【作者】 黄毅；

【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)研究黄芩苷体外对人Burkitt淋巴瘤细胞株CA46增殖、凋亡的影响;黄芩苷联合阿霉素(ADR)、柔红霉素(DNR)、高三尖杉酯碱(HH)、依托泊甙(VP16)体外对CA46细胞的作用效果。(2)观察黄芩苷对CA46细胞增殖、凋亡相关基因及蛋白表达的影响,探讨其作用机制。(3)磷脂酰肌醇-3-羟基激酶(PI3K)/丝氨酸/苏氨酸激酶(Akt)、丝裂原蛋白活化激酶(MAPK)(ERK1/2)信号通路在黄芩苷抑制CA46细胞增殖及诱导凋亡中的作用。(4)研究黄芩苷对CA46细胞裸鼠异种移植瘤的作用。方法(1)四甲基偶氮唑蓝(MTT)比色法绘制细胞生长曲线、克隆形成实验检测黄芩苷对CA46细胞增殖的影响;应用金氏公式评价黄芩苷联合ADR、DNR、HH、VP16对CA46细胞的作用效果;Annexin V FITC/PI双染流式细胞术、细胞DNA片段化检测、TdT酶介导的末端缺失原位标记(TUNEL)法和AO-EB荧光染色法检测黄芩苷对CA46细胞凋亡的影响;DNA倍体分析法检测黄芩苷对CA46细胞周期的影响。(2)RT-PCR法检测黄芩苷作用前后CA46细胞c-myc、bcl-2、bax、hTERT、pim-2、P21、Mcl-1、TGFβ1基因mRNA表达水平的变化;蛋白印迹(Western blot)法检测黄芩苷作用前后CA46细胞c-myc、bcl-2、procaspase-9、procaspase-3、PARP蛋白表达水平的变化以及PI3K/Akt和MAPK(ERK1/2)信号通路蛋白表达水平的变化。(3)构建CA46细胞裸鼠异种移植瘤模型,将荷瘤裸鼠随机分为6组:阴性对照组、15mg/kg黄芩苷组、30mg/kg黄芩苷组、60mg/kg黄芩苷组、4mg/kg VP16阳性对照组、联合用药组(30mg/kg黄芩苷+2mg/kg VP16)。采用腹腔注射方式给药12天后,处死部分裸鼠,称量离体瘤块重量,计算抑瘤率,对瘤组织进行病理组织学检查观察黄芩苷处理对CA46细胞裸鼠异种移植瘤生长的影响,采用透射电镜法检测黄芩苷处理对CA46细胞裸鼠异种移植瘤组织细胞超微结构的影响,提取瘤块组织蛋白,进行Akt、pAkt、mTOR和pmTOR的western blot检测,同时摘除眼球取血检测血常规及肝肾功能,并取肝、脾、肾、心、肺做病理组织学检查以观察黄芩苷处理对裸鼠的毒性作用;继续饲养各组未处死的裸鼠直至全部死亡(从给药时间算起),观察黄芩苷处理对荷瘤裸鼠生存时间的影响。结果(1)细胞生长曲线、克隆形成实验结果显示黄芩苷能有效抑制CA46细胞的增殖,作用48小时的IC50约为10μM;以Q值和协同等级判断,黄芩苷与VP16、ADR、DNR合用时,抗CA46细胞增殖的作用均明显加强,且在低浓度尤其明显,而黄芩苷与HH合用对CA46细胞增殖抑制率的提高作用不明显;Annexin V FITC/PI双染流式细胞术、细胞DNA片段化检测、TUNEL法和AO-EB荧光染色法检测证实黄芩苷能诱导CA46细胞凋亡,且随药物作用浓度的增加,凋亡率也逐渐上升;DNA倍体分析证实黄芩苷能使CA46细胞周期阻滞于G0/G1期。(2)黄芩苷的作用下调了CA46细胞c-myc、bcl-2、hTERT、pim-2、Mcl-1基因mRNA和/或蛋白的表达,上调了bax、P21、TGFβ1基因mRNA的表达;Western blot结果显示procaspase-9、procaspase-3、PARP(116KD)蛋白的表达水平下降,而PARP蛋白的85KD裂解活性片段逐渐升高。(3)黄芩苷的作用下调了CA46细胞的PI3K/Akt信号通路蛋白pAkt、pIκB、NF-κB、mTOR、pmTOR、pGSK-3β的表达,而对Akt、IκB、GSK-3β的表达无明显影响;下调了MAPK(ERK1/2)信号通路蛋白pMAPK(44/42)的表达,而对MAPK(44/42)的表达无明显影响。(4)腹腔注射给药12天后,称量离体瘤块的重量结果显示:当黄芩苷剂量为15mg/kg、30mg/kg和60mg/kg时,瘤块重量分别为1.71±0.17g(P>0.05)、1.35±0.35g(P<0.05)和0.97±0.38g(P<0.01),说明黄芩苷对CA46细胞裸鼠异种移植瘤的生长呈现剂量依赖性的明显抑制作用;将30mg/kg黄芩苷和2mg/kg VP16联合使用时,抑瘤率达60.4%,高于单用4mg/kgVP16阳性对照组的51.1%和单用60mg/kg黄芩苷组的46.7%,提示黄芩苷与VP16具有协同抗CA46细胞裸鼠异种移植瘤的作用。对瘤组织进行病理组织学和透射电镜检查的结果表明:黄芩苷用药组和联合用药组的肿瘤坏死区域和肿瘤细胞凋亡现象较阴性对照组多见,且黄芩苷用药组中以高剂量组的效果最为明显。对提取的瘤块组织蛋白进行western blot检测的结果表明:高剂量黄芩苷组和联合用药组除Akt蛋白的表达无明显改变外,pAkt、mTOR和pmTOR蛋白的表达均出现明显下调,提示黄芩苷体内抗肿瘤作用的机制亦可能与下调PI3K/Akt/mTOR信号通路有关。对荷瘤裸鼠生存时间的观察结果表明:随着黄芩苷剂量的增加,荷瘤裸鼠的生存时间得到提高,低、中、高剂量组的中位生存时间分别为48天、58天、72天,而阴性对照组的中位生存时间仅53天;黄芩苷和VP16合用对荷瘤裸鼠生存时间的提高作用最明显,联合用药组的中位生存时间达88天,与阴性对照组相比较具有显著性差异(P<0.05)。结论(1)黄芩苷体外能有效抑制CA46细胞增殖,阻滞细胞周期于G0/G1期,并通过线粒体途径诱导其凋亡。其机制可能与下调c-myc、bcl-2、h-tert、pim-2、Mcl-1基因mRNA和/或蛋白的表达,上调bax、P21、TGFβ1基因mRNA的表达有关。(2)黄芩苷与一些化疗药物如VP16、ADR和DNR联用,具有体外协同抗CA46细胞的作用。(3)对信号转导通路的研究发现,PI3K/Akt、MAPK(ERK1/2)信号通路参与了黄芩苷抑制CA46细胞增殖、诱导凋亡的过程。(4)黄芩苷可抑制CA46细胞裸鼠异种移植瘤的生长,诱导瘤组织中CA46细胞的凋亡,与VP16合用可明显提高荷瘤裸鼠的生存时间,其机制可能与下调PI3K/Akt/mTOR信号通路有关;黄芩苷与VP16联用具有协同抗CA46细胞裸鼠异种移植瘤的作用。更多还原

【Abstract】 【Objective】(1)To investigate the effects of baicalin on proliferation inhibition and apoptosis induction in human Burkitt lymphoma cell line CA46 cells;the effects of baicalin combined with adriamycin(ADR),daunorubicin(DNR), homoharringtonine(HH) and etoposide(VP16) on CA46 cells respectively.(2)To investigate the effects of baicalin on proliferation and apoptosis related genes and proteins expression in CA46 cells and explore its mechanisms.(3)To investigate the role of PI3K/Akt and MAPK(ERK1/2) signal pathways in the apoptosis of CA46 cells induced by baicalin.(4)To investigate the effects of baicalin on CA46 cell xenografts in nude mice.【Methods】(1)CA46 cells were exposed to various dosages of Baicalin. Proliferation inhibition was detected by both MTT assay and clone formation assay. The effects of baicalin combinating with ADR,DNR,HH and VP16 on CA46 cells were evaluated by Jin formula respectively.The ability of baicalin to induce apoptosis of CA46 cells was examined by Annexin V FITC/PI double staining analysis,DNA fragamentation,TUNEL labeling method and acridine orange/ethidium bromide(AO-EB) double fluorescence staining respectively.The effects of baicalin on cell cycle of CA46 cells were examined by DNA ploid analysis. (2)The expressions of c-myc,bcl-2,bax,hTERT,pim-2,P21,Mcl-1 and TGFβ1 mRNA were detected by RT-PCR.The expressions of c-myc,bcl-2,procaspase-9,procaspase-3,PARP,Akt, pAkt, IκB-α,pIκB-α,NF-κB,mTOR,pmTOR,GSK-3β,pGSK-3β,MAPK(44/42) and pMAPK(44/ 42) proteins were detected by Western-blot.(3)The nude mice with CA46 cell xenografts were divided into six groups:negative control group,15mg/kg baicalin group,30mg/kg baicalin group,60mg/kg baicalin group,4mg/kg VP16 positive control group,and combination group(30mg/kg baicalin+2mg/kg VP16). Drugs were administered via intraperitoneal injection for 12 days. The changes of CA46 cell xenografts’weight and histology were used to evaluate the effects of baicalin on xenografts growth in nude mice.The apoptosis of xenograft cells was tested by tranmission electron microscope assay,and the Akt, pAkt, mTOR and pmTOR proteins extracted from xenografts were detected by western blot.The blood was gained to detected the hematocyte count,liver and renal function.The important organs including liver,spleen,renal,heart and lung were detected by histology assay to evaluate the toxic action of baicalin to nude mice.The other nude mice with CA46 cell xenografts in all groups were fed until died (from the time receiving intraperitoneal injection) in order to evaluate the effects of balcalin on survival time of nude mice with CA46 cell xenografts.【Results】(1)The results of MTT assay and clone formation assay showed that baicalin could remarkably inhibit the CA46 cell proliferation, and the IC50 value at 48h of treatment was about 10μM.The Q value and synergistic grade revealed that baicalin combinated with ADR,DNR and VP16 had the synergistic effects on inhibiting the CA46 cell proliferation,especially in a low dose baicalin.The results of Annexin V FITC/PI double staining analysis,DNA fragamentation,TUNEL labeling method and AO-EB double fluorescence staining revealed that baicalin induced the apoptosis of CA46 cells in dose-dependent manner.The DNA ploid analysis revealed that the cell cycle of CA46 cells treated by baicalin was blocked in G0/G1 phase. (2)The results of RT-PCR and/or western blot showed the expressions of c-myc,bcl-2,hTERT, pim-2,Mcl-1, procaspase-9,procaspase-3 and PARP(116KD) decreased,while bax,P21,TGFβ1 and PARP(85KD) increased after baicalin treatment.(3)Baicalin could downregulate the expressions of pAkt, pIκB, NF-κB,mTOR,pmTOR,pGSK-3βand pMAPK(44/42) proteins in CA46 cells,but the expressions of Akt,IκB,GSK-3βand MAPK(44/42) were not changed remarkably.(4)In vivo baicalin could inhibit the growth of CA46 cell xenografts in a dose-dependent manner with the tumor weight of 1.71±0.17g(P>0.05),1.35±0.35g(P<0.05) and 0.97±0.38g(P<0.01)in the group where baicalin was injected to nude mice with a dose of 15mg/kg,30mg/kg and 60mg/kg for 12 days respectively.Baicalin combined with VP16 had the synergistic effect on inhibiting the growth of CA46 cell xenografts in nude mice with the inhibition rate of 60.4% in combination group(30mg/kg baicalin+2mg/kg VP16),which was higher than 51.1% in 4mg/kg VP16 positive control group and 46.7% in 60mg/kg baicalin group.There were more necrotic and apoptotic cells in baicalin treatment groups and combination group than that in negative control group by histology assay and tranmission electron microscope assay.The expressions of pAkt,mTOR and pmTOR protein in high-dose baicalin group and combination group were downregulated remarkably,which revealed that baicalin could inhibit the proliferation of CA46 cells in vivo by downregulating the PI3K/Akt/mTOR signal pathway.The median survival time in 60mg/kg baicalin group,30mg/kg baicalin group and 15mg/kg baicalin group were 48,58 and 72 days respectively,which revealed that baicalin could raise the survival time of nude mice with CA46 cell xenografts in a dose-dependent manner.The median survival time in combination group(30mg/kg baicalin+2mg/kg VP16) was raised to 88 days,which was higher than that in negative control group significantly(P<0.05). 【Conclusions】(1)Baicalin could efficiently inhibit cell proliferation,block cell cycle in G0/G1 phase,and induce chondriosome pathway-apoptosis in CA46 cells,which could be correlated with the down-regulation expressions of c-myc, bcl-2,hTERT,pim-2 and Mcl-1,and the up-regulation expressions of bax,P21 and TGFβ1.(2)Baicalin combined with ADR,DNR and VP16 could have the synergistic effect on inhibiting the CA46 cell proliferation. (3)PI3K/Akt and MAPK(ERK1/2) signal pathways could be involved in proliferation inhibition and apoptosis induction of CA46 cells after treatment with baicalin. (4)Baicalin could inhibit growth and induce cell apoptosis of CA46 cell xenografts in nude mice,and raise the survival time of nude mice with CA46 cell xenografts significantly if combined with VP16,which mechanism could be correlated with the downregulation of PI3K/Akt/mTOR signal pathway. Baicalin combined with VP16 could have the synergistic effect on inhibiting growth of CA46 cell xenografts in nude mice.更多还原