槲皮素对多药耐药细胞株KB-MRP的耐药逆转研究

目的:研究槲皮素(quercetin)对多药耐药细胞株KB-MRP的耐药逆转作用。方法:使用四甲基偶氮唑盐(MTT)法检测不同浓度槲皮素对细胞株KB-MRP的抑制率,确定其非细胞毒性浓度。用MTT法分别检测阿霉素(ADM)、依立替康(CPT-11)、长春新碱(VCR)、环磷酰胺(CTX)对细胞株KB-3-1、KB-MRP以及使用槲皮素处理后的KB-MRP的半数抑制浓度(IC50)。通过流式细胞术检测KB-3-1、加入槲皮素前后KB-MRP细胞内的化疗药物阿霉素(ADM)的荧光强度以及加入槲皮素前后多药耐药细胞株KB-MRP的MRP1蛋白表达。结果:终浓度为0~40μmol/L的槲皮素对KB-MRP无明显细胞毒作用。KB-MRP对ADM、VCR、CPT-11均有不同程度的耐药但对CTX不耐药。使用20μmol/L槲皮素处理后KB-MRP对上述ADM、CPT-11、VCR的敏感度均有不同程度的提高,胞内的化疗药物阿霉素(ADM)的荧光强度增强。槲皮素的浓度提高至40μmol/L时,KB-MRP对上述药物的敏感程度提高更明显。流式细胞仪结果显示经槲皮素处理后KB-MRP细胞MRP1表达明显下降。结论:槲皮素可以逆转KB-MRP的多药耐药,逆转效果与剂量相关,逆转机制可能与MRP1的表达下调有关。     

十字孢碱促进多柔比星诱导的多药抗药性肿瘤细胞系的凋亡

 【摘要】　目的　明确蛋白激酶C（PKC）活性的抑制是否能促进化疗药物诱导的多药抗药肿瘤细胞系的凋亡。方法　选用口腔鳞癌细胞KB／S及其多药抗药株KB／VCR，常规细胞培养，比较单独或联合PKC抑制剂十字孢碱的情况下，多柔比星（ADM）诱导这2种细胞的凋亡情况。凋亡采用流式细胞术和吖啶橙荧光染色检测，并经电子显微镜观察证实。结果　ADM 0.04 μg／ml，作用36 h有96.68 % KB／S细胞凋亡，作用48 h有64.99 ％的KB／VCR细胞凋亡；将ADM质量浓度增加到0.4 μg／ml和2.0 μg／ml时，KB／VCR细胞凋亡比例分别为69.74 ％和37.18 ％；合用十字孢碱后，凋亡细胞比例分别增加到82.58 ％和47.65 ％，经统计学处理，前者χ 2 = 4.5，P＜0.05；后者χ 2 = 2.2，P＞0.05。这些结果均经电镜和吖啶橙染色证实。结论　耐受凋亡可能是肿瘤细胞多药抗药的机制之一，而PKC抑制剂可解除这种耐受。     

鞣酸对LLC/cMOAT细胞多药耐药性的逆转机制

目的本研究探讨鞣酸(Tannic acid,TA)对LLC/cMOAT细胞多药耐药性的逆转作用及其可能机制。方法采用MTT法检测LLC/CMV和LLC/cMOAT细胞对各种化疗药物的敏感性以及在不同浓度的TA处理下细胞的存活状态;确定TA的非细胞毒性浓度以及在该浓度下各梯度浓度处理时细胞的存活率;采用流式细胞仪技术检测使用非细胞毒性浓度的TA处理前后LLC/cMOAT细胞内柔红霉素(DNR)浓度变化、细胞周期阻滞及对凋亡的影响。结果LLC/cMOAT细胞对羟基喜树碱(CPT-11),阿霉素(ADM),顺铂(DDP)和长春新碱(VCR)均有不同程度的耐药,其耐药倍数分别是2.08、3.02、4.01和9.31,对依托泊苷(VP-16)、紫衫醇(TAX)无明显耐药,10.0μmol/L浓度以下的TA对LLC/CMV和LLC/cMOAT细胞无明显细胞毒性,2.5、5.0、10.0μmol/L浓度的TA能显著降低LLC/cMOAT细胞的IC₅₀(P<0.05),其逆转倍数分别为5.09、6.33、7.76倍;细胞内DNR荧光强度随着TA浓度的增高而明显增强;TA作用细胞12h可使G₁期细胞数百分比由(46.35±3.74) %增至(66. 43 ±5. 87) % , 阻滞细胞于G₁期; TA 与VCR 联合处理细胞24 h 和48 h 时凋亡率分别为12. 1 %和19. 0 % ,与处理前(1. 65 %) 相比提高明显;使用2. 5μmol/ L 、5.0μmol/ L 和10. 0μmol/ L 浓度的TA 与VCR 共同处理细胞24 h 后,凋亡率由处理前的4. 96 %分别提高到11. 2 %、17. 9 %和25. 1 %。结论　TA能部分逆转LLC/ cMOAT 细胞的多药耐药,显著提高耐药细胞内DNR 荧光强度并呈浓度依赖性,阻滞细胞周期于G₁ 期,并可诱导细胞凋亡,呈时间、剂量依赖性,其机制可能与抑制化疗药物外排、增加细胞内药物浓度以及诱导细胞凋亡有关。     

几种化疗药物对肝癌耐药细胞BEL7404/ADM端粒酶活性的影响

目的:研究顺铂(DDP)、丝裂霉素(MMC)、多柔比星(ADM)和长春新碱(VCR)4种化疗药物对人肝癌细胞株BEL-7404、耐阿霉素肝癌细胞株BEL-7404/ADM的细胞毒作用及其对端粒酶活性影响的关系。方法:采用MTT法测定常用4种(DDP、MMC、ADM和VCR)化疗药物对人肝癌细胞株BEL-7404、BEL-7404/ADM的体外抑制作用;以TRAP-PCR-ELISA法检测化疗药物作用后两种细胞端粒酶的活性。结果:DDP、MMC、ADM、VCR对BEL-7404均敏感,各种化疗药物IC50大小比较,DDP和ADM比VCR和MMC均小;而对BEL-7404/ADM这几种化疗药物敏感性均不同程度下降,其中以ADM及VCR尤其明显,耐药倍数分别为100·0倍、60·14倍。未加药前,肝癌亲本细胞BEL-7404和耐药细胞BEL-7404/ADM端粒酶活性均表达,加入化疗药物后,亲本细胞端粒酶活性下降明显,并呈现时间-剂量关系,而对于耐药细胞,只有高浓度的化疗药物才能抑制其端粒酶活性。结论:端粒酶活性的抑制与耐药细胞对药物敏感性存在一定关系,端粒酶可作为化疗敏感的潜在标志。     

氯丙嗪对耐药细胞系K562/AO2多药耐药逆转作用的研究

目的研究氯丙嗪对耐药细胞系K562/AO2多药耐药逆转作用.方法应用免疫组化观察K562/AO2细胞系的耐药蛋白表达情况,用MTT法测定不同浓度的氯丙嗪对K562/AO2细胞系耐药逆转作用,用流式细胞术测定不同浓度的氯丙嗪与K562/AO2细胞作用后细胞内罗丹明的蓄积情况,用半定量RT-PCR法测定氯丙嗪对K562/AO2细胞多药耐药基因(mdr-1)mRNA表达的影响.结果K562/AO2细胞不但P-gp表达阳性,而且肺耐药相关蛋白(lung resistanceelatedprotein,LRP)表达也阳性;氯丙嗪能增强多柔比星对K562/AO2细胞的杀伤作用(单用ADM组、氯丙嗪0.75 μg/mL+ADM组、氯丙嗪1.5μg/m L+ADM组和氯丙嗪3μg/mL+ADM组对K562/AO2的抑制率分别为5.2%、25.9%、39.1%和74.8%)增加K562/AO2细胞内罗丹明的蓄积(对照组、氯丙嗪0.75 μg/mL组、氯丙嗪1.5 μg/mL组和氯丙嗪3μg/mL组细胞内的荧光强度的均值分别为1.87、10.28、48.75和65.63)对K562/AO2细胞mdr-1 mRNA表达无明显影响(对照组mdr-1和β-actin的面积比为0.41,氯丙嗪组为0.42).结论氯丙嗪对K562/AO2细胞的耐药有较强的逆转作用,并呈剂量依赖关系.     

TNFα诱导K562/VCR和K562细胞凋亡及逆转其耐药作用的实验研究

目的探讨肿瘤坏死因子α(TNFα)体外诱导K562/VCR及K562细胞凋亡,及其逆转K562/VCR细胞多药耐药(MDR)的作用机制.方法以光镜、电镜、流式细胞仪观察细胞凋亡;流式细胞仪检测P-gp和bcl-2表达.MTT检测药物敏感性.结果 (1)TNFα浓度>100 U/ml,作用时间>48小时,2株细胞均可见典型凋亡现象,以K562/VCR细胞明显.(2)以TNFα 1 000 U/ml处理后,K562/VCR和K562细胞凋亡率分别为29.4%和10.7%.(3)K562/VCR经TNFα 1 000 U/ml处理72小时P-gp表达率从93.6%降至82.4%,bcl-2表达率从32.9%降至7.0%.(4)K562/VCR和K562细胞分别经TNFα 100 U/ml和1 000 U/ml处理后,VCR、Ara-C和VP-16的药物敏感性增加(P<0.05).结论 (1)TNFα可诱导K562/VCR和K562细胞凋亡,以前者更加敏感,且存在时间和剂量依赖性.(2)高浓度TNFα可下调bcl-2表达,但不显著改变P-gp表达.(3)TNFα能增加K562/VCR和K562细胞对VCR、Ara-C和VP-16的药物敏感性,以K562/VCR细胞更明显.(4)TNFα逆转K562/VCR多药耐药是通过诱导细胞凋亡,降低bcl-2表达,而非下调P-gp表达途径实现.     

miR-135a在调节小细胞肺癌多药耐药中的作用

目的:探讨miR-135a在小细胞肺癌(small cell lung cancer,SCLC)多药耐药中的作用。方法:收集2008年1月至2013年12月本院肿瘤科及呼吸内科就诊的48例SCLC患者外周血液标本,将其分为化疗敏感组(28例)和耐药组(20例),采用QRT-PCR法检测患者血液标本、SCLC敏感细胞株H69及耐药细胞株H69AR中miR-135a的表达,分析其与临床预后的关系,并应用miR-135a模拟物和抑制剂分别上、下调细胞株中miR-135a的表达,采用CCK-8法检测细胞对各种化疗药物(ADM、DDP和VP-16)敏感性的影响。结果:与化疗敏感组相比,miR-135a在化疗耐药患者血液标本中的表达明显增高[(2.174±3.981)vs(-1.963±3.750),P<0.001];在临床患者血液标本中,miR-135a的表达与患者的性别、年龄无关(P>0.05),与疾病的分期、化疗敏感性及患者的生存时间相关(均P<0.01)。miR-135a在H69AR细胞中的表达明显高于H69细胞[(7.841±0.392)vs(1.047±0.081),P<0.01];通过miR-135a抑制剂下调H69AR中miR-135a的表达能够显著增加细胞对化疗药物的敏感性,H69AR细胞对DDP、ADM及VP-16的IC50值明显下降[(247.09±11.55)vs(76.64±10.00)、(150.83±8.03)vs(40.72±5.06)、(436.63±61.19)vs(163.35±20.00);H69细胞转染miR-135a mimics后对化疗药物DDP、ADM和VP-16的IC50值明显增加[(18.58±1.37)vs(159.27±3.29)、(24.37±2.63)vs(129.19±2.64),(48.55±2.59)vs(359.87±2.92)μg/ml,P<0.01]。结论:miR-135a参与调节SCLC的多药耐药,下调miR-135a基因的表达可能增加细胞对化疗药物的敏感性。     

肠胃清药物血清对人口腔表皮癌细胞株KB-A-1多药耐药的逆转作用

目的评价中药复方肠胃清口服液对肿瘤细胞多药耐药的逆转作用.方法采用连续灌胃给药3 d后的SD大鼠的肠胃清药物血清,作用人口腔表皮癌敏感细胞株KB-3-1和耐药细胞株KB-A-1,运用MTT等方法,观察肠胃清对KB-3-1和KB-A-1细胞的生长抑制作用及对KB-A-1细胞的多药耐药的逆转作用.结果肠胃清药物血清在2.5%～20%浓度时对KB-3-1和KB-A-1细胞的生长均有抑制作用,呈剂量依赖性,对KB-A-1和KB-3-1的IC50分别是(8.5±16.8)%和(10±15.6)%.在对KB-3-1和KB-A-1细胞的生长无毒性的含药血清浓度为2.5%、5%的作用下,阿霉素对KB-A-1细胞的IC50从(10.3±0.9)μg/mL分别下降到(4.2±0.2)μg/mL和(2.0±0.1)μg/mL,逆转倍数分别达到了2.5和5.2倍,而对KB-3-1细胞则不产生逆转作用.结论肠胃清可有效逆转多药耐药细胞KB-A-1的耐药作用.     

汉防己甲素逆转白血病细胞株K562/ADM多药耐药性机制研究

目的 研究汉防己甲素(TTD)对慢性粒细胞白血病急性变白血病细胞株K562／ADM多药耐药(MDR)逆转的机理。方法 采用流式细胞仪检测细胞内化疗药物的浓度及细胞表面P糖蛋白(P-gp)的表达；荧光定量PCR法检测MDR1 mRNA；通过流式细胞仪检测Anexin—V判断凋亡细胞的数量。结果 10μmol/L的TTD处理K562／ADM细胞后，细胞内阿霉素(ADM)的浓度明显提高；K562／ADM细胞MDR1 mRNA／P-gp的表达下降；TTD能增强ADM致细胞凋亡的作用。结论 汉防己甲素的耐药逆转机制除了下调MDR1 mRNA／P-gp的表达引起细胞内抗癌药物的积聚外，增加抗癌药物致细胞凋亡也是耐药逆转的重要原因。．     

川芎嗪与β-榄香烯联合应用诱导K562/ADM细胞凋亡及逆转其MDR的实验研究

目的:探讨不同作用机制的川芎嗪(Tetrainethylpyrazine,TMP)与β-榄香烯(β-elemene)联合应用,从多环节逆转耐药细胞K562/ADM的多药耐药(multidrug resistance,MDR),以期进一步提高逆转效果.方法:采用MTT法检测细胞的药敏性及抗药性逆转,流式细胞术及透射电镜检测细胞凋亡,应用SPSS(10.0 Production Pacility)软件包对实验结果进行统计学处理.结果:1)非细胞毒性浓度的TMP(350μg/ml)及β-榄香烯(4.0μg/ml)可显著降低ADM对K562/ADM细胞的IC50(P＜0.01),逆转耐药倍数分别为2.03倍及2.18倍.β-榄香烯(4.0μg/ml)具有诱导该细胞凋亡的作用.2)上述两种药物以非细胞毒性浓度联合应用,对ADM的逆转倍数为4.65倍,明显高于二者单独应用,而且也高于两者单独应用之和.其主要逆转机制是两药的联合应用通过升高细胞内ADM的浓度,诱导该细胞凋亡的途径,实现提高逆转效果的目的.结论:TMP和β-e榄香烯联合应用能明显逆转K562/ADM细胞对ADM的耐药性和诱导该细胞的凋亡.     

顺铂与足叶乙甙对白血病细胞K562的协同杀伤作用及其机制

背景与目的:足叶乙甙(etoposide,VP鄄16)为白血病化疗最常用的化疗药之一,其临床应用日益受到天然与继发耐药的影响。对一些实体瘤的研究发现顺铂(cisplatin,DDP)与VP鄄16联合应用具有协同效应。本研究通过DDP与VP鄄16联合作用杀伤白血病细胞K562,探讨其增强VP鄄16疗效的作用机制。方法:采用VP鄄16(0,5μg/ml)与不同浓度DDP(0,0.3,3,15,30μg/ml)联合对K562细胞进行处理。应用噻唑蓝(MTT)法检测用药后细胞的存活相对数量,计算VP鄄16和DDP应用对K562细胞的抑制作用;AO/EB双荧光法定量分析细胞凋亡率。半定量RT鄄PCR法和Westernblot检测拓扑异构酶(topoisomerase,TOPO)Ⅱα、Ⅱβ、Sp1、Sp3基因的mRNA和蛋白水平。结果:VP鄄16与DDP合用具有明显的协同效应。两药合用后,细胞凋亡率明显增加。单独应用DDP可以明显上调TOPOⅡ和Sp1表达(呈浓度依赖,TOPOⅡα、Ⅱβ、Sp1在30μg/mlDDP时比正常对照分别上调36%,25%和75%),并使Sp3的表达降低49%;单用5μg/mlVP鄄16时TOPOⅡ则下调(TOPOⅡα下降72%),TOPOⅡβ和Sp1的表达未受影响,Sp3的表达上调14%。5μg/mlVP鄄16与DDP联合应用具有协同效应,使TOPOⅡ和Sp1表达水平升高。TOPOⅡα、Ⅱβ、Sp1在5μg/mlVP鄄16与30μg/mlDDP联合应用时比单用VP鄄16分别上升83%,11%,58%,但低于单独应用DDP的表达水平;而Sp3的表达水平与单独应用DDP比较下调61.3%。Western印迹检测结果与RT鄄PCR结果相符。结论:DDP在化疗中与VP鄄16发挥协同作用,通过上调拓扑异构酶Ⅱ的表达水平,为VP鄄16提供更多的靶酶,使VP鄄16杀伤K562细胞效果明显提高。     

RNAi对白血病细胞mdr-1基因和多药耐药表型的影响

目的:探讨RNA干扰技术(RNAi)对慢性粒细胞白血病急变细胞系K562/AO2细胞mdr1基因的抑制和耐药表型的逆转作用。方法:选择合成封闭mdr-1基因的小干扰序列(si-MDR1),以1个碱基突变的si-MDR1-mut为对照序列,在脂质体介导下转染至K562/AO2细胞系。RT-PCR和Western blot检测mdr1 mRNA及P-gp蛋白水平,流式细胞术分析细胞内柔红霉素(daunorubicin,DNR)积累量,并以四甲基唑蓝快速比色法(MTT)反映K562/AO2对阿霉素、长春新碱、足叶乙甙药物敏感性的变化。结果:实验证实该序列能高效封闭K562/AO2细胞内mdr-1基因表达,增加细胞内化疗药物DNR积累量,增强K562/AO2细胞对阿霉素、长春新碱、足叶乙甙的敏感性。结论:RNAi可以通过抑制mdr1基因表达,逆转K562/AO2细胞耐药表型。     

A new quinoline derivative MS-209 reverses multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells.

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.     

Overcoming of multidrug resistance by introducing the apoptosis gene, bcl-Xs, into MRP-overexpressing drug resistant cells

Multidrug resistance associated protein (MRP) is one of drug transport membranes that confer multidrug resistance in cancer cells. Multidrug resistance has been known to be associated with resistance to apoptosis. In this study, using MRP overexpressing multidrug resistant nasopharyngeal cancer cells, we examined the expression of apoptosis related genes including p53, p21WAF1, bax and bcl-Xs between drug sensitive KB and its resistant KB/7D cells. We also examined whether the introduction of apoptosis related gene could increase the sensitivity to anticancer drugs in association with apoptotic cell death. The relative resistances to anticancer drugs in KB/7D cells evaluated by IC50 values were 3.6, 61.3, 10.4 and 10.5 to adriamycin (ADM), etoposide (VP-16), vincristine (VCR) and vindesine (VDS), respectively. The resistance to anticancer drugs in KB/7D cells was associated with the attenuation of internucleosomal DNA ladder formation in apoptosis. Of important, the mRNA expression of bcl-Xs gene in KB/7D cells was decreased in one-fourth as compared to that of KB cells among the apoptosis genes. The mRNA expression of bcl-Xs gene in a bcl-Xs transfected clone (KB/7Dbcl-Xs) was increased about 2-fold compared to that of KB/7Dneo cells, while the mRNA expression of MRP gene was not significantly different in KB/7bcl-Xs and KB/7Dneo cells. The sensitivities to anticancer drugs including ADM, VCR and VDS except VP-16 were increased in KB/7Dbcl-Xs cells, in turn, the relative resistance in KB/7Dbcl-Xs cells was decreased to 1.4, 4.0, and 3.0 in ADM, VCR and VDS, respectively, as compared to those of KB/7Dneo cells. Of interest, the studies on the accumulation of [3H]VCR showed that the decrease of [3H]VCR accumulation in KB/7Dbcl-Xs was not significantly different from that of KB/7Dneo cells. Collectively, these results indicated that the mechanism(s) of drug resistance in KB/7D cells could be explained at least by two factors: a) reduced drug accumulation mediated by MRP; b) resistance to apoptosis due to the decreased expression of the bcl-Xs gene. These results also indicated that the multidrug resistance mediated by MRP might be partially overcome by introducing apoptosis gene into drug resistant cells without modulation of MRP function in drug accumulation.     

A New Quinoline Derivative MS-209 Reverses Multidrug Resistance and Inhibits Multiorgan Metastases by P-glycoprotein-expressing Human Small Cell Lung Cancer Cells

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3/ADM and H69/VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3/ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro , compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3/ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3/ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3/ ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3/ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3/ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3/ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3/ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.     

野生型p53基因对肝癌细胞多药耐药的逆转作用及其相关机制

背景与目的：细胞凋亡的发生机制在肿瘤多药耐药中起重要作用,野生型p53基因是细胞凋亡的激活剂,与肿瘤多药耐药密切相关。本研究旨在探讨野生型p53基因能否实现对肝癌细胞多药耐药的逆转以及逆转的相关机制。方法：构建野生型P53蛋白表达序列的真核表达载体pCR3．1-p53,用脂质体转染技术建立人肝癌细胞Bel-7402的p53转染细胞株,对转染细胞株进行MTT实验,观察p53基因对肿瘤细胞生长的抑制作用和对肝癌细胞对长春新碱（vincristine,VCR）药物敏感性的影响,姬姆萨染色法观察细胞形态,免疫组织化学SP法检测细胞P糖蛋白（P—glycoprotein,P—gp）的表达,逆转录PCR法检测细胞内mdr1、MRP、GSTπ、TopoⅡ mRNA的表达。结果：转染p53的Bel—p53细胞生长明显慢于未转染的Bel-7402细胞。转染野生型p53的Bel-p53细胞在VCR浓度为0．01μg／ml,0．1μg／ml,1．0μg／ml,10μg／ml,25μg／ml时,其药物敏感性增加;VCR作用最佳浓度为1．0μg／ml。形态学检查转染细胞,在VCR作用下细胞数明显减少,细胞散在不成片、细胞高度水肿、胞体不规则突起,可见核固缩、核裂解、核溶解。p53基因对Bel-7402细胞的mdr1／P—gP的表达有明显的抑制作用．对TopoⅡα基因的表达有上调作用,对GSTπ、MRP基因表达没有影响。结论：p53基因对肝癌细胞多药耐药有逆转作用．其逆转机制可能与降低mdr1／P—gp的表达、上调TopoⅡα的表达．从而增加细胞内VCR药物浓度和VCR药效有关。     

Reversal of drug resistance using hammerhead ribozymes against multidrug resistance-associated protein and multidrug resistance 1 gene

We examined the effects of suppressing multidrug resistance-associated protein (MRP) and multidrug resistance 1 (MDR1) gene expression in HCT-8DDP human colon cancer cell lines, which showed both cisplatin and multidrug resistance. Hammerhead ribozymes, designed to cleave MRP mRNA (anti-MRP Rz) and MDR1 mRNA (anti-MDR1 Rz), were transfected into the HCT-8DDP cells. Drug sensitivity was estimated by MTT assay in vitro. The HCT-8DDP/anti-MRP Rz cells were more sensitive to doxorubicin (DOX) and etoposide (VP-16) by 2.5- and 4.1-fold, respectively, compared with HCT-8DDP cells. The HCT-8DDP/anti-MDR Rz cells were more sensitive to DOX and VP-16 by 2.3- and 3.8-fold, respectively. The anti-MRP Rz and anti-MDR1 Rz significantly down-regulated resistance to DOX and VP-16, while anti-MRP Rz and anti-MDR1 Rz did not affect resistance to cisplatin, methotrexate and 5-fluorouracil. The hammerhead ribozyme-mediated specific suppression of MRP or MDR1 was sufficient to reverse multidrug resistance in the human colon cancer cell line.     

Detection of multidrug resistant phenotype in leukemia and lymphoma by monoclonal antibodies

Two monoclonal antibodies, MRK16 and MRK20 that recognize P-glycoprotein and P-85 kd protein on the surface of adriamycin (ADM) resistant cells, respectively, were tested for the reactivity with 40 cultured leukemia/lymphoma cell lines. F(ab')2 form is essential to avoid false reaction through Fc gamma-R. Drug sensitivity of 19 representative cell lines were also examined in vitro. From this study, it was found that these cell lines were classified into 4 groups. Group 1 (4 cell lines) was insensitive to ADM, mitoxantron (MXT), etoposide (VP-16) and vincristine (VCR), and reactive to MRK16 and MRL20. Group II (1 cell line) was insensitive to the 4 drugs, but not reactive to both antibodies. Group III (3 cell lines) was insensitive to ADM, MXT and VP-16, but sensitive to VCR, and reactive to MRK20, but not to MRK16. Group IV (all other cell lines) was sensitive to these drugs, and not reactive to both antibodies. From these results, MRK16 detects P-glycoprotein-associated multidrug resistance (MDR), while MRK20 does P 85-kd-associated another type MDR (cross resistance to ADM, MXT and VP-16, but not to VCR). MRK20 reacted with monocytes, but MRK16 did not with any WBC type. One hundred and ninety eight clinical samples obtained from blood cancer were tested for the reactivity with MRK16. MRK16 did not react with any of 98 samples obtained before treatment, but did with 9 of 100 obtained at relapse or refractory stage after chemotherapy. The results indicate that MRK16 is useful to detect drug resistance phenotype of leukemia and lymphoma.     

5-溴汉防己甲素与汉防己甲素对K562/A02细胞多药耐药性逆转作用的比较

背景与目的:5-溴汉防己甲素(5-bromotetrandrine,BrTet)是汉防己甲素(tetrandrine,Tet)的溴化产物,具有逆转P-糖蛋白(P-glyeoprotein,P-gp)介导的肿瘤多药耐药(muhidrug resistance,MDR)的作用。本研究旨在比较BrTet与Tet对人白血病细胞K562/A02多药耐药的逆转作用。方法:采用四甲基偶氮唑蓝法(MTT)法检测不同浓度BrTet对K562细胞和K562/A02细胞的增殖抑制效应;检测阿霉素(adfiamycin,ADM)对K562细胞和K562/A02细胞增殖的抑制作用,以及加用BrTet、Tet时上述抑制作用的变化,并计算半数抑制浓度(IC50)及逆转倍数。Westernblot法检测各组细胞P-gp的表达,流式细胞仪检测各组细胞内ADM的蓄积。结果:K562/A02细胞对ADM的耐药倍数为49.51倍。2.0μmol/L及更低浓度的BrTet和1.5μmol/L及更低浓度的Tet对K562细胞和K562/A02细胞抑制率均小于10%,无明显细胞毒性作用。加入1.0μmol/L的Tet后,K562/A02细胞对ADM的耐药倍数为12.17倍。加入0.25、0.5和1.0μmol/L的BrTet后,K562/A02细胞对ADM的耐药倍数分别为17.88、9.97和4.24倍。1.0"mol/L的BrTet和Tet分别使K562/A02细胞内ADM浓度提高了69.0%和51.6%,使P-gp表达分别下调了51.1%和43.73%,其差异具有统计学意义(P<0.05)。结论:BrTet及Tet均可逆转K562/A02细胞耐药,且前者较后者逆转作用更强,逆转机制与抑制P-gp的表达、增加细胞内抗肿瘤药物浓度有关。     

食管癌患者外周血淋巴细胞替代肿瘤细胞体外药敏试验相关性研究

背景与目的探讨食管癌患者外周血淋巴细胞与肿瘤细胞体外药敏试验的相关性。方法与材料采用四甲基偶氮唑蓝[3-(4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide,MTT]法体外药敏试验,检测了32例食管癌患者外周血淋巴细胞和肿瘤细胞对9种临床常用化疗药物的敏感性。结果食管癌患者外周血淋巴细胞和肿瘤细胞对5-氟脲嘧啶(5-fluorouracilum,5-Fu)、顺铂(Diamminedich-loroplatinum,DDP)、卡铂(Carboplatinum,CBP)、氨甲喋呤(Methotrexate,MTX)、环磷酰胺(Cyclophosphamide,CTX)敏感;对阿霉素(Adriamycin,ADM)、长春新碱(Vincristin,VCR)不敏感;对足叶乙甙(Etoposide,Vp16)和吡柔比星(Pirarubicin,THP)两者的敏感性不同。结论食管癌患者肿瘤细胞和外周血淋巴细胞对上述化疗药物敏感率间差异无统计学意义。食管癌外周血淋巴细胞化疗药敏检测对临床选择化疗药物具有重要参考价值。对无法取得肿瘤标本的患者,可用外周血淋巴细胞替代癌细胞进行药敏检测。