小檗碱在儿童耐药急性淋巴细胞白血病靶向治疗中的作用机制研究

发布时间：2018-05-19 08:24

本文选题：小檗碱 + 急性淋巴细胞白血病　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分小檗碱诱导儿童p53基因缺失白血病细胞凋亡的实验研究目的肿瘤细胞耐药多由于p53基因异常所致,小檗碱能够诱导多种肿瘤细胞发生细胞周期阻滞及细胞凋亡。本研究主要探讨小檗碱对儿童p53基因缺失的急性淋巴细胞白血病EU-4细胞凋亡诱导作用及其作用靶点。方法选取p53基因缺失的EU-4细胞进行实验,具体如下： (1)终浓度为0、1、10、50、100μmol/L小檗碱作用EU-4细胞72h,采用CCK-8检测细胞增殖,流式细胞仪检测细胞凋亡,荧光显微镜观察凋亡细胞,免疫印迹方法(Western Blot)检测Bcl-2基因家族蛋白(BaxBcl-xl)及X连锁凋亡抑制蛋白(XIAP)的表达,比色法检Caspase-3的活性变化； (2)终浓度为100μmol/L小檗碱分别作用EU-4细胞0、24、48、72h, Western Blot检测PARP、Caspase-3及XIAP蛋白表达； (3)终浓度为50μmol/L Z-VAD-FMK预处理EU-4细胞30min,使用流式细胞仪观察小檗碱诱导EU-4细胞凋亡的变化； (4)终浓度为0、0.1、0.5、1.0μmol/L阿霉素作用EU-4细胞48h,Western Blot检测鼠双微粒体2(MDM2)和XIAP蛋白的变化； (5)使用小干扰RNA(siRNA)或小檗碱下调XIAP蛋白的表达,观察单纯降低XIAP表达或者联合阿霉素对于EU-4细胞凋亡的影响。结果(1)小檗碱作用EU-4细胞后,细胞凋亡率显著升高(P0.05),并呈浓度依赖关系,凋亡相关蛋白表达出现相应变化； (2)小檗碱诱导EU-4细胞凋亡主要依赖Caspase的活性,并且Caspase-3的活性显著增强(P0.05)； (3)小檗碱以浓度和时间依赖的方式下调XIAP蛋白的表达； (4)阿霉素无明显诱导EU-4细胞凋亡的作用,MDM2及XIAP蛋白表达亦无明显变化； (5)单纯下调XIAP蛋白的表达引起EU-4细胞明显凋亡,与阿霉素共同作用后还能够增强EU-4细胞对于阿霉素的敏感性(P0.05)。结论小檗碱能够以不依赖p53的方式诱导白血病EU-4细胞凋亡,XIAP作为小檗碱的作用靶点,参与了EU-4细胞的凋亡。第二部分小檗碱调控白血病细胞X连锁凋亡抑制蛋白表达的机制研究目的小檗碱能够以x连锁凋亡抑制蛋白(XIAP)为作用靶点诱导白血病细胞凋亡。本研究主要探讨小檗碱调控儿童急性淋巴细胞白血病EU-4细胞中XIAP蛋白表达的具体分子机制。方法(1)终浓度为100μmol/L小檗碱分别作用EU-4细胞0、24、48、72h,采用聚合酶链反应(PCR)检钡XIAPmRNA变化；放线菌素D实验观察小檗碱对于XIAPmRNA稳定性的影响； (2)终浓度为0、1、10、50、100μmol/L小檗碱作用EU-4细胞72h,免疫印迹方法(Western Blot)检测细胞中鼠双微粒体2(MDM2)及磷酸化MDM2蛋白量以及亚细胞定位的变化；终浓度为100μmol/L小檗碱分别作用EU-4细胞0、24、48、72h,PCR检钡MDM2mRNA变化； (3)使用小干扰RNA(siRNA)下调EU-4细胞中MDM2蛋白的表达,Western Blot检测降低MDM2表达对于XIAP蛋白的影响； (4)采用放线菌酮实验观察小檗碱对于XIAP蛋白稳定性的影响,免疫共沉淀实验检测小檗碱对于XIAP蛋白泛素化的影响。结果(1)小檗碱不影响)IAP mRNA表达量及其稳定性； (2)小檗碱以浓度和时间依赖的方式下调MDM2蛋白表达,并且该调控可能发生在转录水平,小檗碱能够降低胞浆中MDM2蛋白水平； (3)siRNA下调MDM2表达以后引起XIAP蛋白表达的降低； (4)小檗碱增高XIAP蛋白自身的泛素化水平,降低XIAP蛋白的稳定性,使其通过泛素-蛋白酶体途径降解。结论小檗碱主要在转录后水平调控EU-4细胞中XIAP蛋白的表达,即小檗碱可能通过下调MDM2蛋白表达影响XIAP蛋白翻译,小檗碱诱导XIAP蛋白通过泛素-蛋白酶体途径降解。
[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 .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R733.71

【参考文献】