【作者】 韦艳；

【导师】 梁钢；

【作者基本信息】 广西医科大学 ， 药理学， 2011， 硕士

【摘要】 目的在转录水平上建立抗肝癌及逆转MDR中药的多靶点高通量筛选方法。方法采用体外培养人肝癌细胞BEL-7402及其耐5-氟尿嘧啶细胞Bel-FU,MTT法检测Bel-FU的多药耐药性、5种中药成分的(槲皮素、EGCG、老鼠簕生物碱A、乙酰老鼠簕生物碱A、龙血素B)细胞敏感性以及药物浓度依赖的逆转耐药性;选择与肝癌发生发展相关途径的42个功能基因,采用实时定量PCR微阵列技术检测各中药成分作用于耐药细胞前后的差异表达基因,实时定量PCR检测槲皮素、EGCG、乙酰老鼠簕生物碱A分别作用于Bel-FU后H-ras、MDR1基因表达,以验证微阵列结果。结果Bel-FU对5-氟尿嘧啶、阿糖胞苷、长春新碱、阿霉素、柔红霉素的耐药指数分别为:89.6、3.4、37.2、16.5、3.7倍。槲皮素、EGCG、老鼠簕生物碱A、乙酰老鼠簕生物碱A、龙血素B对Bel-FU的IC50值分别为193.9μM、1230.8μM、507.8μM、244.9μM、163.7μM,对Bel-FU的IC10值分别为58.2μM、159.5μM、75.3μM、64.7μM、25.8μM。43.6、87.2、130.8μM的EGCG对Bel-FU的耐药逆转倍数分别为1.04、1.36、2.10倍;16.5、33、49.5μM的槲皮素对Bel-FU的耐药逆转倍数分别为1.14、1.68、2.38倍;13.2、33、66μM的老鼠簕生物碱A对Bel-FU的耐药逆转倍数分别为1.13、1.54、1.33倍;21、42、63μM的乙酰老鼠簕生物碱A对Bel-FU的耐药逆转倍数分别为1.26、1.78、1.42倍;6、12、24μM的龙血素B对细胞Bel-FU耐药逆转倍数分别为2.25、3.80、5.76倍;阳性对照剂VRP(5μg·mL-1)则为1.11倍。与Bel-7402比较,Bel-FU中的基因H-ras、Bcl-2、PDGFA、MDR1、BCRP表达上调(P<0.05),cyclinB1下调(P<0.05);槲皮素下调Bel-FU中基因H-ras、EGFR、FGF、VEGFA、PDGFA、MDR1、Erk1的表达(P<0.05);EGCG下调Bel-FU中基因H-ras、EGFR、PDGFA、MDR1、VEGFA的表达(P<0.05);乙酰老鼠簕生物碱A下调基因H-ras、VEGFA、PDGFA、C-myc、NF-κB的表达(P<0.05),老鼠簕生物碱A与龙血素B均下调Wnt1、VEGFA的表达(P<0.05)。实时定量PCR结果:槲皮素、EGCG均下调Bel-FU中基因H-ras、MDR1表达(P<0.05),乙酰老鼠簕生物碱A下调Bel-FU中基因H-ras的表达(P<0.05)。结论定量PCR微阵列可作为在转录水平上进行抗肝癌及逆转MDR中药多靶点高通量筛选的方法。目的通过筛选三种中药成分作用于人肝细胞癌MDR细胞前后表达差异的蛋白质点,在检测靶蛋白水平上建立抗肝癌及逆转MDR中药筛选方法。方法采用2-DE及MALDI-TOF-MS技术,检测肝癌敏感细胞Bel-7402与耐药细胞Bel-FU之间的差异蛋白以及三种中药成分作用于Bel-FU前后的差异蛋白,差异倍数为2.5倍以上者定义为差异蛋白。Western blot法检测三种中药成分作用于Bel-FU后蛋白P-gp、FAK、CRT表达。结果与Bel-7402比较, Bel-FU中有24个蛋白表达上调,其中包括FAK、TM3、ORP150、CRT、NSE、80K-H protein、EF-1-D、phosphoprotein等,12个蛋白表达下调;EGCG作用Bel-FU后,下调蛋白FAK、CRT、TM3、80K-H protein、EF-1-D的表达;槲皮素作用Bel-FU后,下调蛋白phosphoprotein、CRT、TM3、EF-1-D的表达;乙酰老鼠簕生物碱A作用Bel-FU后下调蛋白FAK、80K-H protein、CRT、TM3的表达。Western blot检测结果:EGCG、槲皮素均下调P-gp在Bel-FU中的表达(P < 0.05)。EGCG、乙酰老鼠簕生物碱A下调Bel-FU中的FAK的表达(P < 0.05)。经三种中药成分作用后,Bel-FU中的CRT表达均下调。结论EGCG、槲皮素、乙酰老鼠簕生物碱A均具有逆转Bel-FU MDR的作用。运用蛋白质组学技术检测与逆转MDR相关的蛋白质差异表达为MDR逆转剂筛选提供了一个高通量的平台。更多还原

【Abstract】 Objective To establish a high throughput and multi-target method for screening traditional Chinese medicine as to anti-hepatocellular carcinoma and reversing MDR in the course of genetic transcription .Methods Human hepatocellular carcinoma cell line Bel-7402 and 5-FU resistant cell line Bel-FU were cultured in vitro. The multidrug resistance(MDR) of Bel-FU ,drug sensitivity of 5 kinds of traditional Chinese medicine(quercetin,EGCG,IA , ACO-IA,loureirin B)to Bel-FU and their dose-dependen reversal effects were determined by Methyl thiazolyl tetrazolium (MTT) assay.To confine the scope of the hepatocarcinogenesis, 42 gene expression profiles in Bel-FU were detected by q-PCR microarray before and after treatment of traditional Chinese medicine. Then the gene expression of H-ras,MDR1 in Bel-FU treated with quercetin,EGCG, ACO-IA respectively were detected by real-time quantitative PCR to confirm the results of q-PCR microarray.Results In Bel-FU the resistance index (IR)of 5-fluorouracil ,cytarabine, vincristin, doxorubicin, daunorubicin were 89.6,3.4,37.1, 15.0, 3.7respectively. The 50% inhibitory concentration(IC50) of quercetin ,EGCG ,IA ,ACO-IA and loureirin B were 193.9μM,1230.8μM,507.8μM,244.9μΜ,163.7μΜrespectively. The 10% inhibitory concentration(IC10) were 58.2μM,159.5μM,75.3μM, 64.7μM, 25.8μM respectively. 43.6,87.2,130.8μM EGCG reversed the MDR by 1.04,1.36,2.10 folds respectively .16.5,33,49.5μM quercetin reversed the MDR by 1.14,1.68,2.38 folds respectively . 13.2,33,66μM IA reversed the MDR by 1.13,1.54,1.33 folds respectively . 21,42,63μM ACO-IA reversed the MDR by 1.44, 2.87, 1.76 folds respectively . 6, 12, 24μM loureirin B reversed the MDR by 2.25,3.80,5.76 folds respectively . Positive control VRP(5μg·mL-1)was 1.11 folds .Compared with those in Bel-7402, the gene expression of H-ras,Bcl-2,PDGFA,MDR1,BCRP were up-regulated (P<0.05)and cyclinB1 was down-regulated in Bel-FU(P<0.05). H-ras,EGFR,FGF,VEGFA,PDGFA,MDR1,Erk1 were down-regulated in Bel-FU treated with quercetin(P<0.05). The gene expression of H-ras,EGFR,PDGFA,MDR1,VEGFA were down-regulated in Bel-FU treated with EGCG(P<0.05). The gene expression of H-ras, VEGFA,PDGFA,C-myc,NF-κB were down-regulated in Bel-FU treated with ACO-IA ( P < 0.05 ) . The gene expression of Wnt1,VEGFA were down-regulated in Bel-FU treated with both IA and loureirin B(P<0.05). Real-time quantitative PCR showed that quercetin and EGCG could down-regulate the gene expression of H-ras,MDR1 in Bel-FU(P<0.05),then ACO-IA just could down-regulate the gene expression of H-ras in Bel-FU(P<0.05).Conclusion qPCR microarray is a high throughput and multi-target method for screening traditional Chinese medicine as to anti-hepatocellular carcinoma and reversing MDR in the course of genetic transcription . Objective To screen differentially expressed proteins in the human HCC MDR cell line before and after treatment of 3 kinds of traditional Chinese medicine respectively,to found a method for screening traditional Chinese medicine as to anti-hepatocellular carcinoma and reversing MDR by detecting target protein.Methods Detected the differentially expressed proteins between human hepatocellular carcinoma sensitive cell line Bel-7402 and drug -resistant cell line Bel-FU,and that in Bel-FU treated by 3 kinds of traditional Chinese medicine respectively.Differentially expressed proteins were defined as whose absolute ratio values were greater than 2.5. Western blot was used to detect the expression of P-gp ,FAK and CRT in Bel-FU treated with 3 kinds of traditional Chinese medicine.Results Compared with that in Bel-7402,24 proteins expression were up-regulated , including FAK,TM3,ORP150,CRT,NSE ,80K-H protein,EF-1-D,phosphoprotein and so on ,then 12 were down-regulated in Bel-FU.EGCG could down-regulate the proteins expression of FAK,CRT,TM3,80K-H protein,EF-1-D. quercetin could down-regulate the proteins expression of phosphoprotein,CRT,TM3,EF-1-D. COA-IA could down-regulate the proteins expression of FAK,80K-H protein,CRT,TM3 in Bel-FU. The results of Western blot showed that both EGCG and quercetin could down-regulate the proteins expression of P-gp(P < 0.05), EGCG and COA-IA could down-regulate the proteins expression of FAK(P < 0.05), 3 kinds of traditional Chinese medicine could down-regulate the proteins expression of CRT(P < 0.05).Conclusion EGCG , quercetin and COA-IA can reverse the MDR of HCC .Detecting protein differential expression as to anti-HCC and reversing MDR by proteomics technique provides a platform for high throughput drug screen.更多还原