采用shRNA干扰肿瘤细胞Keap1基因表达的研究

发布时间：2018-01-13 20:28

  本文关键词：采用shRNA干扰肿瘤细胞Keap1基因表达的研究　出处：《皖南医学院》2014年硕士论文　论文类型：学位论文

  更多相关文章： Keap1 肿瘤细胞 Real-time PCR 慢病毒 siRNA 滴度 Real-time PCR PC3 细胞 MIO

【摘要】：第一部分Keap1基因在肿瘤细胞中的表达 目的：验证Keap1基因在肿瘤细胞中的表达 方法：用实时荧光定量PCR的方法，测定在人胃癌及前列腺癌细胞中肿瘤抑制因子keap1基因的表达。 结果：肿瘤抑制因子Keap1扩增结果专一；Real-time PCR的实验结果中，Keap1在人胃癌及前列腺癌细胞中ΔCt的值均小于12，说明Keap1在人胃癌及前列腺癌细胞中表达丰度较高；PCR产物电泳图中可见目的基因Keap1片段(240bp)。 结论：肿瘤抑制因子Keap1在人胃癌及前列腺癌细胞中广泛表达且丰度为高，适合做干扰验证实验。 第二部分RNAi慢病毒载体的构建与包装 目的：构建RNAi慢病毒载体，小量包装后测定病毒滴度，确定四组重组慢病毒颗粒的感染复数值，为最佳效率靶点的筛选做准备。 方法：根据GenBank上Keap1的mRNA序列，设计四个有效靶点，制备双链DNA Oligo(Keap1-shRNA)，利用基因重组技术克隆到GV115慢病毒表达载体，双酶切后鉴定克隆子。在脂质体的介导下将包装好的慢病毒载体GV115-Keap1-shRNA转染293T细胞，利用逐孔稀释滴度测定的方法收集病毒原液后测定病毒滴度。 结果：DNA测序证明插入序列正确，，成功构建RNAi慢病毒载体。重组慢病毒载体成功包装，收集病毒原液测定重组慢病毒颗粒的感染复数值为3E+8TU/mL。 结论：RNAi慢病毒载体的成功构建是为后续实验提供了必须的实验工具，为下一步筛选最佳靶点的实验提供了理论基础和前期准备。 第三部分有效RNAi载体的筛选 目的：筛选最佳效率的靶点 方法：用Real-Time PCR检测目的基因mRNA表达水平，数据分析后评价干扰水平，筛选最佳靶点。 结果：Real-Time PCR结果得出，人前列腺癌PC3细胞中，与阴性对照组比较，KD1、KD2、KD3组Keap1基因干扰效率较高(P0.01),KD4组Keap1基因干扰效率无统计学意义(P0.05)，且KD2组Keap1基因干扰效率达到75%(P0.05)。 结论：确定KD2即Keap1-RNAi-2为最有效靶点。
[Abstract]:The first part of the expression of Keap1 gene in tumor cells Objective: to verify the expression of Keap1 gene in tumor cells. Methods: the expression of tumor suppressor keap1 gene in human gastric cancer and prostate cancer cells was detected by real-time fluorescence quantitative PCR. Results: the results of Keap1 amplification of tumor suppressor were specific. The values of 螖 Ct in human gastric cancer and prostate cancer cells were lower than 12 in Real-time PCR. The results showed that Keap1 was highly expressed in human gastric cancer and prostate cancer cells. The Keap1 fragment of the target gene can be seen in the electrophoretogram of PCR products. Conclusion: tumor suppressor Keap1 is widely expressed in human gastric cancer and prostate cancer cells, and its abundance is high. The second part: construction and packaging of RNAi lentivirus vector Aim: to construct a RNAi lentivirus vector and determine the viral titer of the four groups of recombinant lentivirus particles after packaging, so as to prepare for the screening of the best efficiency target. Methods: according to the mRNA sequence of Keap1 on GenBank, four effective targets were designed to prepare double-stranded DNA oligodon Keap1-shRNAs. GV115 lentivirus expression vector was cloned by gene recombination technique. The clones were identified by double enzyme digestion. The packaged lentivirus vector GV115-Keap1-shRNA was transfected into 293T cells under the guidance of liposome. The virus titer was measured by the method of dilution titer-by-hole dilution method. Results the RNAi lentivirus vector was successfully constructed and the recombinant lentivirus vector was successfully packaged. The infection complex of recombinant lentivirus particles was determined to be 3e 8TU / mL. Conclusion the successful construction of the 1: RNAi lentivirus vector provides a necessary experimental tool for further experiments, and provides a theoretical basis and preliminary preparation for the further screening of the best target. Part III screening of effective RNAi vectors Objective: to screen targets for optimal efficiency. Methods: the target gene mRNA expression level was detected by Real-Time PCR, and the interference level was evaluated after data analysis. Results compared with negative control group, KD1 and KD2 were found in PC3 cells of human prostate cancer by using the results of: Real-Time PCR. The interference efficiency of Keap1 gene in KD3 group was higher than that in KD4 group (P 0.05). The interference efficiency of Keap1 gene in KD2 group was 75% (P 0.05). Conclusion: KD2 or Keap1-RNAi-2 is the most effective target.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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