本文選題：PC4 + 非同源末端連接�。� 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的:觀察轉(zhuǎn)錄輔助因子4(PC4)調(diào)控非同源末端連接修復(fù)在非小細(xì)胞肺癌放療中的作用,初步探討其產(chǎn)生作用的發(fā)生機(jī)制。方法:實(shí)驗(yàn)選取了4組不同的非小細(xì)胞肺癌細(xì)胞系進(jìn)行研究,采用四質(zhì)粒共轉(zhuǎn)染方法轉(zhuǎn)染293FT細(xì)胞,從而獲得慢病毒顆粒,經(jīng)過嘌呤霉素篩選,成功的構(gòu)建A549、PC-9干擾PC4表達(dá)、恢復(fù)PC4表達(dá)以及空載對照的穩(wěn)定株細(xì)胞(sh PC4、sh PC4+PC4、Mock)。設(shè)置0-8Gy(分別為0Gy、2Gy、4Gy、6Gy、8Gy)梯度劑量照射細(xì)胞。采用平板克隆實(shí)驗(yàn),檢測細(xì)胞在接受不同輻射劑量照射后,克隆形成能力的變化情況;采用流式細(xì)胞術(shù),檢測PC4不同表達(dá)狀態(tài)下,細(xì)胞接受輻射之后出現(xiàn)凋亡率變化的情況;Western Blot及PCR法觀察PC4對非同源末端連接(NHEJ)相關(guān)蛋白XLF、XRCC4、DNA LigaseⅣ表達(dá)的影響;利用免疫熒光技術(shù),觀察XLF與DNA雙鏈斷裂(DSB)共定位能力的變化及對其修復(fù)的影響;利用免疫共沉淀法(IP),檢測PC4與XLF相互作用及變化情況;通過染色質(zhì)免疫共沉淀法(CHIP),檢測PC4與XLF啟動(dòng)子結(jié)合能力的變化。結(jié)果:相較于支氣管上皮細(xì)胞Beas-2B而言,PC4在A549、PC-9、H1975、H460四組非小細(xì)胞肺癌細(xì)胞中高表達(dá)。干擾PC4的表達(dá)后,A549和PC-9細(xì)胞的克隆形成率降低,輻射敏感性提高,恢復(fù)PC4的表達(dá)后,細(xì)胞的克隆形成率也隨之升高,細(xì)胞對于輻射的敏感性下降。進(jìn)一步的研究發(fā)現(xiàn),干擾了PC4的表達(dá)后,輻射引起的細(xì)胞凋亡增加,此外,抑制了PC4表達(dá)后,XLF表達(dá)下降,且PC4與XLF的相互作用減少,而XRCC4以及DNA LigaseⅣ的表達(dá)未見明顯變化。免疫熒光結(jié)果顯示:細(xì)胞接受照射后,sh PC4組的XLF表達(dá)降低,同時(shí)DNA雙鏈斷裂修復(fù)減少,恢復(fù)PC4表達(dá)后,XLF表達(dá)以及DSB修復(fù)都得到了恢復(fù)。IP及CHIP結(jié)果顯示:PC4在轉(zhuǎn)錄水平調(diào)控XLF的表達(dá),敲低PC4表達(dá)后,PC4與XLF的相互作用減少。結(jié)論:下調(diào)PC4可在轉(zhuǎn)錄水平抑制XLF的表達(dá),通過減少非同源末端連接修復(fù)增加了輻射引起的凋亡,從而增強(qiáng)了非小細(xì)胞肺癌細(xì)胞的輻射敏感性,PC4可能通過參與NHEJ影響細(xì)胞對輻射的敏感性。
[Abstract]:Aim: to investigate the role of transcription cofactor 4 (PC4) in regulating the repair of nonhomologous terminal junctions in radiotherapy of non small cell lung cancer (NSCLC). Methods: four different groups of non-small cell lung cancer cell lines were selected and four plasmids were co-transfected into 293FT cells to obtain lentivirus particles. After purine mycin screening, A549 PC-9 interference PC4 expression was successfully constructed. The expression of PC4 was restored, and the stable cell line of no-load control, shPC4nsh PC4 PC4Mockanus, was restored. The cells were irradiated with a gradient dose of 0 ~ 8 Gy (0 Gy ~ 2 Gy ~ 4 Gy ~ 6 Gy ~ 8 Gy). The colony forming ability of cells was detected by plate cloning assay after irradiation with different doses of radiation. Flow cytometry was used to detect the expression of PC4 in different states. The changes of apoptosis rate after irradiation were observed by Western Blot and PCR to observe the effect of PC4 on the expression of XRCC4DNA Ligase 鈪，

本文編號：1945623