【摘要】：背景:研究表明,WWOX能夠影響卵巢癌干細(xì)胞的生長(zhǎng),其作用機(jī)制是否與Hedgehog信號(hào)通路有關(guān)則未見(jiàn)相關(guān)報(bào)道。目的:研究過(guò)表達(dá)WWOX對(duì)卵巢癌干細(xì)胞凋亡的作用及機(jī)制。方法:轉(zhuǎn)染W(wǎng)WOX過(guò)表達(dá)載體pcDNA3.1-WWOX(pcDNA3.1-WWOX組)和pcDNA4.0-WWOX(pcDNA4.0-WWOX組)至卵巢癌干細(xì)胞中,同時(shí)轉(zhuǎn)染空載體pcDNA3.1(pcDNA3.1組)和pcDNA4.0(pcDNA4.0組),設(shè)置未轉(zhuǎn)染組(只加入轉(zhuǎn)染試劑Lipofectamine2000)。培養(yǎng)48 h后,Western blot檢測(cè)細(xì)胞中WWOX水平,MTT檢測(cè)細(xì)胞增殖情況,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況,Western blot檢測(cè)細(xì)胞中Hedgehog信號(hào)通路相關(guān)蛋白SHH、PTCH1、Gli-1、SMO和凋亡相關(guān)蛋白Cleaved Caspase-3的表達(dá)水平。用Hedgehog信號(hào)通路抑制劑環(huán)巴胺作用于未轉(zhuǎn)染的卵巢癌干細(xì)胞(環(huán)巴胺組)和轉(zhuǎn)染W(wǎng)WOX過(guò)表達(dá)載體的卵巢癌干細(xì)胞(WWOX+環(huán)巴胺組),培養(yǎng)48 h后,再進(jìn)行MTT、流式細(xì)胞術(shù)、Western blot檢測(cè)。結(jié)果與結(jié)論:①pcDNA4.0-WWOX組細(xì)胞中WWOX表達(dá)水平明顯高于pcDNA3.1-WWOX組(t=27.84,P=0.00)。后期選用轉(zhuǎn)染pcDNA4.0-WWOX的卵巢癌干細(xì)胞過(guò)表達(dá)WWOX;②過(guò)表達(dá)WWOX能夠抑制卵巢癌干細(xì)胞增殖,促進(jìn)卵巢癌干細(xì)胞凋亡;③過(guò)表達(dá)WWOX能夠抑制卵巢癌干細(xì)胞中SHH、PTCH1、Gli-1、SMO表達(dá),促進(jìn)Cleaved Caspase-3表達(dá);④環(huán)巴胺能夠抑制SHH、PTCH1、Gli-1、SMO表達(dá),促進(jìn)Cleaved Caspase-3表達(dá)。環(huán)巴胺對(duì)Hedgehog信號(hào)通路具有明顯的抑制作用;⑤環(huán)巴胺能夠增強(qiáng)過(guò)表達(dá)WWOX誘導(dǎo)的卵巢癌干細(xì)胞凋亡,增強(qiáng)過(guò)表達(dá)WWOX對(duì)卵巢癌干細(xì)胞增殖的抑制作用;⑥結(jié)果表明,WWOX對(duì)卵巢癌干細(xì)胞的增殖抑制作用和促凋亡作用可能與抑制Hedgehog信號(hào)通路有關(guān)。
[Abstract]:Background: studies have shown that WWOX can affect the growth of ovarian cancer stem cells and whether its mechanism is related to the Hedgehog signaling pathway has not been reported. Objective: to study the effect and mechanism of overexpression of WWOX on apoptosis of ovarian cancer stem cells. Methods: WWOX overexpression vectors pcDNA3.1-WWOX (pcDNA3.1-WWOX group) and pcDNA4.0-WWOX (pcDNA4.0-WWOX group) were transfected into ovarian cancer stem cells, and the empty vectors pcDNA3.1 (pcDNA3.1 group) and pcDNA4.0 (pcDNA4.0 group) were transfected. After 48 h culture, the level of WWOX in cells was detected by Western blot, and the cell proliferation was detected by flow cytometry. The expression of Hedgehog signal transduction related protein SHH,PTCH1,Gli-1,SMO and apoptosis-related protein Cleaved Caspase-3 was detected by flow cytometry and Western blot was used to detect the expression of Hedgehog signal pathway related protein SHH,PTCH1,Gli-1,SMO and apoptosis-related protein Cleaved Caspase-3. Cyclobaramine, a signal pathway inhibitor of Hedgehog, was used to treat the untransfected ovarian cancer stem cells (cyclophosphamide group) and ovarian cancer stem cells transfected with WWOX overexpression vector (WWOX cyclophosphamide group). After 48 hours of culture, MTT, flow cytometry was used to detect the results of MTT, blot. Results and conclusion the expression of WWOX in the cells of the pcDNA4.0-WWOX group was significantly higher than that in the pcDNA3.1-WWOX group. The over-expression of WWOX;2 and WWOX in ovarian cancer stem cells transfected with pcDNA4.0-WWOX in later stage could inhibit the proliferation of ovarian cancer stem cells, and promote the apoptosis of ovarian cancer stem cells. The overexpression of WWOX could inhibit the expression of SHH,PTCH1,Gli-1,SMO and promote the expression of Cleaved Caspase-3 in ovarian cancer stem cells. 4 Cyclobaramine could inhibit the expression of SHH,PTCH1,Gli-1,SMO and promote the expression of Cleaved Caspase-3. Cyclobaramine has a significant inhibitory effect on Hedgehog signaling pathway. Cyclobaramine can enhance apoptosis of ovarian cancer stem cells induced by overexpression of WWOX and inhibit proliferation of ovarian cancer stem cells induced by overexpression of WWOX. The results suggested that the inhibitory effect of WWOX on the proliferation and apoptosis of ovarian cancer stem cells may be related to the inhibition of Hedgehog signaling pathway.
【作者單位】： 山東大學(xué)臨床醫(yī)學(xué)院;山東大學(xué)齊魯醫(yī)院婦產(chǎn)科;濱州醫(yī)學(xué)院消化內(nèi)科;
【分類號(hào)】：R737.31

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