【作者】 汪选斌；

【导师】 向继洲；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 博士

【摘要】 第一部分人肝癌多药耐药细胞系BEL-7402/ADM的建立一、人肝癌多药耐药细胞株BEL-7402/ADM的建立方法:以文献方法采用体外低浓度梯度递增联合大剂量间断冲击诱导的模式,建立人肝癌BEL-7402/ADM耐药细胞株。取对数生长期的亲本细胞BEL-7402,常规消化细胞,按2×10⁶个/瓶进行分瓶,加入终浓度为50nmol/L的阿霉素（adriamycin,ADM）,24h换液,用PBS冲洗,胰酶进行消化,根据生长情况选择传代周期。低浓度持续诱导,弃去上清和死亡悬浮的细胞,加入新鲜培养液脱药培养,在细胞重新生长并达到覆盖2/3瓶壁时,加入含ADM浓度为2000 nmol/L的培养液进行大剂量冲击诱导,待存活细胞恢复生长增殖后,加入含ADM 200 nmol/L的培养液继续诱导,同上步骤依次建立ADM诱导浓度为600、800、1000、2000nmol/L的人肝癌BEL-7402/ADM耐药细胞,实验最终采用ADM浓度为2000nmol/L的培养液中生长的BEL-7402/ADM耐药细胞为本次实验的耐药细胞组。结果:成功诱导人肝癌耐药细胞BEL-7402/ADM。倒置显微镜下人肝癌耐药细胞BEL-7402/ADM的细胞形态学观察结果,耐药细胞多为分片状、群集性增殖,细胞的多角性改变,棱角变少,呈梭形,胞浆的折光性变大,细胞易被消化。二、MTT法测定人肝癌多药耐药细胞系BEL-7402/ADM的耐药倍数方法:取亲本细胞及耐药细胞,用0.25%胰酶消化处理对数生长期细胞,用含8%FCS的RPMI 1640培养液配成细胞悬液,每孔接种200μL,即2×10⁴个/mL,接种在96孔板上,设有空白对照组、阴性对照组、ADM组、5-FU组、VCR组和CDDP组,以上给药组均设5个浓度。在酶联免疫检测仪上选定490nm波长处测定OD值,参比波长为620nm。按下列公式计算细胞生长抑制率:生长抑制率=（1—给药组平均OD值/对照组平均OD值）×100%.公式计算IC₅₀值:lgIC₅₀=Xm-I[P-（3-Pm-Pn）/4],Xm:1g最大剂量;I:1g（最大剂量/相邻剂量）;P:阳性反应率之和;Pm:最大阳性反应率:Pn:最小阳性反应率。耐药倍数（RI）=（IC Resistance group）/（IC Parental group）×100%.结果:MTT实验结果表明,ADM对亲本的BEL-7402以及耐药的BEL-7402/ADM的IC₅₀值分别是（1.47±0.28）、（7.62±0.51）μmol/L;5—FU的IC₅₀值分别是（4.56±0.72）、（21.53±2.47）μmol/L;VCR的IC₅₀值分别是（8.98±0.30）、（80.27±11.22）μmol/L;CDDP的IC₅₀值分别是（1.2±0.04）、（10.67±2.14）μmol/L,对各药物的耐药倍数分别为:ADM 5.18倍、5-FU 4.72倍、VCR 8.94倍、CDDP 8.89倍。三、ADM不同诱导浓度对人肝癌细胞系BEL-7402中P-gp表达的影响方法:以Western-blot法检测在200、600、800、1000、2000 nmol/L不同ADM浓度诱导至稳定增殖的肝癌BEL-7402细胞的P-gp的表达。结果以各组P-gp/α-tublin与未诱导的亲本细胞（control组）的比值百分率（均值±标准差,X±SD）表示。结果:以200、600、800、1000、2000 nmol/L不同ADM浓度稳定增殖的肝癌BEL-7402细胞,其P-gp的蛋白表达以亲本细胞P-gp/α-tublin（control组）为100%计,分别为（99.37±1.63）、（112.36±1.59）、（105.01±1.93）、（102.12±1.16）、（102.84±2.18）%,其中阿霉素浓度为200nmol/L时,P-gp表达与control组无差异,但当阿霉素浓度为600、800、1000、2000nmol/L时,蛋白表达明显增加（P＜0.05）。四、细胞周期分析方法:取对数生长期的BEL-7402、BEL-7402/ADM细胞于流式细胞仪测定DNA含量并进行细胞周期分析。结果:细胞周期结果显示,耐药细胞G₀/G₁缩短,而G₂/M、S期增加（P＜0.05）（n=3）。五、流式细胞仪检测亲本细胞及耐药细胞内阿霉素的蓄积方法:将对数生长期的BEL-7402、BEL-7402/ADM细胞以8 000 nmol/LADM培养2 h后,流式细胞仪检测细胞内ADM平均荧光强度。结果:FCM检测结果,以BEL-7402内荧光强度为100%计,耐药细胞BEL-7402/ADM内的平均荧光强度为（82.08±6.89）%（P＜0.01）,表明耐药细胞内阿霉素摄入减少。六、细胞贴壁率与平板克隆形成率的检测方法:收集BEL-7402亲本细胞及BEL-7402/ADM多药耐药细胞,制备成单细胞悬液。以载玻片进行台盼蓝染色活细胞计数,各按每瓶1×10⁶个分别接种于6个培养瓶内。每间隔1 h取1组细胞,吸弃未贴壁的细胞,消化贴壁细胞,进行细胞计数。根据以下公式计算细胞贴壁率:细胞贴壁率=（贴壁细胞数/接种细胞数）×100%。细胞平板克隆形成率参考文献实验方法。结果:台盼蓝染色活细胞计数结果,两组细胞贴壁率均随时间增加而增加,在2h时两组有差异,亲本细胞的贴壁率大于耐药细胞（P＜0.05）。平板克隆形成率BEL-7402与耐药细胞BEL-7402/ADM分别为:（68.28±8.06）%、（54.44±5.83）%,两者有统计学差异（P＜0.05）。结论1.本研究采用体外低浓度梯度递增联合大剂量间断冲击诱导的模式,成功建立了人肝癌BEL-7402/ADM耐药细胞株,实验结果表明,该细胞株虽然为ADM所诱导耐药,但该耐药株不仅对ADM产生了较高的耐药性,对5—FU、VCR及CDDP亦有不同程度的耐药性,证实了其类型为多药耐药。耐药细胞株经冻存后复苏并连续脱药培养1个月后,其耐药性可有所有下降,但通过短暂的药物再次诱导,耐药性仍能恢复至冻存之前的水平,与文献报道一致。2.从Western blot结果看,当阿霉素诱导浓度在200 nmol/L时,对BEL—7402细胞P-gp的表达无影响。当浓度在600 nmol/L以上时,P-gp表达显著升高,但P-gp的表达量并不随诱导剂量增加而显著增加,而是耐药表型随着药物的增加而逐渐稳定。由于肿瘤多药耐药机制非常复杂,可能存在多机制联合作用导致耐药,有待下一步实验证实。3.有关肿瘤耐药细胞周期与其亲本细胞的区别,文献报道不一。本研究中细胞周期实验结果显示,耐药细胞与亲本细胞存在一定差异。表现在耐药细胞G0/G1比值减小,而G2/M、S期增加。各实验室结果差异除了可能与实验条件不尽相同、诱导方法、操作误差等都有关系。此外,耐药的肝癌细胞在贴壁率、平板克隆形成率、细胞形态上与亲本细胞还存在差异。4.流式细胞仪检测结果,说明阿霉素诱导的多药耐药细胞耐药的机制与外排药物有关。结合Western blot结果,考虑可能P-gp参与了这一机制。第二部分川芎嗪逆转人肝癌多药耐药细胞BEL-7402/ADM的作用及机制一、MTT法测定川芎嗪对人肝癌多药耐药细胞BEL-7402/ADM的逆转倍数方法:将BEL-7402/ADM以TMP（400和600μmol/L）、VRP（5和10μmol/L）分别培养于96孔板至24 h,然后分别加入浓度为0.3,0.6,1.2,2.4,4.8μmol/L的ADM至72 h。其中维拉帕米（Verapamil,VRP）为阳性对照。分别计算IC₅₀值、耐药倍数、逆转倍数。其中IC₅₀值的计算按第一部分“MTT法测定人肝癌多药耐药细胞系BEL-7402/ADM的耐药倍数”计算。逆转倍数=(IC₅₀ Resistance group)/(IC₅₀ TMP/VRP group)×100%.结果:MTT实验证明,TMP对耐药细胞的逆转倍数分别为3.79（P＜0.01）（TMP浓度为400μmol/L）、14.65（P＜0.01）（TMP浓度为600μmol/L）,与VRP结果相似,并呈浓度依赖性。二、荧光显微镜观察川芎嗪对人肝癌多药耐药细胞BEL-7402/ADM中ADM蓄积的影响方法:将生长良好的BEL-7402/ADM及其亲本细胞于培养瓶中培养过夜,分为6组,阴性亲本组、阴性耐药组、亲本组、耐药组、TMP逆转组、阳性对照组（VRP）。接种于培养瓶中,逆转组加入川芎嗪至终浓度为600μmol/L,阳性对照组（VRP）,加入VRP至终浓度为5μmol/L,逆转组及阳性对照组在TMP或VRP中预培养3 h,再与亲本组、耐药组平行加入8 000 nmol/L ADM培养2 h,阴性亲本组、阴性耐药组加入新鲜培养液,不加任何药物;各组以冰冷的PBS洗3次,荧光显微镜下直接观察细胞内阿霉素的含量。激发光（λex）为480nm,发射光（λem）为575 nm。结果:荧光显微镜下观察,ADM在荧光镜下发红色荧光。镜下可见,ADM红色荧光信号强弱顺序依次为:Resistance +VRP+ADM组＞Resistance +TMP+ADM组＞亲本细胞＞Resistance+ADM组。实验表明,未加药的亲本细胞及耐药细胞无阿霉素的红色荧光,各组中亲本细胞中的阿霉素荧光最强,耐药组有所减弱,经TMP、VRP预处理后的耐药组荧光有所增强,说明TMP、VRP可使细胞内阿霉素的蓄积量增加。三、流式细胞仪检测川芎嗪对人肝癌多药耐药细胞BEL-7402/ADM中ADM蓄积的影响方法:设阴性组、耐药组、TMP组、VRP组。接种于培养瓶中,阴性对照组加入新鲜培养液,逆转组加入川芎嗪至终浓度为600μmol/L,阳性对照组（VRP）,加入VRP至终浓度为5μmol/L,继续培养24h,加入ADM 8000nmol/L,培养2 h,以冰冷的PBS洗3次,胰酶消化为单个细胞悬液（不低于10⁶个/mL）,流式细胞仪检测细胞内ADM平均荧光强度。激发光（λex）为480 nm,发射光（λem）为575 nm.结果:FCM检测结果,Parental组,Resistance+TMP组和Resistance+VRP组平均荧光强度分别为耐药细胞Resistance组的（121.83±23.2）%（P＜0.01）、（163.78±39.5）%（P＜0.01）、（320.1±47.18）%（P＜0.01）。表明TMP能使细胞内的阿霉素蓄积增加,与VRP相似。四、HPLC法检测TMP对细胞内ADM浓度的影响方法:设亲本组、耐药组、TMP组、VRP组。将TMP组、VRP组分别在TMP（600μmol/L）或VRP（5μmol/L）中预培养3 h,所有组再以8 000 nmol/LADM培养2h,以HPLC检测细胞内ADM浓度。以目标药与内标的峰面积的比值为横坐标,以药物的浓度为纵坐标,做标准曲线,并考察重现性、精密度、加样回收率、稳定性等。实验结果以盐酸阿霉素（分子量579.99）浓度单位折算为nmol/L。结果:HPLC结果显示,ADM在0.1-40000ng/mL的范围内具有良好的线性关系。检测限（LOD）为0.05ng/mL,RSD为0.407。标准曲线为:C=47.73A+23.62（r=0.998）,其中C为阿霉素浓度,A为阿霉素与alprazolam的峰面积比值。实验数据显示,亲本细胞摄入阿霉素的量为（2.45±0.52）μmol/L,耐药细胞对阿霉素摄入减少至（2.06±0.40）μmol/L,TMP可逆转耐药情况而使阿霉素的蓄积量超过亲本细胞的水平。实验数据还表明,川芎嗪在使细胞内阿霉素浓度蓄积增加的同时,川芎嗪本身在细胞内的蓄积并未增加,说明川芎嗪并非P-gp底物,它使细胞内阿霉素浓度增加并非属于与阿霉素竞争受体结合位点所致。五、川芎嗪对人肝癌多药耐药细胞BEL-7402/ADM中MDR1、MRP2、MPP3、MRP5 mRNA表达的影响方法:设耐药组、TMP组、VRP组。将TMP组、VRP组分别在TMP（600μmol/L）或VRP（5μmol/L）中预培养3 h,所有组再以8 000 nmol/L ADM培养2 h,采用PCR检测MDR1,MRP2,MRP3和MRP5 mRNA表达水平。结果:实验结果以耐药组（resistance）为100%,计算得各组值。结果显示,TMP可使MDR1,MRP2,MRP3和MRP5 mRNA表达分别达到耐药组的（0.67±0.03）、（0.85±0.10）、（0.90±0.04）、（0.63±0.11）,与耐药组比较均有统计学差异（P＜0.05）,但VRP组除MDR1 mRNA表达与阴性对照组有差异外,其余组无差异（P＜0.05）。六、川芎嗪对人肝癌多药耐药细胞BEL-7402/ADM中P-gp、MRP2、MRP3、MRP5蛋白表达的影响方法:细胞分设耐药组、TMP组、VRP组。将TMP组、VRP组分别在TMP（600μmol/L）或VRP（5μmol/L）中预培养3 h,所有组再以8 000 nmol/LADM培养2 h,采用免疫印迹检测P-gp、MRP2、MRP3、MRP5蛋白表达水平。结果:结果表明TMP可使MDR1,MRP2,MRP3和MRP5蛋白表达分别下调至阴性对照组（以1.0计）的（0.49±0.04）、（0.61±0.13）、（0.38±0.14）、（0.68±0.07）（P＜0.01）。P-gp、MRP2、MRP3及MRP5蛋白表达下降。（P＜0.05）但VRP组除P-gp表达下降（0.44±0.08）（P＜0.01）外,其余蛋白表达无差异,与mRNA结果一致。结论1.川芎嗪（Tetramethylpyrazine,TMP）自中药川芎中分离,有研究表明其对HCC的耐药有作用,可能与逆转P—gp有关,但仍然未见是否与逆转其它蛋白有关。本研究探讨了TMP对人肝癌多药耐药细胞BEL-7402/ADM MDR的逆转作用。研究发现:TMP可浓度依赖性逆转肝癌多药耐药细胞BEL-7402/ADM的MDR。2.TMP作用后,耐药细胞内的ADM蓄积增加,推测可能是由于TMP影响了ABC家族成员的一种或数种蛋白的表达及功能所致。3.Realtime PCR及Western blot结果证实,TMP作用于多药耐药细胞后MDR1、MRP2、MRP3及MRP5在肝癌耐药株细胞中mRNA及蛋白高表达可能发挥联合耐药作用,并可被TMP所抑制。4.研究还发现:TMP和第一代逆转剂VRP均可以抑制MDR1,但川芎嗪对MRP2,MRP3,MRP5亦有效,不同于VRP;研究还发现:TMP和第一代逆转剂VRP均可以抑制MDR1,但川芎嗪对MRP2,MRP3,MRP5亦有效,不同于VRP;TMP在使细胞内阿霉素浓度蓄积增加的同时,川芎嗪本身在细胞内的蓄积并未增加,说明川芎嗪并非P-gp底物,它使细胞内阿霉素浓度增加并非属于与阿霉素竞争受体结合位点所致。其机制值得进一步研究。综上所述,川芎嗪（Tetramethylpyrazine,TMP）对HCC的多药耐药有逆转作用,其机制与抑制P—gp的表达有关;实验还表明MDR1,MRP2,MRP3及MRP5在肝癌耐药株细胞中高表达可能发挥联合耐药作用,并能被TMP浓度依赖性逆转。研究还发现:TMP和第一代逆转剂VRP均可以抑制MDR1/P-gp,但川芎嗪对MRP2,MRP3,MRP5亦有效,不同于VRP;川芎嗪并非P-gp底物,它使细胞内阿霉素浓度增加并非属于与阿霉素竞争受体结合位点所致。其机制值得进一步研究。更多还原

【Abstract】 PartⅠEstablishment of ADM-induced resistant human hepatocellular carcinoma cell line BEL-7402/ADM1.Establishment of ADM-induced resistant human hepatocellular carcinoma cell line BEL-7402/ADMMethods:High-dose pulse therapy combined with continuous stepwise exposure to adriamycin（ADM） to induce resistant HCC cell line BEL-7402/ADM was conducted. The parental human hepatocellular carcinoma cells BEL-7402 was cultured,digested, and washed,and then cultured at the population of 2×10⁶ per culture bottle,ADM was added at the end concentration of 50nmol/L,the culture media was changed after 24 hours,washed by phosphate buffer solution（PBS）,digested by 0.25%trypsin, subculture period was ensured according to the proliferation of cells.When the HCC had been induced continuously with a low concentration of ADM for a few months, the suspended and the dead cells and the media had been discarded before the fresh media without drug was added.At the time of that HCC cells were covered on the two-thirds area of the plate,the media containing 2000 nmol/L ADM was added for pulse therapy,followed by continuous stepwise exposure in the media containing 200 nmol/L ADM.The steps repeated by using 600,800,1000 and 2000nmol/L of ADM, until drug-resistant BEL-7402/ADM cells were well grown in the media with 2000nmol/L ADM.Results:HCC cell line BEL-7402/ADM was established.BEL-7402/ADM cells were clustered and scattered,with less multiangular,increased refractivity in cytoplasm,more shuttle,and more easily digested compared with BEL-7402.2.Determination of reversal index of human hepatocellular carcinoma cells /ADM by MTT（3-（4,5-dimethylthiazolyl-2）-2, 5-diphenyltetrazolium bromide）Methods:Parental BEL-7402 and resistant BEL-7402/ADM cells in logarithmic phase of growth were digested by 0.25%trypsin,suspended in RPMI 1640 medium supplemented with 8%FCS,then transferred in 96-well plate,for 200μL（2×10⁴ cells/mL） per well,and cells were divided into 6 groups,blank,sham,ADM,5-FU, VCR and CDDP.Blank group was RPMI 1640 medium supplemented with 8%FCS without any drug and cells;Sham group were BEL-7402/ADM cells without any drug. Each group of drugs contained different concentrations,and there were 4 wells for each concentration of drugs.The absorbance was measured with a microplate reader at a test wavelength of 490 nm,and a reference wavelength of 620 nm.The inhibition rate of drugs was calculated as IR=(1-A_drug/A_sham)×100%.The IC₅₀ value was calculated as lgIC₅₀=Xm-Ⅰ[P-（3-Pm-Pn）/4](Xm:lg C_max;Ⅰ:lg(C_max/C_submax);P:sum of effects;Pm:E_max;Pn:E_min.Reversal index（RI）=(IC_50Resistance)/(IC_50Parental)×100%..Results:The data of MTT showed that IC₅₀ value of ADM on BEL-7402 and BEL-7402/ADM were（1.47±0.28）,（7.62±0.51）μmol/L,respectively;while data for 5 -FU were（4.56±0.72） and（21.53±2.47）μmol/L,respectively;VCR（8.98±0.30） and （80.27±11.22）μmol/L;CDDP（1.2±0.04） and（10.67±2.14）μmol/L,the reversal index of drugs were:ADM 5.18,5-FU 4.72,VCR 8.94,CDDP 8.89,respectively.3.Relationship between the concentration of ADM and the expression of P-gp in BEL-7402 by western blotMethods:Treated with 200,600,800,1000 and 2000 nmol/L of ADM,proteins in BEL-7402 and induced BEL-7402 cells were detected by western blot,andα-tublin as a reference.Blots were then exposed to a computer scanner and detected by ImageJ 1.38x（NIH.USA）.Data presented are mean±SD values compared with the percentage of the level of P-gp/α-tublin in parental group（control）.Results:The P-gp expression in well-cultured cells BEL-7402 in media containing 200,600,800,1000 and 2000 nmol/L ADM were（99.37±1.63）,（112.36±1.59）, （105.01±1.93）,（102.12±1.16） and（102.84±2.18）%compared with parental groups （the ratio of P-gp/α-tublin of parental calculated as 100%）,respectively,in which the expression of P-gp in 200nmol/L ADM had no statistic difference from control,but that in 600,800,1000 and 2000nmol/LADM did（P<0.05）.4.Analysis of cell cycle-phaseMethods:Parental BEL-7402 and resistant BEL-7402/ADM cells in logarithmic phase of growth were harvested,digested,centrifuged and washed.1×10⁶ per group was fixed by adding 70%ethanol.Samples were detected using 250μg/mL propidium iodide by flow cytocemetry.Results:Analysis of cell-cycle phase distribution was carried out on BEL-7402 and BEL-7402/ADM cells.The results showed that BEL-7402/ADM cells appeared lower value of G₀/G₁ and higher S and G₂/M（P<0.01） than that of BEL-7402.5.Intracellular accumulation of Adriamycin（ADM） in BEL-7402 and BEL-7402/ADM cells by flow cytometry（FCM）Methods:To assess the steady accumulation of ADM,BEL-7402 cells and BEL-7402/ADM cells were incubated with 8 000 nmol/L ADM for 2 h.The fluorescence intensity of intracellular ADM was recorded by FCM with an excitation wavelength（λex） of 480 nm and emission wavelength（μem） of 575 nm.Results:The mean fluorescence intensity of ADM in Resistance group was （82.08±6.89）%of Parental group（P<0.01）,indicating that there was a decreased intake of ADM in BEL-7402/ADM. 6.The adherence rate and the plate cloning efficiency of BEL-7402/ADMMethods:Parental BEL-7402 and resistant BEL-7402/ADM cells in logarithmic phase of growth were harvested and resuspended to be single-cell solution, respectively.The live cells were calculated on glass slide by trypan blue staining, 1×10⁶ cells per culture bottle was distributed for 6 groups,respectively.The adherent cells was calculated every 1 h as following:The adherence rate=(A_adherent/A_inoculated)×100%.Results:The data showed that the longer the time lasted,the greater the adherence rate of BEL-7402 and BEL-7402/ADM were.There was a statistic difference between the two groups at 2h,when the adherence rate of BEL-7402 was more than that of BEL-7402/ADM（P<0.05）.The plate cloning efficiency of BEL-7402 cells（54.44±5.83%） was more than that of the parental cells（68.28±8.06%）.Conclusion1.In this study,resistant human hepatocellular carcinoma cell line BEL-7402/ADM was established by adriamycin（ADM） using the model of high-dose pulse therapy combined with continuous stepwise exposure.The data indicated that ADM-induced resistant human hepatocellular carcinoma cell line BEL-7402/ADM was resistant to not only ADM,but also 5-FU,VCR and CDDP.The resistant cell line with such a phenotype would be weaker slightly after stored,but recovered and cultured for about 1 month,given induction again,the level ofphenotype would appear as before.2.The result of western blot showed that there was no effect of ADM at the concentration of 200 nmol/L on the P-gp expression in the cell line but at 600 nmol/L and more.3.As to the difference of cell cycle-phase between the parental cell line and the resistant one,the experimental results showed BEL-7402/ADM cells appeared lower value of G₀/G₁ and higher of S and G₂/M（P<0.01） than that of BEL-7402,while phase S had no change,there were differences on the adherence rate and the plate cloning efficiency between BEL-7402/ADM and BEL-7402,this result seemed to be different from other references,it maybe occur since different conditions,methods or/and deviations.4.The mechanism of the BEL-7402/ADM resistant to agents may associate with P-gp acting as a pump to the drugs according to the results of FCM and western blot.5.According to references as described previously,tumor cells are often cultured in the media containing 10%FCS,in this study,we used 8%FCS and obtained the same results,and saved a lot of agents. PartⅡReversal and mechanism of tetramethylpyrazine on HCC cell line BEL-7402/ADM1.Measurement of reversal of TMP on BEL-7402/ADM by methylthiazoletetrazolium（MTT） assayMethods:To assess the reversal effect,a measurement of cells proliferation by methylthiazoletetrazolium（MTT） assay was conducted as described previously. BEL-7402/ADM cells were preconditioned with TMP（400 and 600μmol/L） or VRP （5 and 10μmol/L） in 96-well plate for 24 h,respectively,then treated with 0.3,0.6,1.2, 2.4,4.8μmol/L of ADM in 96-well plate for 72 h,respectively.Verapamil（VRP） was conducted as positive control,and then the medium was discarded and the cells were washed by PBS for 3 times.IC₅₀ value,resistance index and reversal index were calculated as described previously.The resistance index（RI） is calculated as RI= (IC₅₀ Resistance group)/(IC₅₀ Parental group)×100%.The reversal index is calculated as RI=(IC₅₀ Resistance group)/(IC₅₀ TMP/VRP group)×100%.Results:Results from the experiment showed that the reversal index were 3.79（P<0.01） by TMP at the concentration of 400μmol/L and 14.65（P<0.01） at the concentration of 600μmol/L,respectively,similar to VRP,indicating that the reversal effects of regulators took place in a dose-dependent manner.2.Intracellular accumulation of Adriamycin（ADM） by fluorescence microscopyMethods:With/without TMP（600μmol/L） or VRP（5μmol/L） preconditioning in 3 hours,BEL-7402 cells and BEL-7402/ADM were incubated with 8 000 nmol/L ADM for 2h at 37℃,washed three times with ice-cold PBS.With an excitation（^λex） wavelength of 488 nm and emission（^λem） wavelength of 575 nm,the fluorescence intensity of intracellular ADM was directly observed by fluorescence microscopy. Results:By the photos taken with fluorescence microscopy,red fluorescence was caught,increased accumulation of ADM in BEL-7402/ADM cells treated with TMP or VRP compared with that in BEL-7402/ADM cells treated without TMP or VRP, following the parental group,was observed directly.3.Intracellular accumulation of ADM in HCC cell lines by FCMMethods:With/without TMP（600μmol/L） or VRP（5μmol/L） preconditioning in 3 hours,BEL-7402 cells and BEL-7402/ADM were incubated with 8 000 nmol/L ADM for 2 h at 37℃,washed three times with ice-cold PBS.With an excitation（^λex） wavelength of 488 nm and emission（^λem） wavelength of 575 nm,the mean fluorescence intensity of intracellular ADM was recorded by FCM.Results:The mean fluorescence intensity of intracellular ADM in Parental group, TMP group and VRP group were（121.83±23.2）%（P<0.01）,（163.78±39.5）%（P<0.01） and（320.1±47.18）%（P<0.01） compared to Resistance group,indicating that TMP can increase accumulation of ADM in BEL-7402/ADM.4.Intracellular accumulation of ADM by high performance liquid chromatography（HPLC）Methods:the parental and resistant cell lines were divided into Parental,Resistance, TMP（with 600μmol/L TMP） and VRP（with 5μmol/L VRP）.With/without TMP or VRP preconditioning for 3 hours,BEL-7402 cells and BEL-7402/ADM were incubated with 8 000 nmol/L ADM for 2 h,washed three times with ice-cold PBS. The accumulation of ADM in cells was also quantified by the HPLC method.The linear,reproducibility,recovery,precision,accuracy,stability were observed and calculated.Results:The peak area ratio of ADM and internal standard alprazolam was obtained and calculated by HPLC.The calibration curve was obtained as the following with the content ranging from o.1 ng/mL to 2 000 ng/mL.The limit of detection（LOD） was 0.05ng/mL,and RSD was 0.407.The standard curve was C=47.73A+23.62 （correlation factor:0.99 8）,where C refers to the concentration of ADM,A refers to the peak area ratio of ADM and alprazolam.The mean concentration of ADM in Parental group,Resistance group,TMP group and VRP group were （2.45±0.52）,（2.06±0.40）μmol/L,（3.63±0.59） and（3.92±0.97）,respectively,indicating that TMP can increase accumulation of ADM in BEL-7402/ADM as same as the result by FCM.The study also indicated that TMP increased the level of ADM without increased itself accumulation in the resistant cells,inferring TMP was not the substrate of P-gp like ADM/VRP and did not compete the ABC proteins with ADM.5.Effects of TMP on expression levels of MDR1,MRP2,MRP3 and MRP5 mRNA by real-time reverse-transcription-PCR analysisMethods:Total RNA was isolated from BEL-7402 and BEL-7402/ADM in RNA clean solution by Trizole Reagent according to the manufacturer’s protocol.All primer pairs and their appropriate fluorescent hybridization probes were designed and produced by Shanghai Sangon Biological Engineering Technology and Services CO., Ltd.（China）.The mRNA levels of MDR1,MRP2,MRP3 and MRP5 were measured by real-time RT-PCR and quantitated by SLAN Real-time PCR Detection System.In addition,the mRNA level of the reference geneβ-actin was measured and used to normalize the mRNA levels of the drug resistance genes.Results:The data were calculated as the expression level of mRNA of Resistance group was 100%.It was showed TMP decreased the expression of MDR1,MRP2, MRP3 and MRP5 mRNA to（0.67±0.03）,（0.85±0.10）,（0.90±0.04） and （0.63±0.11） compared to Resistant group（P<0.05）,but this only took place on the expression of MDR1 mRNA in VRP group,as there was no statistical difference in the expression of MRP2,MRP3 and MRP5 mRNA in VRP group.6.Effects of TMP on expression levels of P-gp MRP2,MRP3 and MRP5 proteins by Western blot analysis（WB） Methods:BEL-7402 and BEL-7402/ADM cells were divided into Resistance,TMP and VRP group,and preconditioned with TMP（600μmol/L） and VRP（5μmol/L） for 3 hours,then treated with 8 000 nmol/L ADM for 2 hours.The proteins level of P-gp, MRP2,MRP3 and MRP5 were detected andβ-actin as a reference by western blot.Results:We found that compared to Resistance group,the expression of P-gp, MRP2,MRP3 and MRP5 was entirely decreased to（0.49±0.04）,（0.61±0.13）, （0.38±0.14） and（0.68±0.07）（P<0.01） in TMP group,however,except the expression of P-gp in VRP group,there was no statistical difference in the expression of MRP2, MRP3 and MRP5 in VRP group.Conclusion1.Tetramethylpyrazine（TMP） is a bioactive constituent isolated from the root of Ligusticum chuanxiong Hort,a Chinese herb（Chuanxiong）,which can enhance the chemosensitivity effects of drug on human hepatocellular carcinoma BEL-7402 cells, acting as a MDR modulator.However,it is unknown till now if it has effect on other transporters till now.In this study,the reversal effect of TMP on MDR in multidrug resistant human hepatocellular carcinoma BEL-7402/ADM was investigated and the relationships among TMP and MDR1,MRP2,MRP3 and MRP5 were explored.It was concluded that TMP can be reverse the resistant level of BEL-7402/ADM in a dose-dependent manner.2.The Intracellular accumulation of ADM preconditioned by TMP decreased in resistant BEL-7402/ADM suggested that TMP would have effects on one and/or more ABC super family proteins.3.MDR1/P-gp,MRP2,MRP3,and MRP5 are of significance to contribute to MDR mechanism jointly in resistant HCC,and can be reversed by TMP.4.Both TMP and the first generation modulator,VRP have effects on MDR1/P-gp, TMP also has effects on MRP2,MRP3 and MRP5,but VRP has not,which deserves further study.We also found that the Intracellular accumulation of TMP in resistant cells did not alternate indicating that unlike ADM and VRP,TMP is not the substrate of P-gp/ABC family members and not resulting in competitive inhibition to ADM.更多还原