本文選題：皮膚鱗癌 + 跨膜接頭蛋白��； 參考：《中國(guó)癌癥雜志》2017年07期

【摘要】：背景與目的:跨膜接頭蛋白(Csk-binding protein,CBP)是新發(fā)現(xiàn)的Src家族成員,與多種腫瘤的發(fā)生有關(guān)。該研究旨在觀察CBP基因過(guò)表達(dá)對(duì)皮膚鱗癌細(xì)胞系A(chǔ)431增殖及細(xì)胞凋亡的影響,探討其相關(guān)的分子機(jī)制。方法:構(gòu)建CBP-增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)融合蛋白的慢病毒過(guò)表達(dá)載體,采用反轉(zhuǎn)錄病毒轉(zhuǎn)染的方法建立CBP過(guò)表達(dá)的A431細(xì)胞株。實(shí)驗(yàn)分為親本細(xì)胞組(未進(jìn)行基因轉(zhuǎn)染的A431細(xì)胞)、對(duì)照組(A431細(xì)胞轉(zhuǎn)染僅含EGFP陰性對(duì)照病毒)和實(shí)驗(yàn)組(A431細(xì)胞轉(zhuǎn)染CBP-EGFP病毒)。在激光共聚焦顯微鏡下觀察細(xì)胞轉(zhuǎn)染率,驗(yàn)證轉(zhuǎn)染成功與否;CCK-8法檢測(cè)CBP過(guò)表達(dá)對(duì)A431細(xì)胞增殖能力的影響,并采用流式細(xì)胞術(shù)(flow cytometry,FCM)檢測(cè)對(duì)細(xì)胞凋亡的影響;實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[質(zhì)]印跡法(Western blot)檢測(cè)Lck、Csk和Fyn三種上游信號(hào)轉(zhuǎn)導(dǎo)分子分別在m RNA和蛋白中的表達(dá)水平變化。結(jié)果:建立了穩(wěn)定過(guò)表達(dá)CBP的A431細(xì)胞株;CCK-8法結(jié)果提示,CBP過(guò)表達(dá)明顯抑制細(xì)胞生長(zhǎng),第2~6天的組間差異有統(tǒng)計(jì)學(xué)意義(P0.05);FCM檢測(cè)顯示,實(shí)驗(yàn)組細(xì)胞凋亡率顯著增加,與親本細(xì)胞組及對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.001);RTFQ-PCR結(jié)果顯示,實(shí)驗(yàn)組A431細(xì)胞Lck m RNA的相對(duì)表達(dá)水平顯著下調(diào)(P0.001),實(shí)驗(yàn)組細(xì)胞Csk和Fyn m RNA的表達(dá)分別約為親本細(xì)胞組的1.6倍和3.8倍,表達(dá)顯著上調(diào)(P0.001);Western blot結(jié)果表明,實(shí)驗(yàn)組Lck蛋白的相對(duì)表達(dá)水平明顯下降(P0.001),實(shí)驗(yàn)組細(xì)胞Csk和Fyn蛋白與親本細(xì)胞組和對(duì)照組相比表達(dá)明顯增加(P0.001)。結(jié)論:CBP過(guò)表達(dá)可抑制皮膚鱗癌細(xì)胞增殖,誘導(dǎo)細(xì)胞凋亡及壞死。CBP通過(guò)調(diào)節(jié)上游信號(hào)轉(zhuǎn)導(dǎo)通路中的蛋白酪氨酸激酶Lck、Csk和Fyn來(lái)調(diào)控細(xì)胞的增殖活性。
[Abstract]:Background & AIM: transmembrane junction protein Csk-binding protein (CBP) is a newly discovered member of Src family, which is involved in the development of many kinds of tumors. The aim of this study was to investigate the effects of CBP gene overexpression on the proliferation and apoptosis of cutaneous squamous cell carcinoma cell line A431, and to explore its molecular mechanism. Methods: the lentivirus overexpression vector of CBP- enhanced green fluorescent protein (EGFP) fusion protein was constructed, and a CBP overexpression A431 cell line was established by retrovirus transfection. The experiment was divided into parent cell group (A431 cells without gene transfection and control group with EGFP negative control virus only) and experimental group with CBP-EGFP virus transfection. The transfection rate of A431 cells was observed under confocal laser microscope, and the effect of CBP overexpression on the proliferation of A431 cells was detected by CCK-8 method, and the apoptosis was detected by flow cytometry (FCM). Real-time fluorescence quantitative polymerase chain reaction- PCR (RTFQ-PCR) and Western blot were used to detect the expression levels of LckCsk and Fyn in mRNA and protein, respectively. Results: CCK-8 assay was established for stable overexpression of CBP in A431 cell line. The results showed that the overexpression of CBP significantly inhibited the growth of A431 cells. The results of FCM analysis showed that the apoptosis rate of the experimental group was significantly higher than that of the control group. The results of RTFQ-PCR showed that the relative expression level of Lck mRNA in A431 cells of experimental group was significantly down-regulated, and the expression of Csk and Fyn mRNA in experimental group was about 1.6 times and 3.8 times as much as that in parent cell group, respectively. The results of Western blot showed that the relative expression level of Lck protein in the experimental group was significantly lower than that in the control group. The expression of Csk and Fyn protein in the experimental group was significantly higher than that in the parent cell group and the control group, and the expression of Csk and Fyn protein in the experimental group was significantly higher than that in the parent cell group and the control group. Conclusion the overexpression of CBP can inhibit the proliferation of skin squamous cell carcinoma cells, induce apoptosis and necrosis. CBP regulates the proliferation activity of the cells by regulating the protein tyrosine kinase LckCsk and Fyn in the upstream signal transduction pathway.
【作者單位】： 青島大學(xué)附屬醫(yī)院燒傷整形外科;青島大學(xué)醫(yī)學(xué)院免疫教研室;
【分類(lèi)號(hào)】：R739.5
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本文編號(hào)：2029911