本文選題：喉癌 + Jab1　； 參考：《鄭州大學》2010年碩士論文

【摘要】： 喉鱗狀細胞癌(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)生過程,發(fā)病機制復雜,治療困難。目前,喉癌治療以手術切除或放療為主,盡管取得了較大進展,但手術致殘率高、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,轉移率高,病人生存率低。因此,急需尋找有效、簡便的治療方法。隨著喉癌相關基因及分子生物學研究的發(fā)展,喉癌基因治療的相關研究也在不斷深入。 對腫瘤相關基因在轉錄、轉錄后和翻譯等水平特異性的阻斷能夠誘導腫瘤細胞向成熟方向轉化或凋亡。RNA干擾((RNA interference, RNAi)現(xiàn)象廣泛存在于各種生物體中,能夠在哺乳動物細胞內(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用,是分子靶向抗癌治療的分子學基礎。最近,RNA干擾作為一種抗癌治療的方法已經(jīng)進入實驗階段,預計將發(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)信號復合體的第五亞單位,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Jab1和多種蛋白的出核轉運降解有關,例如p27kip1(kinase inhibit protein 1, P27)、P53、Smad4。Jab1作為一個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是細胞周期素依賴的激酶抑制因子,通常在細胞核內(nèi)發(fā)生作用,Jab1可以與P27發(fā)生作用并改變其細胞定位使其出核降解,從而導致腫瘤細胞無限增殖,Jab1與P27在各種腫瘤細胞的研究中表明存在負相關性。 在我們前期的實驗中發(fā)現(xiàn)Jab1在人類喉癌細胞中高表達,與P27的表達存在負相關性,并利用RNAi技術沉默喉癌細胞中活躍的癌基因Jab1的表達,通過RT-PCR、流式細胞學、MTT的方法檢測到其能達到阻止或減緩腫瘤細胞生長的目的。在本研究中,我們通過應用RNA干擾技術來研究Jab1的低表達對喉癌細胞的細胞株(Hep-2細胞)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，，通過WST-8檢測細胞增殖的能力,通過Western Blotting檢測基因的表達。為了觀察shRNA質(zhì)粒在體內(nèi)引起Jab1的下調(diào)對腫瘤生長的影響,我們將Hep-2腫瘤細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)粒和對照組治療,腫瘤的生長受到了抑制。這些結果為人類喉癌治療提供了新的實驗數(shù)據(jù)。 目的：利用RNA干擾技術,構建Jab1基因靶向shRNA質(zhì)粒,觀察目的基因的沉默效應及對Hep-2腫瘤細胞的影響。 方法：設計合成以Jab1基因為靶向目標的shRNA,將其定向克隆到真核表達載體pGenesill.1中形成重組質(zhì)粒；利用脂質(zhì)體介導的方法將質(zhì)粒轉染至人喉癌細胞株(Hep-2),通過WST-8、Western Blotting的方法對細胞凋亡及蛋白水平的表達變化進行檢測,分析shRNA干擾質(zhì)粒對喉癌細胞的影響。 結果：成功構建了Jab1基因靶向的pJab1以及陰性對照pKB真核重組質(zhì)粒。WST-8結果示pJab1重組質(zhì)粒能顯著抑制Hep-2細胞的增殖活性,與對照組相比,顯著差異(p0.001)。Western Blotting結果示Hep-2-pJab1細胞Jab1蛋白表達量明顯降低,P27蛋白表達量明顯升高,與對照組相比,差異顯著(p0.001)。 結論：構建靶向Jab1癌基因的shRNA干擾質(zhì)粒能夠下調(diào)Jab1基因的表達,上調(diào)p27基因的表達,抑制人喉癌細胞株Hep-2細胞在體外的增殖。 目的：探討Jab1重組質(zhì)粒對人喉癌Hep-2細胞裸鼠移植瘤生長的影響。 方法：建立了喉癌Hep-2細胞裸鼠皮下移植瘤模型,通過瘤體內(nèi)注射pJab1, pKB, PBS,觀察瘤體增長情況,通過免疫組化方法、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。 結果：成功建立喉癌Hep-2細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,顯著低于錯意序列對照組和空白對照組；免疫組化結果顯示實驗組瘤內(nèi)的Jabl蛋白的表達水平降低,顯著低于對照組,P27蛋白的表達水平增高,顯著高于對照組；RT-PCR結果顯示實驗組瘤內(nèi)Jab1的mRNA水平顯著降低,P27的mRNA無明顯變化；Western Blotting結果顯示實驗組瘤內(nèi)Jab1蛋白表達量明顯減少,P27蛋白表達量明顯增高。 結論：建立了喉癌Hep-2細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基因的表達并抑制瘤體的生長；pJab1質(zhì)粒能有效抑制Jab1基因的表達,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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R739.65

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