本文選題：shRNA + Survivin基因　； 參考：《海南大學(xué)》2014年碩士論文

【摘要】：目的：構(gòu)建具有干擾效應(yīng)的靶向Survivin和CDK1基因及兩者串聯(lián)的短發(fā)夾樣RNA(short hairpin RNA, shRNA)表達(dá)載體,并研究其對鼻咽癌細(xì)胞CNE-2干擾后,癌細(xì)胞的生物學(xué)行為(增殖、凋亡)的改變。 方法：根據(jù)Genbank記錄的Survivin及CDK1序列,遵循shRNA設(shè)計(jì)原則并合成相應(yīng)的寡核苷酸鏈SurvivinshRNA、 CDK1shRNA’構(gòu)建pU6-SurvivinshRNA、 pU6-CDK1shRNA重組載體,用脂質(zhì)體Lipofectamine2000方法將其轉(zhuǎn)染入CNE-2細(xì)胞株中,RT-PCR初步篩選具有干擾效應(yīng)的pU6-SurvivinshRNA、 pU6-CDK1shRNA;將上述具有干擾效應(yīng)的SurvivinshRNA和CDK1shRNA串聯(lián)進(jìn)行pU6-SurvivinshRNA-CDK1shRNA表達(dá)載體的構(gòu)建,經(jīng)酶切及測序鑒定后,以脂質(zhì)體Lipofectamine2000轉(zhuǎn)染CNE-2細(xì)胞株后；收集轉(zhuǎn)染單基因及聯(lián)合基因重組質(zhì)粒的CNE-2細(xì)胞,進(jìn)行逆轉(zhuǎn)錄實(shí)時(shí)熒光定量PCR(RT-qPCR)檢測癌細(xì)胞Survivin和CDK1的mRNA表達(dá)水平,Western blot檢測Survivin和CDK1蛋白表達(dá)的變化,噻唑藍(lán)溴化四唑(]methyl thiazolyl tetrazolium,MTT)法檢測癌細(xì)胞增殖活性的變化,流式細(xì)胞儀檢測癌細(xì)胞凋亡的變化。 結(jié)果：(1)經(jīng)酶切及測序結(jié)果分析：pU6-SurvivinshRNA、pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA重組載體均成功構(gòu)建；(2)RT-qPCR的結(jié)果顯示：pU6-SurvivinshRNA和pU6-SurvivinshRNA-CDK1shRNA各干擾組均下調(diào)Survivin mRNA的表達(dá),分別為52.7%和82.4%,pU6-CDK1shRNA干擾組的Survivin mRNA的表達(dá)沒有下調(diào)；pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1ShRNA各干擾組均下調(diào)CDK1mRNA的表達(dá),分別為56.7%和85.0%, pU6-SurvivinshRNA干擾組的CDK1mRNA的表達(dá)沒有下調(diào)；(3) pU6-SurvivinshRNA和pU6-SurvivinshRNA-CDK1shRNA干擾組均下調(diào)Survivin蛋白的表達(dá),pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA干擾組均下調(diào)CDK1蛋白的表達(dá)。(4)MTT法檢測癌細(xì)胞增殖活性的結(jié)果顯示：pU6-Survivin,hRNA、 PU6-CDKlshRNA和pU6-SurvivinshRNA-CDK1shRNA干擾組均能抑制細(xì)胞的生長,抑制率分別為25.60%、15.62%和32.54%；(5)流式細(xì)胞儀檢測癌細(xì)胞凋亡的結(jié)果顯示：pU6-SurvivinshRNA、 pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA干擾組均促進(jìn)癌細(xì)胞凋亡,凋亡率分別為10.27%,8.00%和14.87%。 結(jié)論：(1)pU6-SurvivinshRNA、 pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA重組載體均成功構(gòu)建；(2) pU6-SurvivinShRNA、pU6-CDK1ShRNA和pU6-SurvivinshRNA-CDK1shRNA重組載體干擾鼻咽癌細(xì)胞,均可以抑制癌細(xì)胞的增殖,促進(jìn)癌細(xì)胞的凋亡；(3)Survivin基因和CDK1基因有望成為鼻咽癌腫瘤基因治療的靶基因,多基因聯(lián)合干擾可以提高效應(yīng)。
[Abstract]:Aim: to construct the expression vector of survivin and CDK1 gene targeting survivin and CDK1 gene and short hairpin (short hairpin RNA (shRNA) in tandem, and to study the changes of biological behavior (proliferation and apoptosis) of nasopharyngeal carcinoma cell line CNE-2 after the interference of survivin and CDK1 gene. Methods: according to the sequence of survivin and CDK1 recorded in Genbank, following the shRNA design principle and synthesizing the corresponding oligonucleotide chain survivshRNAs, CDK1shRNAs' constructed pU6-survivin RNA, pU6-CDK1shRNA recombinant vector. Lipofectamine2000 was transfected into CNE-2 cell line by RT-PCR to screen interference pU6-survivin shRNAs (pU6-CDK1shRNAs), the interfering survivshRNA and CDK1shRNA were connected in tandem to construct pU6-survivshRNA-CDK1shRNA expression vector. CNE-2 cells transfected with liposome Lipofectamine2000 were collected and transfected into CNE-2 cells, and the expression levels of survivin and CDK1 were detected by reverse transcription-real-time fluorescence quantitative PCR (RT-qPCR). The expression of survivin and CDK1 protein was detected by Western blot. Methyl thiazolyl tetrazoliumium tetrazolium tetrazol@@ Results: (1) the recombinant vectors of pU6-survivshRNA-CDK1shRNA and pU6-survivvinshRNA-CDK1shRNA were successfully constructed by restriction endonuclease digestion and sequencing, (2) the results of RT-qPCR showed that the expression of survivin mRNA was down-regulated in the two interference groups (52.7% and 82.4pU6-CDK1shRNA respectively). Both pU6-CDK1shRNA and pU6-survivvinshRNA-CDK1ShRNA down-regulated the expression of CDK1 mRNA in 56.7% and 85.0%, respectively. The expression of CDK1 mRNA was not down-regulated in pU6-survivin RNA interference group. (3) both pU6-survivvinshRNA-CDK1shRNA interference group and pU6-survivvinshRNA-CDK1shRNA interference group down-regulated the expression of survivin protein. (5) the apoptosis of cancer cells was detected by flow cytometry. The results showed that the cell apoptosis was promoted by cell apoptosis in the groups of cell apoptosis induced by the interference of cell apoptosis, the percentage of apoptosis was 10.277.00% and the percentage of apoptosis was 14.87% (P < 0.05), respectively, in the interference groups of pU6-CDK1shRNA and pU6-survivin shRNA-CDK1shRNA. Conclusion: (1) the recombinant vectors of pU6-survivvinshRNA, pU6-CDK1shRNA and pU6-survivvinshRNA-CDK1shRNA were successfully constructed, (2) pU6-survivin shRNA-CDK1shRNA and pU6-survivvinshRNA-CDK1shRNA interference vector could inhibit the proliferation of cancer cells and promote the apoptosis of cancer cells. (3) survivin gene and CDK1 gene are expected to be the target genes for gene therapy of nasopharyngeal carcinoma.
【學(xué)位授予單位】：海南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R739.63

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本文編號：2110903