GSK-3β抑制劑增強(qiáng)前列腺癌22RV1細(xì)胞化療敏感性的體外研究
發(fā)布時(shí)間：2019-01-07 20:15
【摘要】：[目的]在體外對(duì)GSK-3β抑制劑調(diào)控DTX誘導(dǎo)的前列腺癌22RV1細(xì)胞化療敏感性的作用機(jī)制進(jìn)行初步研究。 [方法] 1.DTX聯(lián)合GSK-3β抑制劑對(duì)前列腺癌細(xì)胞的毒性作用 22RV1細(xì)胞經(jīng)DTX單獨(dú)處理或聯(lián)合GSK-3β抑制劑LiCl處理后,經(jīng)結(jié)晶紫染色分析細(xì)胞毒性作用。 2. GSK-3β抑制劑對(duì)DTX誘導(dǎo)的細(xì)胞自噬活性的調(diào)控 22RV1細(xì)胞經(jīng)DTX單獨(dú)處理或聯(lián)合GSK-3β抑制劑處理后,細(xì)胞經(jīng)NP40液裂解,Western Blot檢測(cè)自噬相關(guān)蛋白表達(dá)(LC-3、Beclin-1, ATG14L, ATG5及Bif-1蛋白等)及GSK-3β活性(總GSK-3β蛋白、pGSK-3β-S9磷酸化蛋白)。 3.GSK-3p抑制劑對(duì)自噬復(fù)合體與下游分子(ATG14L或Rubicon)結(jié)合的調(diào)控 22RV1細(xì)胞經(jīng)DTX單獨(dú)或聯(lián)合GSK-3β抑制劑處理,收集細(xì)胞經(jīng)NP40液裂解后,細(xì)胞裂解液分別通過(guò)Beclin-1抗體免疫沉淀,蛋白A/G瓊脂糖孵育、洗脫后,4×SDS上樣緩沖液重懸,通過(guò)ATG14L及Rubicon抗體Western Blot檢測(cè),明確各處理組VPS34/Beclin-1自噬復(fù)合體與上述蛋白分子的結(jié)合情況。 [結(jié)果]在DTX基礎(chǔ)上聯(lián)合應(yīng)用LiCl,可以下調(diào)GSK-3β活性；上調(diào)自噬促進(jìn)蛋白ATG14L, Beclin-1水平,下調(diào)自噬抑制蛋白R(shí)ubicon水平；上調(diào)自噬促進(jìn)復(fù)合物beclin-1/ATG14的形成,下調(diào)自噬抑制復(fù)合物beclin-1/Rubicon的形成。 [結(jié)論]GSK-3β抑制劑Lic1可以通過(guò)上調(diào)DTX誘導(dǎo)的前列腺癌細(xì)胞自噬水平,促進(jìn)自噬性死亡,提高化療敏感性。
[Abstract]:[objective] to study the mechanism of GSK-3 尾 inhibitor regulating chemosensitivity of prostate cancer 22RV1 cells induced by DTX in vitro. [methods] the toxic effects of 1.DTX combined with GSK-3 尾 inhibitor on prostate cancer cells were analyzed by crystal violet staining after 22RV1 cells were treated with DTX alone or GSK-3 尾 inhibitor LiCl. 2. Regulation of GSK-3 尾 inhibitor on DTX induced autophagy activity of 22RV1 cells treated with DTX alone or in combination with GSK-3 尾 inhibitor, the expression of autophagy associated protein (LC-3,Beclin-1, ATG14L,) was detected by NP40 cleavage, Western Blot. ATG5 and Bif-1 protein) and GSK-3 尾 activity (total GSK-3 尾 protein, pGSK-3 尾 -S9 phosphorylated protein). Regulation of autophagy complex combined with downstream molecules (ATG14L or Rubicon) by 3.GSK-3p inhibitor, 22RV1 cells were treated with DTX alone or in combination with GSK-3 尾 inhibitor, and the collected cells were lysed in NP40 solution. The cell lysate was immunoprecipitated by Beclin-1 antibody and incubated with protein A / G agarose. After elution, the buffer on 4 脳 SDS was resuspended, and detected by ATG14L and Rubicon antibody Western Blot. To determine the binding of VPS34/Beclin-1 autophagy to the above protein molecules in each treatment group. [results] combined application of LiCl, on the basis of DTX could down-regulate the activity of GSK-3 尾, up-regulate the ATG14L, Beclin-1 level of autophagy promoter protein and down-regulate the Rubicon level of autophagy inhibitor protein. Upregulated autophagy promoted the formation of beclin-1/ATG14 and down-regulated the formation of autophagy inhibitory complex beclin-1/Rubicon. [conclusion] GSK-3 尾 inhibitor Lic1 can promote autophagic death and chemosensitivity by up-regulating DTX induced autophagy of prostate cancer cells.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25
本文編號(hào)：2404107
[Abstract]:[objective] to study the mechanism of GSK-3 尾 inhibitor regulating chemosensitivity of prostate cancer 22RV1 cells induced by DTX in vitro. [methods] the toxic effects of 1.DTX combined with GSK-3 尾 inhibitor on prostate cancer cells were analyzed by crystal violet staining after 22RV1 cells were treated with DTX alone or GSK-3 尾 inhibitor LiCl. 2. Regulation of GSK-3 尾 inhibitor on DTX induced autophagy activity of 22RV1 cells treated with DTX alone or in combination with GSK-3 尾 inhibitor, the expression of autophagy associated protein (LC-3,Beclin-1, ATG14L,) was detected by NP40 cleavage, Western Blot. ATG5 and Bif-1 protein) and GSK-3 尾 activity (total GSK-3 尾 protein, pGSK-3 尾 -S9 phosphorylated protein). Regulation of autophagy complex combined with downstream molecules (ATG14L or Rubicon) by 3.GSK-3p inhibitor, 22RV1 cells were treated with DTX alone or in combination with GSK-3 尾 inhibitor, and the collected cells were lysed in NP40 solution. The cell lysate was immunoprecipitated by Beclin-1 antibody and incubated with protein A / G agarose. After elution, the buffer on 4 脳 SDS was resuspended, and detected by ATG14L and Rubicon antibody Western Blot. To determine the binding of VPS34/Beclin-1 autophagy to the above protein molecules in each treatment group. [results] combined application of LiCl, on the basis of DTX could down-regulate the activity of GSK-3 尾, up-regulate the ATG14L, Beclin-1 level of autophagy promoter protein and down-regulate the Rubicon level of autophagy inhibitor protein. Upregulated autophagy promoted the formation of beclin-1/ATG14 and down-regulated the formation of autophagy inhibitory complex beclin-1/Rubicon. [conclusion] GSK-3 尾 inhibitor Lic1 can promote autophagic death and chemosensitivity by up-regulating DTX induced autophagy of prostate cancer cells.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 潘半舟;封冰;宋海珠;;自噬在調(diào)控抗腫瘤藥物耐藥中的研究進(jìn)展[J];醫(yī)學(xué)研究生學(xué)報(bào);2012年12期
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