构库 >学院待认领
颗粒溶素与穿孔素的表达及其对肺癌细胞影响的研究
Alternative TitleExpression of Human Granulysin and Perforin and their Effects on Lung neoplasms
王晚霞
Thesis Advisor居军
2009-05-13
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword颗粒溶素 穿孔素 表达 人肺癌A549细胞 细胞凋亡
Abstract颗粒溶素(Granulysin,GNLY)和穿孔素(Perforin,PFP)是存在于NK细胞和CTL细胞中的细胞毒蛋白,广泛参与机体抗病原微生物感染和抗肿瘤免疫,有望成为理想的抗肿瘤、抗结核药物。颗粒溶素和穿孔素的表达与癌症、感染、器官移植等多种疾病都有密切关系,深入进行颗粒溶素和穿孔素的研究对许多重大疾病的诊断及治疗都有非常重要的意义。本实验运用基因工程的方法体外获取颗粒溶素和穿孔素基因片段,利用原核表达体系合成不同分子量的颗粒溶素融合蛋白,为颗粒溶素的后续研究创造条件;构建颗粒溶素和穿孔素真核共表达载体并转染人肺腺癌A549细胞,体外观察颗粒溶素和穿孔素在肺癌细胞中的表达情况,为进一步探索颗粒溶素和穿孔素在肿瘤治疗中的应用提供依据。 主要研究方法及结果: 1. 体外分离并培养外周血单个核细胞,抽提总RNA,RT-PCR扩增人颗粒溶素和穿孔素基因片段,将其插入pMD18-T构建克隆载体,测序结果表明人颗粒溶素和穿孔素基因片段与Genbank中序列一致,构建了pMD-GNLY和pMD-PFP。 2. 构建原核表达质粒pET-GNLY9K和pET-GNLY15K,将重组质粒转入大肠杆菌Rosetta(DE3),经IPTG诱导在原核表达系统中高效表达了相应分子质量约为31和37kD的融合蛋白,Western-blotting结果表明重组颗粒溶素融合蛋白能与羊抗人颗粒溶素抗体发生良好的抗原抗体反应。 3. 以FMDV 2A代替IRES,构建真核共表达载体pGNLY-2A-PFP并转染人肺癌A549细胞, RT-PCR和间接免疫荧光检测目的蛋白表达,流式细胞Annexin V/PI检测转染后细胞凋亡情况。重组质粒转染A549细胞后检测出了目的基因mRNA及蛋白的表达,pGNLY-2A-PFP转染后细胞死亡率高于其他对照组(P<0.05),死亡细胞以凋亡为主。 结论及研究意义: 1. 成功获取了颗粒溶素和穿孔素基因片段,为颗粒溶素和穿孔素的后续研究创造了条件。 2. 利用原核表达体系成功地表达了不同分子量的颗粒溶素融合蛋白,为颗粒溶素的后续研究奠定了基础。 3. 人颗粒溶素和穿孔素基因可以在人肺癌A549细胞中表达,二者共表达能够促进细胞凋亡,这将有助于颗粒溶素和穿孔素在肿瘤治疗中应用的后续研究。
Other AbstractGranulysin and perforin are cytolytic protein presented in the granules of human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, which exhibit cytolytic activity against a broad range of microbes and tumors.Granulysin and perforin are expected to be ideal drugs in the use of anti-tumor and anti-tuberculosis.Granulysin and perforin are implicated in a myriad of diseases including infection, cancer, transplantation. Researches of granulysin and perforin will be very helpful to diagnosis and treatment of many diseases. In this study, granulysin and perforin gene segments were cloned from cultured PBMC. Different molecular weight granulysin fusion protein were recombinanted using prokaryotic expression system. Co-expression eukaryotic vector of granulysin and perforin was constructed and transfected into A549 cells. Effects which granulysin and perforin co-operated in A549 cells were observed after transfection. The methods and results were as follow. 1. Total RNA was extracted from cultured PBMC. Granulysin and perforin gene segements were obtained with specific primers by RT-PCR and then respectively inserted into pMD18-T plasmid for sequencing. Sequencing results showed that the cloned granulysin and perforin gene segements were in the same with the sequences in Genbank.Vectors pMD-GNLY and pMD-PFP were successfully constructed. 2. To construct prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K and transfer them into E. coli Rosetta (DE3).Fusion protein were expressed under the induction of IPTG.SDS-PAGE and Western-blotting showed that the corresponding molecular weight of 31kD and 37kD granulysin fusion protein,which had satisfactory antigen-antibody reaction with anti-granulysin antibody, were highly expressed in E. coli after induction. 3. To construct co-expression eukaryotic vector pGNLY-2A-PFP and transfect them into A549 cells. The expression of target genes was detected by RT-PCR and indirect immunofluorescence. Target protein expression was detected after transfection.FCM results showed that the death rate of pGNLY-2A-PFP transfected cells was markedly increased (P<0.05). Conclusion 1. Granulysin and perforin gene segements were successfully obtained in vitro, which would be helpful for the further studies of granulysin and perforin. 2. Different molecular weight granulysin were successfully expressed using prokaryotic expression system, which would be helpful for the further study of granulysin. 3. Granulysin and perfo...
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223155
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
王晚霞. 颗粒溶素与穿孔素的表达及其对肺癌细胞影响的研究[D]. 兰州. 兰州大学,2009.
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