我們前期研究發(fā)現(xiàn)血清和糖皮質(zhì)激素調(diào)節(jié)激酶3（Serum and glucocorticoid kinase 3,SGK3）能夠促進(jìn)肝癌細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化（epithelial-mesenchymal transition,EMT）進(jìn)而促進(jìn)肝癌細(xì)胞的侵襲和遷移,但其是否參與調(diào)控肝癌干細(xì)胞干性還不清楚。PI3K信號(hào)通路（通常指I型PI3K）異常調(diào)節(jié)在各種惡性腫瘤中非常普遍,Akt一直被認(rèn)為是PI3K信號(hào)通路中至關(guān)重要的中轉(zhuǎn)信號(hào)分子,近年來,有研究證實(shí)SGK3是I型PI3K通路下游不依賴Akt的獨(dú)立調(diào)控分子,而SGK3在I型PI3K信號(hào)通路中的作用及機(jī)制仍未闡明。Ⅲ型PI3K（PIK3C3）又被稱作Vps34,在細(xì)胞自噬和胞吞過程中發(fā)揮重要作用。PIK3C3還是生成PtdIns（3）P的重要場(chǎng)所,PtdIns（3）P是細(xì)胞內(nèi)的重要第二信使,能夠激活含PX和FYVE結(jié)構(gòu)域的蛋白質(zhì)。有報(bào)道稱,PIK3C3在結(jié)腸癌、乳腺癌等多種腫瘤的發(fā)生發(fā)展過程中發(fā)揮重要作用,但是其是否參與了腫瘤干細(xì)胞的調(diào)控還未見相關(guān)報(bào)道。第一部分抑制PI3K對(duì)肝癌干細(xì)胞自我更新的影響及機(jī)制研究目的:探究SGK3在肝癌干細(xì)胞自... 

【文章來源】：重慶醫(yī)科大學(xué)重慶市

【文章頁數(shù)】：103 頁

【學(xué)位級(jí)別】：博士

【部分圖文】：

磁珠分選細(xì)胞分選架組裝示意

圖 1. SGK3 在肝癌干細(xì)胞中磷酸化程度明顯增高Fig 1. SGK3 is preferentially activated in liver CSCs(A) MHCC-97H cells were cultured in monolayer or ultra-low attachment conditions. ThemRNA expression of liver CSC-related genes and SGK3 in spheroids and attached cells wascompared by RT-PCR. (B) Western blot analysis for levels of CD133, active Akt (phosphorylated atSer473)/total Akt and active SGK3 (phosphorylated at Thr320)/total SGK3 between spheroids andattached cells. (C) Flow cytometry analysis of CD133+ cell distribution in CD133- and CD133+cells isolated using CD133 MicroBead Kit. (D) Representative images of CD133+ and CD133- cellssorted from MHCC97H HCC cells cultured in serum-free culture medium after 7 days. Scale bars,100 μm. (E) CD133+/CD133- cells were subcutaneously injected into 6-week-old female athymicnude mice, and tumourigenicity was evaluated 5 weeks post-inoculation. (F) Levels of CD133,active Akt (phosphorylated at Ser473)/total Akt and active SGK3 (phosphorylated at Thr320)/total

重慶醫(yī)科大學(xué)博士研究生學(xué)位論文SGK3 were compared by western blot analysis between CD133+ and CD133- cells. β-actin was usedas a loading control. All experiments were performed in triplicate. *P < 0.05.2.2 SGK3 促進(jìn)肝癌干細(xì)胞自我更新為了深入探究 SGK3 對(duì)肝癌干細(xì)胞自我更新的調(diào)控作用，我們使用慢病毒載體轉(zhuǎn)染 MHCC97H 和 Huh7 細(xì)胞并構(gòu)建了 SGK3 穩(wěn)定過表達(dá)細(xì)胞株。如圖 2.A 和 B所示，慢病毒轉(zhuǎn)染成功后 SGK3 在 MHCC97H 和 Huh7 細(xì)胞中明顯過表達(dá)，并且SGK3 過表達(dá)明顯地促進(jìn)了肝癌干細(xì)胞相關(guān)基因 CD133、CD90、Oct4、Nanog、Bmi-1 和 Sox2 的表達(dá)。我們?cè)诘鞍姿津?yàn)證了 SGK3 對(duì)干細(xì)胞相關(guān)基因表達(dá)的促進(jìn)作用（圖 2.C）。為了進(jìn)一步證實(shí) SGK3 對(duì)肝癌干細(xì)胞自我更新的影響，我們檢測(cè)了兩組細(xì)胞的成球能力，發(fā)現(xiàn) SGK3 過表達(dá)明顯促進(jìn)肝癌細(xì)胞的成球能力（圖2.D）。

【參考文獻(xiàn)】：
期刊論文
[1]Cancer stem cell plasticity and tumor hierarchy[J]. Marina Carla Cabrera,Robert E Hollingsworth,Elaine M Hurt.  World Journal of Stem Cells. 2015(01)



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