本文選題：小檗堿 + 急性淋巴細(xì)胞白血病�。� 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分小檗堿誘導(dǎo)兒童p53基因缺失白血病細(xì)胞凋亡的實(shí)驗(yàn)研究 目的腫瘤細(xì)胞耐藥多由于p53基因異常所致,小檗堿能夠誘導(dǎo)多種腫瘤細(xì)胞發(fā)生細(xì)胞周期阻滯及細(xì)胞凋亡。本研究主要探討小檗堿對兒童p53基因缺失的急性淋巴細(xì)胞白血病EU-4細(xì)胞凋亡誘導(dǎo)作用及其作用靶點(diǎn)。 方法選取p53基因缺失的EU-4細(xì)胞進(jìn)行實(shí)驗(yàn),具體如下： (1)終濃度為0、1、10、50、100μmol/L小檗堿作用EU-4細(xì)胞72h,采用CCK-8檢測細(xì)胞增殖,流式細(xì)胞儀檢測細(xì)胞凋亡,熒光顯微鏡觀察凋亡細(xì)胞,免疫印跡方法(Western Blot)檢測Bcl-2基因家族蛋白(BaxBcl-xl)及X連鎖凋亡抑制蛋白(XIAP)的表達(dá),比色法檢Caspase-3的活性變化； (2)終濃度為100μmol/L小檗堿分別作用EU-4細(xì)胞0、24、48、72h, Western Blot檢測PARP、Caspase-3及XIAP蛋白表達(dá)； (3)終濃度為50μmol/L Z-VAD-FMK預(yù)處理EU-4細(xì)胞30min,使用流式細(xì)胞儀觀察小檗堿誘導(dǎo)EU-4細(xì)胞凋亡的變化； (4)終濃度為0、0.1、0.5、1.0μmol/L阿霉素作用EU-4細(xì)胞48h,Western Blot檢測鼠雙微粒體2(MDM2)和XIAP蛋白的變化； (5)使用小干擾RNA(siRNA)或小檗堿下調(diào)XIAP蛋白的表達(dá),觀察單純降低XIAP表達(dá)或者聯(lián)合阿霉素對于EU-4細(xì)胞凋亡的影響。 結(jié)果(1)小檗堿作用EU-4細(xì)胞后,細(xì)胞凋亡率顯著升高(P0.05),并呈濃度依賴關(guān)系,凋亡相關(guān)蛋白表達(dá)出現(xiàn)相應(yīng)變化； (2)小檗堿誘導(dǎo)EU-4細(xì)胞凋亡主要依賴Caspase的活性,并且Caspase-3的活性顯著增強(qiáng)(P0.05)； (3)小檗堿以濃度和時(shí)間依賴的方式下調(diào)XIAP蛋白的表達(dá)； (4)阿霉素?zé)o明顯誘導(dǎo)EU-4細(xì)胞凋亡的作用,MDM2及XIAP蛋白表達(dá)亦無明顯變化； (5)單純下調(diào)XIAP蛋白的表達(dá)引起EU-4細(xì)胞明顯凋亡,與阿霉素共同作用后還能夠增強(qiáng)EU-4細(xì)胞對于阿霉素的敏感性(P0.05)。 結(jié)論小檗堿能夠以不依賴p53的方式誘導(dǎo)白血病EU-4細(xì)胞凋亡,XIAP作為小檗堿的作用靶點(diǎn),參與了EU-4細(xì)胞的凋亡。 第二部分小檗堿調(diào)控白血病細(xì)胞X連鎖凋亡抑制蛋白表達(dá)的機(jī)制研究 目的小檗堿能夠以x連鎖凋亡抑制蛋白(XIAP)為作用靶點(diǎn)誘導(dǎo)白血病細(xì)胞凋亡。本研究主要探討小檗堿調(diào)控兒童急性淋巴細(xì)胞白血病EU-4細(xì)胞中XIAP蛋白表達(dá)的具體分子機(jī)制。 方法(1)終濃度為100μmol/L小檗堿分別作用EU-4細(xì)胞0、24、48、72h,采用聚合酶鏈反應(yīng)(PCR)檢鋇XIAPmRNA變化；放線菌素D實(shí)驗(yàn)觀察小檗堿對于XIAPmRNA穩(wěn)定性的影響； (2)終濃度為0、1、10、50、100μmol/L小檗堿作用EU-4細(xì)胞72h,免疫印跡方法(Western Blot)檢測細(xì)胞中鼠雙微粒體2(MDM2)及磷酸化MDM2蛋白量以及亞細(xì)胞定位的變化；終濃度為100μmol/L小檗堿分別作用EU-4細(xì)胞0、24、48、72h,PCR檢鋇MDM2mRNA變化； (3)使用小干擾RNA(siRNA)下調(diào)EU-4細(xì)胞中MDM2蛋白的表達(dá),Western Blot檢測降低MDM2表達(dá)對于XIAP蛋白的影響； (4)采用放線菌酮實(shí)驗(yàn)觀察小檗堿對于XIAP蛋白穩(wěn)定性的影響,免疫共沉淀實(shí)驗(yàn)檢測小檗堿對于XIAP蛋白泛素化的影響。 結(jié)果(1)小檗堿不影響)IAP mRNA表達(dá)量及其穩(wěn)定性； (2)小檗堿以濃度和時(shí)間依賴的方式下調(diào)MDM2蛋白表達(dá),并且該調(diào)控可能發(fā)生在轉(zhuǎn)錄水平,小檗堿能夠降低胞漿中MDM2蛋白水平； (3)siRNA下調(diào)MDM2表達(dá)以后引起XIAP蛋白表達(dá)的降低； (4)小檗堿增高XIAP蛋白自身的泛素化水平,降低XIAP蛋白的穩(wěn)定性,使其通過泛素-蛋白酶體途徑降解。 結(jié)論小檗堿主要在轉(zhuǎn)錄后水平調(diào)控EU-4細(xì)胞中XIAP蛋白的表達(dá),即小檗堿可能通過下調(diào)MDM2蛋白表達(dá)影響XIAP蛋白翻譯,小檗堿誘導(dǎo)XIAP蛋白通過泛素-蛋白酶體途徑降解。
[Abstract]:Experimental study on the apoptosis of p53 gene - deleted leukemia cells induced by berberine in the first part

Objective To investigate the effect of berberine on cell cycle arrest and apoptosis in various tumor cells due to the abnormal p53 gene .

Methods EU - 4 cells with p53 gene deletion were selected for the experiment , which was as follows :

( 1 ) At the end concentration of 0 , 1 , 10 , 50 and 100 渭mol / L berberine for 72h , CCK - 8 was used to detect cell proliferation , apoptosis was detected by flow cytometry , apoptotic cells were observed by fluorescence microscope , Western Blot was used to detect the expression of Bcl - 2 gene family protein ( Bax Bcl - xl ) and X - linked apoptosis protein ( XIAP ) , and the activity of Caspase - 3 was detected by colorimetry .


( 2 ) At the final concentration of 100 渭mol / L berberine , the expressions of parp , Caspase - 3 and XIAP were detected by Western Blot in the presence of 0 , 24 , 48 , 72 h of EU - 4 cells .


( 3 ) The final concentration was 50 渭mol / L Z - VAD - FMK pretreatment of EU - 4 cells for 30 min , and the apoptosis of EU - 4 cells induced by berberine was observed by flow cytometry .


( 4 ) At the final concentration of 0 , 0.1 , 0.5 and 1.0 渭mol / L for 48h , Western Blot was used to detect the changes of the mouse ' s double microsome 2 and XIAP proteins .


( 5 ) The expression of XIAP protein was downregulated by small interfering RNA ( siRNA ) or berberine , and the effect of XIAP expression or combination of adriamycin on the apoptosis of EU - 4 cells was observed .

Results ( 1 ) After the action of berberine on EU - 4 cells , the apoptosis rate was significantly increased ( P0.05 ) .


( 2 ) The apoptosis of EU - 4 cells induced by berberine mainly depended on the activity of Caspase - 3 , and the activity of Caspase - 3 was significantly increased ( P0.05 ) .


( 3 ) berberine downregulates the expression of XIAP protein in a concentration and time - dependent manner ;


( 4 ) There was no obvious effect of adriamycin on the apoptosis of EU - 4 cells .


( 5 ) The apoptosis of EU - 4 cells was induced by downregulating the expression of XIAP protein , and the sensitivity of EU - 4 cells to adriamycin ( P0.05 ) could be enhanced .

Conclusion berberine can induce apoptosis of leukemia EU - 4 cells in a manner independent of p53 , XIAP plays a role as a target for berberine , and is involved in the apoptosis of EU - 4 cells .

Study on the mechanism of the second part berberine in regulating the expression of X - linked apoptosis protein in leukemia cells

Objective To investigate the specific molecular mechanism of berberine in controlling XIAP protein expression in children with acute lymphoblastic leukemia ( EU - 4 ) .

Methods ( 1 ) The final concentration of berberine ( 100 渭mol / L ) was 0 , 24 , 48 and 72 h in EU - 4 cells , and the changes of XIAP mRNA were detected by polymerase chain reaction ( PCR ) .
Effect of berberine on the stability of XIAP mRNA was observed by actinomycin D experiment .


( 2 ) At the final concentration of 0 , 1 , 10 , 50 and 100 渭mol / L berberine for 72 h , Western Blot was used to detect the amount of mouse double microsome 2 and phosphorylates the protein and the changes of sub - cell localization in the cells .
The final concentration was 100 渭mol / L berberine in the presence of 0 , 24 , 48 , 72 h of EU - 4 cells , and the mRNA of MDM2mRNA was detected by PCR .


( 3 ) Using siRNA to downregulate the expression of the protein in the EU - 4 cells , Western Blot was used to detect the effect of the expression of p53 on the expression of XIAP protein .


( 4 ) The effect of berberine on the stability of XIAP protein was observed by means of the experiment of actinomycin , and the effect of berberine on the ubiquitination of XIAP protein was detected by immune co - precipitation experiment .

Results ( 1 ) The expression of IAP mRNA and its stability were not affected by berberine .


( 2 ) berberine down - regulates the protein expression in the concentration and time - dependent manner , and the regulation may occur at the transcription level , and berberine can reduce the level of the protein in the cytoplasm ;


( 3 ) siRNA down - regulates the expression of XIAP protein after down - regulation of the protein ;


( 4 ) berberine increases the ubiquitination level of XIAP protein itself , reduces the stability of XIAP protein , and causes it to be degraded by ubiquitin - proteasome pathway .

Conclusion The expression of XIAP protein in EU - 4 cells is regulated by berberine mainly after transcription , that is , berberine may affect XIAP protein translation by down - regulation of the protein expression , and berberine - induced XIAP protein can be degraded by ubiquitin - proteasome pathway .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R733.71

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本文編號(hào)：1909395