本文選題：喉癌 + Jab1　； 參考：《鄭州大學(xué)》2010年碩士論文

【摘要】： 喉鱗狀細(xì)胞癌(Laryngeal Squamous Cell Carcinoma, LSCC)和其它腫瘤一樣,是一種多基因性、多步驟、多階段的發(fā)生過(guò)程,發(fā)病機(jī)制復(fù)雜,治療困難。目前,喉癌治療以手術(shù)切除或放療為主,盡管取得了較大進(jìn)展,但手術(shù)致殘率高、并發(fā)癥多,常規(guī)放療的毒副作用大,療效差,特別是晚期腫瘤病人,轉(zhuǎn)移率高,病人生存率低。因此,急需尋找有效、簡(jiǎn)便的治療方法。隨著喉癌相關(guān)基因及分子生物學(xué)研究的發(fā)展,喉癌基因治療的相關(guān)研究也在不斷深入。 對(duì)腫瘤相關(guān)基因在轉(zhuǎn)錄、轉(zhuǎn)錄后和翻譯等水平特異性的阻斷能夠誘導(dǎo)腫瘤細(xì)胞向成熟方向轉(zhuǎn)化或凋亡。RNA干擾((RNA interference, RNAi)現(xiàn)象廣泛存在于各種生物體中,能夠在哺乳動(dòng)物細(xì)胞內(nèi)產(chǎn)生特異性的基因沉默,在后基因組研究中得到廣泛應(yīng)用,是分子靶向抗癌治療的分子學(xué)基礎(chǔ)。最近,RNA干擾作為一種抗癌治療的方法已經(jīng)進(jìn)入實(shí)驗(yàn)階段,預(yù)計(jì)將發(fā)展成為一種基因治療的藥物。 Jab1 (C-Jun activation domain- binding protein 1)位于COP9(COP9signalosome, CSN)信號(hào)復(fù)合體的第五亞單位,早期研究證明Jabl是c-Jun激活區(qū)的輔助激活因子,后來(lái)研究發(fā)現(xiàn)Jab1也是激活蛋白-1(activator protein 1, AP-1)的輔助因子,最近研究證實(shí)Jab1和多種蛋白的出核轉(zhuǎn)運(yùn)降解有關(guān),例如p27kip1(kinase inhibit protein 1, P27)、P53、Smad4。Jab1作為一個(gè)潛在的致癌基因在多種腫瘤疾病的發(fā)展中起著調(diào)節(jié)器的作用。 P27是細(xì)胞周期素依賴的激酶抑制因子,通常在細(xì)胞核內(nèi)發(fā)生作用,Jab1可以與P27發(fā)生作用并改變其細(xì)胞定位使其出核降解,從而導(dǎo)致腫瘤細(xì)胞無(wú)限增殖,Jab1與P27在各種腫瘤細(xì)胞的研究中表明存在負(fù)相關(guān)性。 在我們前期的實(shí)驗(yàn)中發(fā)現(xiàn)Jab1在人類喉癌細(xì)胞中高表達(dá),與P27的表達(dá)存在負(fù)相關(guān)性,并利用RNAi技術(shù)沉默喉癌細(xì)胞中活躍的癌基因Jab1的表達(dá),通過(guò)RT-PCR、流式細(xì)胞學(xué)、MTT的方法檢測(cè)到其能達(dá)到阻止或減緩腫瘤細(xì)胞生長(zhǎng)的目的。在本研究中,我們通過(guò)應(yīng)用RNA干擾技術(shù)來(lái)研究Jab1的低表達(dá)對(duì)喉癌細(xì)胞的細(xì)胞株(Hep-2細(xì)胞)在體外增殖的影響,使用一系列的干擾方式,例如,短發(fā)夾施工干擾RNA (shRNA)的質(zhì)粒和非特異性質(zhì)粒抑制Jab1的表達(dá)，，通過(guò)WST-8檢測(cè)細(xì)胞增殖的能力,通過(guò)Western Blotting檢測(cè)基因的表達(dá)。為了觀察shRNA質(zhì)粒在體內(nèi)引起Jab1的下調(diào)對(duì)腫瘤生長(zhǎng)的影響,我們將Hep-2腫瘤細(xì)胞在裸鼠皮下注射建立人喉癌異種移植瘤模型。模型建立后經(jīng)與Jab1shRNA質(zhì)粒和對(duì)照組治療,腫瘤的生長(zhǎng)受到了抑制。這些結(jié)果為人類喉癌治療提供了新的實(shí)驗(yàn)數(shù)據(jù)。 目的：利用RNA干擾技術(shù),構(gòu)建Jab1基因靶向shRNA質(zhì)粒,觀察目的基因的沉默效應(yīng)及對(duì)Hep-2腫瘤細(xì)胞的影響。 方法：設(shè)計(jì)合成以Jab1基因?yàn)榘邢蚰繕?biāo)的shRNA,將其定向克隆到真核表達(dá)載體pGenesill.1中形成重組質(zhì)粒；利用脂質(zhì)體介導(dǎo)的方法將質(zhì)粒轉(zhuǎn)染至人喉癌細(xì)胞株(Hep-2),通過(guò)WST-8、Western Blotting的方法對(duì)細(xì)胞凋亡及蛋白水平的表達(dá)變化進(jìn)行檢測(cè),分析shRNA干擾質(zhì)粒對(duì)喉癌細(xì)胞的影響。 結(jié)果：成功構(gòu)建了Jab1基因靶向的pJab1以及陰性對(duì)照pKB真核重組質(zhì)粒。WST-8結(jié)果示pJab1重組質(zhì)粒能顯著抑制Hep-2細(xì)胞的增殖活性,與對(duì)照組相比,顯著差異(p0.001)。Western Blotting結(jié)果示Hep-2-pJab1細(xì)胞Jab1蛋白表達(dá)量明顯降低,P27蛋白表達(dá)量明顯升高,與對(duì)照組相比,差異顯著(p0.001)。 結(jié)論：構(gòu)建靶向Jab1癌基因的shRNA干擾質(zhì)粒能夠下調(diào)Jab1基因的表達(dá),上調(diào)p27基因的表達(dá),抑制人喉癌細(xì)胞株Hep-2細(xì)胞在體外的增殖。 目的：探討Jab1重組質(zhì)粒對(duì)人喉癌Hep-2細(xì)胞裸鼠移植瘤生長(zhǎng)的影響。 方法：建立了喉癌Hep-2細(xì)胞裸鼠皮下移植瘤模型,通過(guò)瘤體內(nèi)注射pJab1, pKB, PBS,觀察瘤體增長(zhǎng)情況,通過(guò)免疫組化方法、RT-PCR、Western Blotting等觀察瘤體內(nèi)Jab1、P27的mRNA水平及蛋白水平的變化。 結(jié)果：成功建立喉癌Hep-2細(xì)胞裸鼠皮下移植瘤模型；pJab1質(zhì)粒組的腫瘤體積為(267.60±88.19)mm3,瘤重為(0.49±0.03)g,顯著低于錯(cuò)意序列對(duì)照組和空白對(duì)照組；免疫組化結(jié)果顯示實(shí)驗(yàn)組瘤內(nèi)的Jabl蛋白的表達(dá)水平降低,顯著低于對(duì)照組,P27蛋白的表達(dá)水平增高,顯著高于對(duì)照組；RT-PCR結(jié)果顯示實(shí)驗(yàn)組瘤內(nèi)Jab1的mRNA水平顯著降低,P27的mRNA無(wú)明顯變化；Western Blotting結(jié)果顯示實(shí)驗(yàn)組瘤內(nèi)Jab1蛋白表達(dá)量明顯減少,P27蛋白表達(dá)量明顯增高。 結(jié)論：建立了喉癌Hep-2細(xì)胞裸鼠皮下移植瘤模型；pJab1干擾質(zhì)粒明顯降低裸鼠腫瘤組織內(nèi)Jab1基因的表達(dá)并抑制瘤體的生長(zhǎng)；pJab1質(zhì)粒能有效抑制Jab1基因的表達(dá),有望成為臨床治療喉癌的新途徑。
[Abstract]:Laryngeal Squamous Cell Carcinoma (LSCC), like other tumors, is a multi gene, multistep, multi-stage process, complicated and difficult to treat. At present, surgical resection or radiotherapy is the main treatment for larynx cancer. Although great progress has been made, there are high morbidity, complications and conventional radiotherapy. Therefore, it is urgent to find effective and simple treatment methods. With the development of the related genes and molecular biology of larynx cancer, the related research on the gene therapy of larynx cancer is also in deep.
Specific blocking of tumor related genes at transcriptional, post transcriptional and translation levels can induce tumor cells to transform or apoptotic.RNA interference (RNA interference, RNAi) in various organisms, which can produce specific gene silencing in mammalian cells, and can be widely used in post genome research. Ubiquitous application is the sub molecular basis for molecular targeting anticancer therapy. Recently, RNA interference, as a method of anticancer therapy, has entered the experimental stage and is expected to develop into a drug for gene therapy.
Jab1 (C-Jun activation domain- binding protein 1) is located in the fifth subunit of the COP9 (COP9signalosome, CSN) signal complex. Early studies showed that Jabl was an auxiliary activating factor in the c-Jun activation region. Nuclear transport and degradation, such as p27kip1 (kinase inhibit protein 1, P27), P53, Smad4.Jab1, as a potential oncogene, play the role of regulators in the development of a variety of tumor diseases.
P27 is a cyclin dependent kinase inhibitor, which usually acts in the nucleus. Jab1 can interact with P27 and change its cell location to cause nuclear degradation, which leads to the proliferation of tumor cells. Jab1 and P27 have negative correlation in the study of various tumor cells.
In our previous experiments, we found that Jab1 was highly expressed in human larynx cancer cells and had a negative correlation with the expression of P27. The expression of active oncogene Jab1 in laryngeal cancer cells was silenced by RNAi technique. The purpose of detecting the growth of tumor cells could be detected by RT-PCR, flow cytology and MTT. In this study, We used RNA interference to study the effect of low expression of Jab1 on the proliferation of cell line (Hep-2 cells) in laryngeal cancer cells in vitro. We use a series of interference methods, for example, the plasmid and non specific plasmids that interfere with RNA (shRNA) and non specific plasmids to suppress Jab1, and the ability to detect cell proliferation through WST-8 and through Western Blo. The expression of the gene was detected by tting. In order to observe the effect of the downregulation of the shRNA plasmid on the tumor growth in the body, we injected the Hep-2 tumor cells into the nude mice to establish a xenograft tumor model of human larynx. After the establishment of the model, the growth of the tumor was inhibited by the treatment of the Jab1shRNA plasmid and the control group. These results were the human larynx. Cancer treatment provides new experimental data.
Objective: to construct Jab1 gene targeting shRNA plasmid using RNA interference technology, observe the silencing effect of target gene and its effect on Hep-2 tumor cells.
Methods: the shRNA was designed and synthesized with the target of Jab1 gene. The recombinant plasmid was cloned into the eukaryotic expression vector pGenesill.1, and the plasmid was transfected into the human larynx cell line (Hep-2) by liposome mediated method. The expression of apoptosis and protein level was detected by WST-8 and Western Blotting. The effects of shRNA interference plasmids on laryngeal cancer cells were analyzed.
Results: the Jab1 gene targeted pJab1 and the negative control pKB eukaryotic recombinant plasmid.WST-8 showed that the pJab1 recombinant plasmid could significantly inhibit the proliferation activity of Hep-2 cells. Compared with the control group, the significant difference (p0.001).Western Blotting results showed that the Jab1 protein expression of Hep-2-pJab1 cells decreased obviously, and the expression of P27 protein was obvious. The difference was significant compared with the control group (p0.001).
Conclusion: the construction of shRNA interfering plasmid for targeting Jab1 oncogene can downregulate the expression of Jab1 gene, up regulate the expression of p27 gene and inhibit the proliferation of Hep-2 cells in human larynx cell line in vitro.
Objective: To investigate the effect of recombinant plasmid Jab1 on the growth of human laryngeal carcinoma Hep-2 cells transplanted in nude mice.
Methods: a subcutaneous tumor model of Hep-2 cells in larynx cancer cells was established. The tumor growth was observed by injection of pJab1, pKB and PBS in the tumor. The changes of Jab1 in the tumor, mRNA level and protein level in the tumor were observed by immunohistochemical method, RT-PCR, Western Blotting and so on.
Results: the model of subcutaneous transplantation of Hep-2 cells in laryngeal carcinoma was successfully established. The tumor volume of the pJab1 plasmid group was (267.60 + 88.19) mm3, and the tumor weight was (0.49 + 0.03) g, significantly lower than the wrong sequence control group and the blank control group. The immunohistochemical results showed that the expression level of Jabl protein in the experimental group was lower, significantly lower than the control group, P27 egg. The level of white expression was higher, significantly higher than that in the control group. The RT-PCR results showed that the mRNA level of Jab1 in the experimental group was significantly decreased and the mRNA of P27 was not significantly changed. The Western Blotting results showed that the expression of Jab1 protein in the experimental group was significantly reduced and the expression of P27 protein was significantly increased.
Conclusion: the subcutaneous transplanted tumor model of Hep-2 cells in larynx cancer cells was established. The pJab1 interference plasmid obviously reduced the expression of Jab1 gene in the tumor tissues of nude mice and inhibited the growth of the tumor. The pJab1 plasmid could effectively inhibit the expression of Jab1 gene, and it is expected to be a new way to treat the cancer of the larynx.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.65

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