【作者】 曾辉；

【导师】 伍钢；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2011， 博士

【摘要】 第一部分SMAC RNAi载体制备与靶点筛选及慢病毒包装目的：构建针对目的基因SMAC的RNAi质粒载体,并在筛选出有效干扰靶点后进行慢病毒包装及细胞感染稳定株的制备。材料和方法：1、针对目的基因靶基因序列,利用公用网站按照RNA干扰序列设计原则,设计多个RNA干扰靶点序列,根据设计软件进行评估测定,选择最佳的动力学参数靶点进入后续实验流程；由吉凯基因公司合成含干扰序列的双链]DNA oligo,其两端含酶切位点粘端,直接连入酶切后的RNA干扰载体上。将连接好后的产物转入制备好的细菌感受态细胞,对长出的克隆进行PCR鉴定,在进行测序对比后,比对正确的克隆即为构建成功的目的质粒。2、为了确定复合载体是否转染到人肺癌细胞中,在荧光显微镜下观察感染Smac基因-RNAi-慢病毒载体后72小时A549细胞中报告基因GFP的表达。RT-PCR和Western印迹分析转染Smac基因-RNAi-慢病毒载体前后,Smac在mRNA和蛋白水平的沉默效果。3、将编码慢病毒颗粒的重组病毒颗粒及其两种辅助包装载体质粒分别进行高纯度无内毒素抽提,按lipofectamine2000试剂盒使用说明进行共转染T293细胞,转染后按说明书进行培养,之后收集、浓缩富含慢病毒颗粒的细胞上清液,在T293细胞中测定并标定病毒滴度。结果：1、含有vshRNA片段的psc-1克隆为我们所成功构建的含有siRNA靶序列载体。2、A549细胞在转染了上述慢病毒72小时后,超过80%的A549细胞表达了marker基因GFP,表明该转染系统的高效和稳定、SMAC基因在敲除后在mRNA(抑制效率约为70%,P值<0.01)和蛋白水平都较敲除前有显著意义于,表明RNAi有效靶点筛选成功。3、工具细胞与病毒载体进成功行包装并制备细胞感染的稳定株。结论：SMAC RNAi干扰质粒载体成功构建并筛选出RNAi有效靶点,对其进行慢病毒包装与滴度测定后制备了细胞感染的稳定株,这为我们后续对SMAC基因进行功能研究奠定了基础。第二部分目的基因过表达慢病毒载体构建目的：针对目的基因SMAC构建过表达的慢病毒载体,为后续基因功能研究准备载体工具。材料和方法：1、从含目的基因的质粒或cDNA文库中,用PCR钓取目的基因。由吉凯基因公司进行目的基因的引物合成与鉴定,对目的基因进行PCR扩增、酶切,表达载体酶切、纯化,定向克隆连接重组后与感受态细胞共转染,PCR鉴定转化子,送invitrogen公司进行阳性克隆测序和比对分析,比对正确的克隆即为构建成功目的质粒。2、在目的细胞A549中,通过转染含有Smac全长编码序列的pGC-FU载体来实现Smac高表达,转染pGC-FU载体的siRNA在C-末端含有marker基因GFP,通过western blot观察转染前后Smac蛋白水平的变化对过表达慢病毒载体的效果进行验证。3、按lipofectamine2000使用说明进行共转染T293细胞,转染后按说明书进行培养并进行收集、浓缩病毒液并进行滴度的测定。结果：含有Smac全长编码序列的pGC-FU载体高表达目的基因SMAC,并在蛋白水平得到验证。结论：成功构建目的基因SMAC过表达慢病毒载体,为后续SMAC基因进行功能研究提供了可靠工具。第三部分SMAC基因对肺癌细胞生长及顺铂耐药性影响的实验研究目的：探讨慢病毒介导RNAi敲除Smac基因前后对肺癌细胞生长及顺铂耐药性的影响,并且通过转染含有Smac全长编码序列的pOE载体来实现Smac的高表达,以来进一步验证Smac对肺癌细胞生长和对顺铂耐药性的影响。从正反两方面来相互验证,以来为进一步研究Smac在肺癌细胞生长及对顺铂耐药性的可能机制奠定基础。材料和方法：1、用慢病毒介导RNAi敲除Smac基因,用MTT,细胞克隆形成试验,Annexin V-PI双染流式细胞仪检测凋亡等手段来检测A549和95D细胞敲除Smac基因前后增殖能的力变化。2、用MTT、Annexin V-PI双染流式细胞仪检测顺铂干预前后和Smac基因敲除前后细胞生长情况和凋亡比率的变化来研究其对顺铂耐药的影响。3、用转染含有Smac全长编码序列的pOE载体来实现Smac高表达的A549细胞系,用MTT实验、Brdu增殖试验来检测顺铂干预前后细胞生长能力的变化来进一步验证Smac基因对肺癌细胞生长及顺铂耐药性的影响。结果：1、MTT分析法和克隆形成试验观察Smac基因敲除后对肺癌细胞系A549和95D生长的影响,与pNC-转染细胞相比,pKD-转染细胞的增殖显著增加(p=0.01,p=0.013),说明Smac基因敲除后促进人肺癌细胞生长。2、在克隆形成试验中,pKD一转染细胞的细胞克隆量和大小与pNC-转染细胞相比有显著增加,提示Smac基因敲除促进肺癌细胞系A549的集落形成能力。3、流式细胞计数检测也表明Smac基因敲除可抑制顺铂诱导的细胞调亡。在A549和95D两种细胞系中,Smac下调时,早期和晚期调亡细胞的比例(LR和UR)大大下降(p=0.01,p=0.01)；细胞存活率则显著增加。4、pOE-转染的细胞系A549 Smac蛋白表达明显增加,证明了慢病毒介导的Smac高表达。与Smac基因敲除相反,Smac高表达显著抑制A549细胞生长,并且增强其对顺铂的敏感性,从另一方面证明了Smac在肺癌细胞生长和药物耐药中的重要作用。结论：Smac抑制肺癌细胞生长并且增强其对顺铂的敏感性,Smac有可能成为预测肺癌对顺铂敏感性的分子标志之一。研究证明了Smac在抑制肺癌细胞生长和化疗药物增敏中的重要作用,因此Smac是肺癌治疗中极有希望的一个靶标,而且小分子Smac类似物也可用于人类肺癌细胞的治疗。更多还原

【Abstract】 Part I Preparation of the Vector Expressing SMAC RNAi, Screening of the Target Genes and Package of LentivirusPurpose:The aim was to construct RNAi Plasmid Vector containing siRNA sequences targeting Smac, and to make package lentivirus and preparations of stable cell strains infected with lentivirus.Materials and Methods:1. multiple RNA interference target sequences were designed aiming target gene sequences and according to the design principles of RNA interference from public websites;and then they were assessed by professional software and some targets with the best kinetic parameters were chosen for subsequent experiments. The double strand DNA oligo with cohesive ends of restriction site at both ends containing interference constructed by Genechem Co., Ltd was directly connected to vector containing siRNA. The binding products were transformed to the prepared competent cells. The cell clones were chosen as those with successfully constructed plasmid after PCR analysis and comparisons in sequence analysis.2. A549 cells were infected with lentivirus gene transfer vector (pGC-FU vector) containing siRNA sequences targeting Smac. The efficiency of gene silencing of shRNA-lentivirus for the target SMAC was verified not only under light microscope, but also from mRNA and protein levels by RT-PCR and Western Blot method respectively.3. The recombinant virus particles encoding the lentivirus and the two auxiliary packed vectors were extracted with endotoxin-free large number of high-purity plasmid method. According to the instructions of lipofectamine2000 kit, T293 cells were cotransfected, cultured and then cell supernatant full of lentiviral particles was collected and concentrated. The virus titers were tested and demarcated.Results:1.The psc-1 clones containing vshRNA fragments were the successfully constructed vectors with target siRNA.2. The efficiency of gene silencing for the target SMAC was verified not only under light microscope, but also from mRNA and protein levels after the induction of shRNA-lentivirus into the target A549 cells, which indicated the useful target for RNA interference was successfully identified.3. The cells used for tools and virus vectors were successfully packed and stable cell strains infected with lentivirus were prepared successfully.Conclusions:The RNAi Plasmid Vector containing siRNA sequences targeting Smac were successfully constructed and the useful target for RNA interference was successfully identified. The titers of virus were tested after package and the stable cell strains infected with lentivirus were prepared successfully, which laid the foundation of our subsequent study on the functions of SMAC. PartⅡConstruction of a Lentivirus Vector over-expressing SMACPurpose:The aim was to a Lentivirus Vector over-expressing SMAC as a tool for our subsequent study on the gene functions.Materials and Methods:1. The target gene was obtained from the plasmid containing the gene or from the cDNA library. Genechem Co., Ltd carried out the followings:the primer synthesis and identification of the target gene, and its PCR amplification, purification and enzyme digestion, and direct connection to recombinant clone, confections to the competent cells, and PCR identification of recombinant transformants. The company performed the sequencing and alignment of the positive clones and so the correct clones were chosen as the successfully constructed plasmid.2. A549 cells were infected with lentivirus gene transfer vector (pGC-FU vector) containing full-length coding region of human Smac (for overexpression). pGC-FU vector containing siRNA with a green fluorescent protein (GFP) sequence at the C-termination and the control vector were also transfected. The expressions of the vectors were verified from protein levels by western blot method.3. according to the instructions of lipofectamine2000 kit, T293 cells were cotransfected, cultured and then cell supernatant full of lentiviral particles was collected and concentrated. The virus titers were tested and demarcated.Results:SMAC was over expressed in the pGC-FU vector containing full-length coding region of human Smac, which was verified from protein levels.Conclusions:The Lentivirus Vector over-expressing SMAC was successfully constructed providing a reliable tool for our subsequent study on the gene functions. PartⅢStudy of the effects of SMAC on the growth and cisplatin resistance of human lung cancer cellsPurpose:We investigated the effects of the Smac knockdown through lentivirus-mediated Smac RNAi on growth and drug resistance to cisplatin in lung cancer cells, while the effects of the Smac on growth and drug resistance to cisplatin in lung cancer cells are further proved by Smac over-expression through transfection with pOE vector containing complete encoding sequence of Smac. Thus the potential mechanism of Smac is verified pro and con, which laid the foundation of our further study on the mechanisms on the effects of the Smac on growth and drug resistance to cisplatin of lung cancer cells.Materials and Methods:1. the knockdown of Smac expression was established by knockdown of Smac gene. The changes of proliferation ability of A549 and 95D cells were tested after and before the Smac knockdown by the method of MTT, colony-formation assay, and amphochromophil AnnexinⅤ-PI flow cytometry after the Smac knockdown mediated by RNAi.2. the changes of growth and apoptosis of A549 and 95D cells were tested after and before the intervention of cisplatin and the Smac knockdown by the method of MTT and amphochromophil AnnexinⅤ-PI flow cytometry so as to test the effect of cisplatin-resistance.3. Smac over-expression was achieved through transfection with pOE vector containing complete encoding sequence of Smac, and the changes of cellular growing ability were checked by the method of MTT, Brducell proliferation assay so as to further test the effect of cisplatin-resistance.Results:1. To assess the effect of Smac knock-down on proliferation and growth of human lung cancer cell lines A549 and 95D, MTT assay and colony-forming assay were performed. We found that pKD-transfected cells had a significantly increase in proliferation as compared with that of pNC-transfected cells in both cell lines, which indicates that knockdown of Smac promotes the proliferation ability of human lung cancer cells. In addition, the results from colony-forming assay showed that the colony amounts and size of pKD-transfected cells had an obvious increase by comparing with pNC-transfected cells, providing evidence for that Smac knockdown significantly promoted colony-formation of lung cancer cell A459.2. flow cytometry showed that the apoptosis induced by cisplatin was also inhibited by Smac knockdown. In both lung cancer cells lines, the proportion of cells at early and late stages of apoptosis (LR and UR) was largely reduced when Smac was down-regulated, while at the same the proportion of living cells was dramatically increased.3. Protein content of Smac was greatly increased in pOE-transfected A549 cells which validated the effectiveness of lentivirus mediated Smac overexpression. In contrast to Smac knockdown, overexpression of Smac significantly inhibited cell growth and increased cisplatin sensitivity of A549 cells. These data demonstrated the important roles of Smac in cell growth and drug sensitivity of lung cancer cells from another aspect.Conclusions:In our study, Smac significantly stimulated cell proliferation and enhanced susceptibility to cisplatin. Smac may be a useful target to predict the cisplatin sensitivity in lung cancer cells. Our results provided robust evidence for the important roles of Smac in suppressing lung cancer cells growth and sensitizing them to chemotherapeutic drugs. Therefore, Smac is also a promising target for the development of effective drugs for lung cancer treatment, and small molecule mimics of Smac will also function in the treatment of human lung cancer.更多还原