【摘要】：實(shí)驗(yàn)背景：食管癌是世界第八大腫瘤,占據(jù)腫瘤導(dǎo)致死亡的第六位,其中絕大部分食管癌的病理類型為鱗癌,但是食管鱗癌的五年生存率只有不足15%,近年來(lái)總生存期也沒有明顯的延長(zhǎng),這跟目前缺少有效的治療有關(guān)。隨著高通量測(cè)序技術(shù)的發(fā)展,在多個(gè)食管鱗癌隊(duì)列當(dāng)中發(fā)現(xiàn)AJUBA基因突變,而相關(guān)研究是匱乏的,探索AJUBA突變?cè)谑彻荀[癌發(fā)生發(fā)展中的分子機(jī)制及臨床意義,可能改善患者的預(yù)后,提供新的治療靶點(diǎn)。實(shí)驗(yàn)方法：本論文收集了五個(gè)食管鱗癌的隊(duì)列,共545例患者,都有臨床信息及全外顯子組測(cè)序數(shù)據(jù),通過統(tǒng)計(jì)學(xué)分析AJUBA突變與患者TNM分期的相關(guān)性,分別繪制AJUBA突變型與野生型患者的Kaplan-Meier曲線,通過Log-Rank比較兩者是否有顯著差異,以獲得可能的AJUBA突變的臨床意義,指導(dǎo)進(jìn)一步的機(jī)制探索及臨床應(yīng)用。同時(shí),運(yùn)用293T細(xì)胞包裝慢病毒,慢病毒感染目的細(xì)胞構(gòu)建可供研究AJUBA突變的穩(wěn)轉(zhuǎn)細(xì)胞系,這包括穩(wěn)定敲降的細(xì)胞系,過表達(dá)野生型AJUBA的細(xì)胞系及過表達(dá)突變型的細(xì)胞系,通過流式熒光分選、潮霉素/嘌呤霉素進(jìn)行篩選,RT-PCR及蛋白印跡進(jìn)行驗(yàn)證。在構(gòu)建細(xì)胞系基礎(chǔ)上,進(jìn)行CCK-8、Transwell細(xì)胞功能實(shí)驗(yàn),體外驗(yàn)證AJUBA突變與細(xì)胞增殖、遷移及侵襲的關(guān)系。實(shí)驗(yàn)結(jié)果：在545例患者當(dāng)中,AJUBA突變型及野生型的TNM分期沒有顯著差異(Fisher確切概率p=1.000),AJUBA突變型患者Kaplan-Meier曲線有生存時(shí)間更長(zhǎng)的趨勢(shì)(Log-Rank檢驗(yàn)p=0.13)。構(gòu)建穩(wěn)轉(zhuǎn)敲降細(xì)胞株,感染后10日通過潮霉素/流式熒光分選進(jìn)行篩選,RT-PCR及蛋白印跡驗(yàn)證有一定的敲降效率,但是敲降效率不高。構(gòu)建過表達(dá)野生型及突變型細(xì)胞株,通過嘌呤霉素進(jìn)行篩選,運(yùn)用蛋白印跡技術(shù)驗(yàn)證,可見野生型AJUBA條帶及截短的突變型AJUBA條帶,構(gòu)建穩(wěn)轉(zhuǎn)過表達(dá)細(xì)胞系成功。通過CCK-8比較過表達(dá)細(xì)胞系與野生型細(xì)胞系的增殖能力,發(fā)現(xiàn)三個(gè)過表達(dá)細(xì)胞系的增殖能力都強(qiáng)于野生型細(xì)胞系(t檢驗(yàn)48h:p0.001,p0.001, p0.001; 72h:p=0.008, p0.001, p0.001)。Transwell實(shí)驗(yàn)比較過表達(dá)細(xì)胞系與野生型細(xì)胞系的遷移及侵襲能力,三個(gè)過表達(dá)細(xì)胞系的遷移能力強(qiáng)于野生型細(xì)胞系(t檢驗(yàn)：p0.001,p0.001,p0.001),過表達(dá)野生型AJUBA及突變1的侵襲能力強(qiáng)于野生型細(xì)胞系(t檢驗(yàn)：p0.001,p=0.035,p=0.576),其中過表達(dá)野生型AJUBA細(xì)胞系的遷移及侵襲能力增強(qiáng)最為顯著。結(jié)論：AJUBA突變型的食管鱗癌患者預(yù)后相對(duì)于AJUBA野生型的患者有更好的趨勢(shì)。構(gòu)建穩(wěn)轉(zhuǎn)敲降細(xì)胞系可作為進(jìn)一步研究的對(duì)照,并為構(gòu)建敲降后過表達(dá)突變型AJUBA的細(xì)胞系打下基礎(chǔ)。過表達(dá)突變型細(xì)胞系可供研究AJUBA突變,作為進(jìn)一步體外體內(nèi)實(shí)驗(yàn)的基礎(chǔ)。過表達(dá)野生型及突變型AJUBA的KYSE450細(xì)胞系具有更強(qiáng)的增殖能力。過表達(dá)野生型及突變型AJUBA的KYSE450細(xì)胞系具有更強(qiáng)的遷移和侵襲能力(除了突變2的侵襲能力),其中以過表達(dá)野生型AJUBA的細(xì)胞系增強(qiáng)的更為明顯。
[Abstract]:Background: esophageal cancer is the eighth largest tumor in the world, occupying the sixth place in the death caused by tumor. The majority of esophageal cancer is squamous cell carcinoma. However, the 5-year survival rate of esophageal squamous cell carcinoma is less than 15%, and the total survival time has not been significantly prolonged in recent years, which is related to the lack of effective treatment. With the development of high-throughput sequencing technique, AJUBA gene mutations were found in many esophageal squamous cell carcinoma cohorts, but the related studies were scarce. The molecular mechanism and clinical significance of AJUBA mutation in the carcinogenesis and development of esophageal squamous cell carcinoma were explored. It may improve the prognosis of patients and provide new therapeutic targets. Methods: five cohorts of esophageal squamous cell carcinoma were collected in this paper. All 545 patients had clinical information and total exon sequence data. The correlation between AJUBA mutation and TNM staging was analyzed statistically. The Kaplan-Meier curves of AJUBA mutation and wild-type patients were drawn, and the differences between the two were compared by Log-Rank, in order to obtain the clinical significance of the possible AJUBA mutation and to guide the further exploration of the mechanism and clinical application. At the same time, using 293T cells to package lentivirus, lentivirus infected target cells to construct stable cell lines for studying AJUBA mutation, including stable knockdown cell line, overexpression of wild-type AJUBA cell line and over-expression mutant cell line. By flow cytometry, hygromycin / purine mycin was screened by RT-PCR and Western blotting. On the basis of the constructed cell line, CCK-8 transwell cell function test was carried out to verify the relationship between AJUBA mutation and cell proliferation, migration and invasion in vitro. Results: there was no significant difference in the TNM stages of AJUBA mutation and wild type (Fisher exact probability p1.000) among 545 patients with AJUBA mutation, the Kaplan-Meier curve of AJUBA mutation had a longer survival time (Log-Rank test p0.13). The stable knockdown cell line was constructed and screened by hygromycin / flow fluorescence sorting on 10 days after infection. RT-PCR and Western blotting showed that the knockdown efficiency was not high but the knockdown efficiency was not high. Overexpression wild-type and mutant cell lines were constructed and screened by purine mycin. The results showed that the wild-type AJUBA bands and truncated mutant AJUBA bands were successfully constructed. The proliferative ability of overexpression cell line and wild type cell line was compared by CCK-8. It was found that the proliferation ability of the three overexpression cell lines was stronger than that of wild type cell lines (t test 48h: p0.001p0.001, p0.001; 72h: p0.008, p0.001, p0.001) .Transwell experiment was used to compare the migration and invasion ability between overexpression cell lines and wild type cell lines. The migration ability of three overexpression cell lines was stronger than that of wild type cell lines (t test: p0.001 + p0.001p0.001p0.001), and the invasion ability of overexpression of wild type AJUBA and mutant 1 was stronger than that of wild type cell lines (t test: 0. 001 p0. 001 p0. 035 p0. 576), among them, overexpression of wild type AJUBA cell line was stronger than that of wild type AJUBA cell line. And the most significant enhancement of invasion ability. Conclusion the prognosis of the esophageal squamous cell carcinoma with the mutation of: AJUBA is better than that of the wild type of AJUBA. The construction of stable knockdown cell line could be used as a control for further study and lay a foundation for the construction of mutant AJUBA cell line after knockdown. Overexpression mutant cell line can be used to study AJUBA mutation as a basis for further in vitro experiments. Overexpression of wild-type and mutant AJUBA KYSE450 cell lines have stronger proliferative ability. Overexpression of wild-type and mutant AJUBA in KYSE450 cell lines had stronger migration and invasion ability (except for the invasion ability of mutant 2), especially in the over-expression of wild-type AJUBA cell lines.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.1

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