【作者】 郑静；

【导师】 胡建达；

【作者基本信息】 福建医科大学 ， 内科学， 2009， 博士

【摘要】 目的(1)研究中药黄芩苷(Baicalin)对耐阿霉素人髓系白血病HL-60/ADR细胞株增殖、凋亡的影响。观察黄芩苷对HL-60/ADR细胞增殖、凋亡相关基因、凋亡相关蛋白表达及在PI3K/Akt/mTOR、MAPK信号转导通路中蛋白表达水平的影响,探讨其作用机制。(2)研究黄芩苷对HL-60/ADR细胞耐药相关基因和相关蛋白表达的影响,探讨其是否具有逆转耐药的作用及可能机制。(3)研究黄芩苷对高致瘤HL-60细胞裸鼠异种移植瘤的作用。方法(1)应用MTT法绘制细胞生长曲线、细胞克隆形成实验观察黄芩苷对HL-60 /ADR细胞增殖的影响;Annexin V/PI双染流式细胞检测、DNA倍体分析、末端缺口原位标记(TUNEL)法及DNA凝胶电泳分析细胞凋亡及细胞周期变化;RT- PCR检测黄芩苷作用前后c-myc、bcl-2、hTERT、pim-2、mcl-1、p21等凋亡相关基因mRNA表达水平的变化,蛋白印迹法(Western-Blot)检测c-myc、bcl-2、bad、caspase-3、PARP等凋亡相关蛋白和PI3K/Akt/mTOR、MAPK信号通路相关蛋白表达水平的变化。(2)采用MTT法分别检测阿霉素、柔红霉素、阿糖胞苷、足叶乙甙对HL-60细胞和HL-60/ADR细胞增殖的抑制效应,以及加用黄芩苷时上述抑制作用的变化,并计算逆转倍数。RT-PCR和Western Blot检测黄芩苷作用后HL-60/ADR细胞耐药相关基因和相关蛋白的表达,流式细胞术检测黄芩苷作用前后HL-60/ADR细胞阿霉素蓄积水平。(3)构建高致瘤HL-60细胞裸鼠移植瘤模型,将荷瘤裸鼠随机分为6组:阴性对照组、25mg/kg黄芩苷组、50mg/kg黄芩苷组、100mg/kg黄芩苷组、联合用药组(50mg/kg黄芩苷+2mg/kg VP16)和VP16阳性对照组(4mg/kg)。计算平均瘤重及抑瘤率。通过电镜观察瘤组织超微结构,病理组织学观察裸鼠各主要脏器结构改变,瘤块组织蛋白检测信号转到通路指标。观察裸鼠生存时间。结果(1)细胞生长曲线结果显示黄芩苷能明显抑制HL-60/ADR细胞增殖,半数抑制浓度(IC50)约为28μmol/L;较低浓度黄芩苷即可抑制HL-60/ADR细胞集落的形成;Annexin V FITC/PI法检测到早期凋亡细胞,DNA片段化、TUNEL法检测到晚期凋亡细胞,细胞凋亡率呈浓度依赖性。细胞周期分析显示,与对照组比较,40μmol/L、80μmol/L浓度组细胞被阻滞于G0/G1期比率明显增高(P<0.01),S期细胞比率降低(P<0.01),各浓度组均检测到典型的亚二倍体峰(凋亡峰)。黄芩苷作用后HL-60/ADR细胞c-myc、bcl-2、hTERT、pim-2、mcl-1基因mRNA表达随着作用时间的延长而逐渐减少;p21基因mRNA表达随着作用时间的延长而逐渐升高。Western-Blot的结果显示c-myc、bcl-2、caspase-3(35kD)和PARP(116kD)蛋白的表达水平呈时间依赖性下降,而caspase-3(17kD、19kD)、PARP(85kD)及bad蛋白的表达则随着作用时间的延长逐渐升高。黄芩苷下调了HL-60/ADR细胞的PI3K/Akt/mTOR和MAPK信号通路蛋白中p-Akt、NF-κB、p-NF-κB、p-IκB-α、mTOR、p-mTOR、p-GSK-3β、p-MAPK的表达,而Akt、IκB-α、GSK-3β和MAPK的表达与对照组相比无明显改变。(2)黄芩苷能部分逆转HL-60/ADR细胞的多药耐药性,阿霉素和黄芩苷联合应用对HL-60/ADR细胞的增殖抑制率明显低于单用阿霉素者,阿霉素单用及联合黄芩苷对HL-60细胞的增殖抑制率没有影响。黄芩苷能下调mrp-1、lrp、GSTπ基因mRNA和mrp-1、GSTπ蛋白的表达,上调TopoⅡα和TopoⅡβ基因mRNA的表达(p<0.05)。而TopoⅠ基因在各组间表达无明显差别。同时观察到黄芩苷能使染上阿霉素荧光的HL-60/ADR细胞比例增加。(3)黄芩苷可抑制裸鼠HL-60细胞皮下移植瘤的生长,呈剂量依赖性。100mg/kg黄芩苷组和联合用药组(50mg/kg黄芩苷+2mg/kg VP16)的瘤块重量明显低于阴性对照组(p<0.01)。瘤组织病理组织学和透射电镜检查结果显示:黄芩苷用药组和联合用药组的肿瘤坏死和细胞凋亡现象较阴性对照组多见。黄芩苷可能通过抑制Akt活性,下调p-Akt、mTOR和p-mTOR的表达来抑制裸鼠异种移植瘤的增长;联合用药组中位生存期明显高于阴性对照组(p<0.05),提示黄芩苷与VP16具有协同抗HL-60细胞裸鼠异种移植瘤的作用。黄芩苷对裸鼠一般状况和体重无不良影响,对裸鼠无明显毒副作用。结论(1)黄芩苷能有效抑制HL-60/ADR细胞增殖,诱导其凋亡。其机制可能与下调c-myc、bcl-2、hTERT、pim-2、mcl-1 mRNA和/或蛋白表达,上调p21、bad的表达有关。PI3K/Akt/mTOR、MAPK信号通路的改变可能参与了黄芩苷抑制HL-60/ADR细胞增殖、诱导凋亡的过程。(2)黄芩苷能部分逆转HL-60/ADR细胞的耐药性,其机制可能与下调mrp-1、lrp、GSTπ基因mRNA和/或蛋白表达,上调TopoⅡα和TopoⅡβ基因mRNA的表达,增加细胞内药物蓄积水平有关。(3)黄芩苷可抑制高致瘤HL-60细胞裸鼠异种移植瘤的生长,诱导瘤组织中HL-60细胞的凋亡,提高荷瘤裸鼠的中位生存期,其机制之一可能与下调PI3K/Akt/mTOR信号通路有关;黄芩苷与VP16联用具有协同抗HL-60细胞裸鼠异种移植瘤的作用。更多还原

【Abstract】 Objective(1)To study the effects of baicalin on proliferation inhibition and apoptosis induction on adriamycin-resistant human myeloid leukemia cell line HL-60/ADR cells and on expressions of related apoptotic proteins and mRNA,and to study the change of PI3K/Akt/mTOR,MAPK signal pathway in baicalin-induced HL-60/ADR cells.(2)To investigate the effects of baicalin on the expression of drug-resistance related genes in HL-60/ADR cells and explore its reversal mechanism (.3)To observe the effects of baicalin on HL-60 cell xenografts in nude mice.Methods(1)HL-60/ADR cells were in vitro cultured and exposed to baicalin at different dosages.Its proliferation inhibition and colony formation were detected by MTT assay and colony formation assay respectively.The cell apoptosis was tested by Annexin V FITC/PI double staining analysis, cell cycle analysis, DNA fragmentation and TUNEL labeling method.RT-PCR was used to analyze the c-myc, bcl-2, hTERT, pim-2, mcl-1 and p21 mRNA expression.The expressions of apoptosis related proteins and signaling molecular PI3K/AKT proteins were examined by western blot.(2)The effect of Adriamycin (ADR),Daunorubicin (DNR) ,Cytarabine (Ara-C) and Etoposide (VP16) on the proliferation of HL-60 and HL-60/ADR cells were observed by MTT assay.The inhibitory effects of all these chemotherapeutic agents used alone or incomebination with baicalin on the proliferation of HL-60 and HL-60/ADR cells were evaluated by MTT assay. The reversal fold of drug resistance was calculated. The expressions of mrp-1, lrp,GSTπ,TopoⅠ,TopoⅡαand TopoⅡβmRNA were measured by RT-PCR.The proteins level of mrp-1 and GSTπwere detected by western blot.The positive rate of ADR fluorescence was analyzed by flow cytometry.(3)Xenograft tumor model of HL-60 cell in nude mice was established,which was divided randomly into six groups:negative control group (5%NaHCO3 group),25mg/ kg baicalin group,50mg/kg baicalin group,100mg/kg baicalin group,combination group (50mg/kg baicalin+2mg/kg VP16),VP16 positive control group(4mg/kg). Nude mice growth state was observed, average weigh and inhibition rate of transplanted tumor were calculated, and the ultramicrostructure change of xenografts cells were tested by transmission electron microscope .Histopathology were used to observed the change of chief organs in nude mice.The expression of Akt, p-Akt, mTOR and p-mTOR proteins extracted from xenografts were detected by western blot. The effects of balcalin on survival rate and median survival time in nude mice with HL-60 cell xenografts were evaluated.Result(s1)Baicalin effectively inhibited HL-60/ADR cells, proliferation, with an IC50 value of 28μmol/L.Cell colony formation was obviously inhibited by baicalin. Apoptosis occurred with dose dependent manners(20μmol/L, 40μmol/L,80μmol/L), and its earlier and later stages were both detected by Annexin V FITC/PI double staining analysis, DNA fragmentation and TUNEL labeling method. G0/G1 phase cell population increased while S phase cells decreased in 40μmol/L and 80μmol/L groups compared with control group(P<0.01).The typical hypo-diploid peak (apoptotic peak) appeared in each dose group.The mRNA expressions of c-myc, bcl-2, hTERT,pim-2 and mcl-1 in baicalin treated cells decreased in a time-dependent manner(12h,24h, 48h), while the p21 mRNA expression increased. Meanwhile, protein expressions of c-myc, bcl-2, caspase-3(35kD)and PARP(116kD)were down-regulated in a time- dependent manner, and the expressions of caspase-3(17kD,19kD),PARP(85kD) and bad were up-regulated.Moreover,western blot showed the expressions of p-Akt, NF-κB, p-NF-κB, p-IκB-α, mTOR, p-mTOR, p-GSK-3βand p-MAPK protein decreased in HL-60/ ADR cells after baicalin treatment , but the expression of Akt , IκB-α, GSK-3βand MAPK showed no difference compared to control group(.2)The multidrug resistance (MDR) in HL-60/ADR cells can be partially reversed by baicalin. Proliferation inhibition of ADR incombination with baicalin on the HL-60/ADR cells were significantly lower than that of ADR used alone, there was no difference of proliferation inhibition on HL-60 cells between ADR used alone and ADR incombination with baicalin.Baicalin could down-regulate the expression level of mrp-1, lrp,GSTπmRNA and mrp-1, GSTπprotein, and up-regulate TopoⅡαand TopoⅡβmRNA(p<0.05), but TopoⅠdid not change obviously. Furthermore,it was found that baicalin could elevated positive rate of ADR fluorescence in HL-60/ADR cells.(3)Baicalin could inhibit the growth of transplanted tumors in dose dependent manner.The transplanted tumor weight in 100 mg/kg baicalin group and combination group(50mg/kg baicalin+2mg/kg VP16) were significantly lower than that in negative control group(p<0.01) .There were more necrotic and apoptotic cells in baicalin treatment groups and combination group than that in negative control group by histopathology and transmission electron microscope methods.Baicalin could inhibit the proliferation of HL-60 cells in vivo by down-regulating the PI3K/Akt/mTOR signal pathway, where the expressions of p-Akt, mTOR and p-mTOR proteins decreased compared to negative control group, and Akt did not change significantly. Baicalin could prolong the median survival time of nude mice.Compared with negative control group, the median survival time in combination group was significantly different(p<0.05), which illustrated that baicalin combinated with VP16 have the synergistic effect on inhibiting growth of HL-60 cell xenografts in nude mice. The baicalin administration was well tolerated by the nude mice, and no significantly adverse effect was shown.Conclusion(1)Baicalin could efficiently inhibit proliferation and induce apoptosis in HL-60/ADR cells, which could be correlated with the down-regulated expressions of c-myc,bcl-2,hTERT, mcl-1, and up-regulated expressions of p21and bad. PI3K/Akt /mTOR and MAPK signal pathways may have involved in the process of proliferation inhibition and apoptosis in HL-60/ADR cells induced by baicalin.(2)Baicalin could partially reverse the multidrug resistance of HL-60/ADR cell, and the reversal effect may be related with the reduction of the expression of mrp-1, lrp, GSTπmRNA or protein, increase of TopoⅡαand TopoⅡβmRNA expression level, and elevation of drug accumulation in HL-60/ADR cells.(3)Baicalin could inhibit growth and induce apoptosis of HL-60 cell xenografts in nude mice, and prolong its median survival time. The possible mechanisms may be related to inhibition of Akt activity and down- regulation ation of the PI3K/Akt /mTOR signal pathway. The combination of baicalin and VP16 shows a synergistic effect on inhibiting growth in HL-60 cell xenografts in nude mice.更多还原