本文選題：腫瘤　切入點(diǎn)：口腔癌　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究生物鐘基因PER2對人類口腔鱗癌SCC15細(xì)胞凋亡、增殖、侵襲、遷移的影響與機(jī)理。方法:利用RNAi技術(shù)構(gòu)建出3組靶向PER2基因shRNA慢病毒質(zhì)粒,感染人類口腔鱗狀細(xì)胞癌SCC15細(xì)胞;經(jīng)過測序、實(shí)時(shí)熒光定量PCR以及Western blot檢測方法鑒定并篩選出PER2基因沉默效果最佳組為實(shí)驗(yàn)組,對照組為不包含PER2片段的scramble質(zhì)粒病毒,空白組為未經(jīng)過任何的處理的SCC15細(xì)胞。應(yīng)用實(shí)時(shí)熒光定量PCR檢測Ki-67、MDM2、P53、Bcl-2、Bax、c-Myc、MMP-2、Timp-2和VEGFmRNA的表達(dá)改變;CCK-8試驗(yàn)用于檢測SCC15細(xì)胞體外生長能力,流式細(xì)胞儀用于檢測基因沉默后細(xì)胞增殖、凋亡水平、細(xì)胞周期分布,平板克隆形成實(shí)驗(yàn)用于檢測細(xì)胞克隆形成率,Transwell小室用于檢測細(xì)胞遷移、侵襲能力改變,裸鼠的體內(nèi)成瘤實(shí)驗(yàn)用于檢測人口腔鱗癌細(xì)胞SCC15體內(nèi)的成瘤情況。結(jié)果:成功的構(gòu)建了3組靶向PER2基因的PER2-shRNA慢病毒質(zhì)粒,PER2-shRNA-I組沉默效果為最佳,即被作為實(shí)驗(yàn)組。沉默PER2基因后,Ki-67、MDM2、Bcl-2、c-Myc、MMP-2和VEGF mRNA的表達(dá)水平顯著升高(均P0.05),p53、Bax和Timp-2 mRNA的表達(dá)水平顯著降低(均P0.05)。PER2沉默后,SCC15細(xì)胞的生長能力、細(xì)胞增殖指數(shù)、細(xì)胞遷移和侵襲能力顯著升高(均P0.05),凋亡指數(shù)顯著降低(P0.05)。體內(nèi)實(shí)驗(yàn)也證明PER2沉默后SCC15細(xì)胞的成瘤能力顯著增強(qiáng)(P0.05)。結(jié)論:表明鐘基因PER2通過對下游許多重要腫瘤相關(guān)基因調(diào)控而發(fā)揮重要抑癌作用,對生物鐘PER2基因的深入研究有望成為治療癌癥的新分子靶點(diǎn)。
[Abstract]:Objective: to study the effect and mechanism of clock gene PER2 on apoptosis, proliferation, invasion and migration of human oral squamous cell carcinoma (SCC15) cells. Methods: three groups of shRNA lentivirus plasmids targeting PER2 gene were constructed by RNAi technique. SCC15 cells infected with human oral squamous cell carcinoma were identified and screened by sequencing, real-time fluorescence quantitative PCR and Western blot detection methods. The best silencing effect of PER2 gene was identified as experimental group, and the control group was scramble plasmid virus without PER2 fragment. The expression changes of Ki-67M2MDM2P53P53Bcl-2Bax-2MP-2Timp-2 and VEGFmRNA were detected by flow cytometry. Flow cytometry was used to detect the proliferation and apoptosis of SCC15 cells after gene silencing, and CCK-8 assay was used to detect the proliferation and apoptosis of SCC15 cells after gene silencing. Cell cycle distribution, flat plate clone formation assay was used to detect cell clone formation rate and Transwell chamber was used to detect cell migration and invasion ability. Results: three groups of PER2-shRNA lentivirus plasmids targeting PER2 gene, PER2-shRNA-I, were successfully constructed, and the silencing effect of PER2-shRNA-I group was the best. After silencing the PER2 gene, the expression levels of Ki-67m2m2mcl-2c-Mycnc-MMP-2 and VEGF mRNA were significantly increased (both P0.05P53BX and Timp-2 mRNA were significantly decreased after silencing the PER2 gene (all P0.05PER2) after silencing SCC15 cells, the growth ability and proliferation index of SCC15 cells were significantly decreased. The ability of cell migration and invasion was significantly increased (all P0.05N, apoptotic index significantly decreased P0.050.The in vivo experiments also proved that the tumorigenic ability of SCC15 cells after PER2 silencing was significantly enhanced. Conclusion: it is suggested that the clock gene PER2 can significantly enhance the tumorigenesis of SCC15 cells in many important tumor phases downstream. Gene regulation plays an important role in the inhibition of cancer, Further study on PER2 gene of biological clock is expected to be a new molecular target for cancer treatment.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.8

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本文編號：1623796