目的:B7-H4和自噬可以通過PI3K信號通路調(diào)節(jié)或誘導(dǎo)。然而,在肝細(xì)胞癌中B7-H4與自噬的關(guān)系仍未清楚。本課題的目的是研究肝細(xì)胞癌中B7-H4是否通過PI3K信號通路調(diào)節(jié)自噬。方法:siRNA敲低Huh7和Hep3B細(xì)胞B7-H4后,采用CCK-8法檢測細(xì)胞增殖,流式細(xì)胞術(shù)檢測細(xì)胞凋亡,western blot檢測cleaved-caspase3、cleaved-PARP、BAX、BCL-2、LC3、P62、P-AKT、P-PI3K、P-m TOR和總AKT、PI3K、m TOR蛋白水平,MDC法檢測自噬體。結(jié)果:敲低Huh7和Hep3B細(xì)胞B7-H4后,siRNA組與NC組相比,細(xì)胞增殖減少,細(xì)胞凋亡率增加;凋亡相關(guān)指標(biāo)cleaved-caspase3、cleaved-PARP、BAX蛋白增加,P62蛋白降低;自噬相關(guān)指標(biāo)LC3Ⅱ增加,Bcl2蛋白降低;MDC法可見siRNA組更多自噬體;AKT、PI3K、m TOR的磷酸化被抑制;siRNA組和NC組分別使用CQ抑制LC3降解后發(fā)現(xiàn)LC3Ⅱ仍然增加,使用3MA抑制PI3K后,發(fā)現(xiàn)細(xì)胞凋亡和自噬減弱。結(jié)論:B7-H4通過PI3K信... 

【文章來源】：重慶醫(yī)科大學(xué)重慶市

【文章頁數(shù)】：37 頁

【學(xué)位級別】：碩士

【部分圖文】：

7-H4表達(dá)下調(diào)抑制HCC細(xì)胞增殖Figure1AandB.AftersiRNAinterference,theexpressionofB7-H4RNAlevelwas

重慶醫(yī)科大學(xué)碩士研究生學(xué)位論文153.2B7-H4表達(dá)下調(diào)促進(jìn)HCC細(xì)胞凋亡用流式細(xì)胞儀檢測Huh7和Hep3B細(xì)胞凋亡，發(fā)現(xiàn)siB7-H4作用后，細(xì)胞凋亡率增加。siRNA組較NC組凋亡存在差異顯著Huh7:6.11%vs2.96%,P<0.05;Hep3B:7.25%vs3.54%,P<0.05。在C-D圖中，siB7-H4組較NC組凋亡相關(guān)蛋白cleavedcaspase3、cleavedPARP與BAX增加，BCL-2減少。ABCD圖2B7-H4表達(dá)下調(diào)促進(jìn)HCC細(xì)胞凋亡Figure2CellapoptosisratewasdeterminedbyflowcytometrywithAnnexinV-FITC(A-B).Theassayswererepeatedthrice,andillustratedasthemean±SD.**siRNAvsNC,P<0.05,***P<0.05.Expressionchangeofapoptosis-relatedgenesdetectedbywesternblotanalysisinHuh7andHep3BcellsafterB7-H4siRNAtreatment(CandD).*siRNAvsNC,P<0.05,**P<0.05,***P<0.05.

重慶醫(yī)科大學(xué)碩士研究生學(xué)位論文17圖3B7-H4表達(dá)下調(diào)促進(jìn)HCC細(xì)胞自噬Figure3Expressionchangeofautophagy-relatedgenesdetectedbywesternblotanalysisinHuh7andHep3BcellsafterB7-H4siRNAtreatment(A-B).*siRNAvsNC,P<0.05,**P<0.05,***P<0.05.Expressionchangeofautophagy-relatedgenesdetectedbywesternblotanalysisinHuh7andHep3BcellsaftersiRNAand/orchloroquine(CQ)treatment(C-F).MDCstainingobservedbyfluorescencemicroscopeshowedthepresenceofalargenumberofautophagosomesinB7-H4(G).*siRNAvsNC,P<0.05,**P<0.05,***P<0.05.3.4B7-H4調(diào)節(jié)HCC細(xì)胞中的AKT/PI3K/mTOR途徑用siRNA抑制B7-H4表達(dá)后，p-AKT,p-PI3Kandp-mTOR表達(dá)水平減少，而且，p-AKT/AKT,p-PI3K/PI3Kandp-mTOR/mTOR與NC組相比顯著減少。AB圖4B7-H4表達(dá)下調(diào)引起HCC細(xì)胞中的AKT/PI3K/mTOR改變Figure4ExpressionchangeofPI3K/AKT/mTORsignalingpathwayinHuh7andHep3BcellsafterB7-H4siRNAtreatment.**siRNAvsNC,P<0.05.3.5B7-H4調(diào)節(jié)自噬與凋亡通過PI3K信號通路

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期刊論文
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