本文關(guān)鍵詞：采用shRNA干擾腫瘤細(xì)胞Keap1基因表達(dá)的研究　出處：《皖南醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Keap1 腫瘤細(xì)胞 Real-time PCR 慢病毒 siRNA 滴度 Real-time PCR PC3 細(xì)胞 MIO

【摘要】：第一部分Keap1基因在腫瘤細(xì)胞中的表達(dá) 目的：驗證Keap1基因在腫瘤細(xì)胞中的表達(dá) 方法：用實時熒光定量PCR的方法，測定在人胃癌及前列腺癌細(xì)胞中腫瘤抑制因子keap1基因的表達(dá)。 結(jié)果：腫瘤抑制因子Keap1擴(kuò)增結(jié)果專一；Real-time PCR的實驗結(jié)果中，Keap1在人胃癌及前列腺癌細(xì)胞中ΔCt的值均小于12，說明Keap1在人胃癌及前列腺癌細(xì)胞中表達(dá)豐度較高；PCR產(chǎn)物電泳圖中可見目的基因Keap1片段(240bp)。 結(jié)論：腫瘤抑制因子Keap1在人胃癌及前列腺癌細(xì)胞中廣泛表達(dá)且豐度為高，適合做干擾驗證實驗。 第二部分RNAi慢病毒載體的構(gòu)建與包裝 目的：構(gòu)建RNAi慢病毒載體，小量包裝后測定病毒滴度，確定四組重組慢病毒顆粒的感染復(fù)數(shù)值，為最佳效率靶點的篩選做準(zhǔn)備。 方法：根據(jù)GenBank上Keap1的mRNA序列，設(shè)計四個有效靶點，制備雙鏈DNA Oligo(Keap1-shRNA)，利用基因重組技術(shù)克隆到GV115慢病毒表達(dá)載體，雙酶切后鑒定克隆子。在脂質(zhì)體的介導(dǎo)下將包裝好的慢病毒載體GV115-Keap1-shRNA轉(zhuǎn)染293T細(xì)胞，利用逐孔稀釋滴度測定的方法收集病毒原液后測定病毒滴度。 結(jié)果：DNA測序證明插入序列正確，，成功構(gòu)建RNAi慢病毒載體。重組慢病毒載體成功包裝，收集病毒原液測定重組慢病毒顆粒的感染復(fù)數(shù)值為3E+8TU/mL。 結(jié)論：RNAi慢病毒載體的成功構(gòu)建是為后續(xù)實驗提供了必須的實驗工具，為下一步篩選最佳靶點的實驗提供了理論基礎(chǔ)和前期準(zhǔn)備。 第三部分有效RNAi載體的篩選 目的：篩選最佳效率的靶點 方法：用Real-Time PCR檢測目的基因mRNA表達(dá)水平，數(shù)據(jù)分析后評價干擾水平，篩選最佳靶點。 結(jié)果：Real-Time PCR結(jié)果得出，人前列腺癌PC3細(xì)胞中，與陰性對照組比較，KD1、KD2、KD3組Keap1基因干擾效率較高(P0.01),KD4組Keap1基因干擾效率無統(tǒng)計學(xué)意義(P0.05)，且KD2組Keap1基因干擾效率達(dá)到75%(P0.05)。 結(jié)論：確定KD2即Keap1-RNAi-2為最有效靶點。
[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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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