人结肠癌HCTv2000细胞系耐药机制的初步研究

ＨＣＣＴｖ２０００是人结肠癌ＨＣＴ－８细胞系经长春新碱选择而建立的耐药系。该系细胞对抗Ｐ－糖蛋白单抗呈阳性反应。编码Ｐ－糖蛋白的多向耐药基因ｍｄｒｌ未见扩增或重排，但ｍｄｒｌ ｍＲＮＡ过度表达。异博定使细胞内Ｈ－长春新碱蓄积增多，可部分逆转耐药。据此认为，ｍｄｒｌａｄｌｄｒ ｌｆｎｙｖｉｕ ｎｔｇ ｉｒｙｔｉｔｄｇｊ ＨＣＴｖ２０００细胞耐药的重要原因，但有其他机制参与。     

人结肠癌耐药细胞系HCTv2000的建立

ＨＣＴ－８是一种体外培养的人结肠癌细胞系，本文用长春新碱选择得到对多种抗肿瘤药物交叉耐药的ＨＣＴｖ２０００细胞系，与ＨＣＴ－８细胞相比，ＨＣＴｖ２０００对长春碱的耐药程度为１４７倍，秋水仙碱为６５倍，高三尖杉酯碱为４７倍，紫杉醇为３１倍，阿霉素为２０倍，对鬼臼乙叉甙等也产生了耐药性，但对５－氟尿嘧啶的敏感性无明显改变，符合我向耐药表型特点。ＨＣＴｖ２０００细胞生长状态良好，耐药性能稳定，可以用作分     

人肝癌原发性耐药细胞亚系的建立及耐药机制的初步探讨

目的从人肝癌细胞系HeFG2中筛选出原发性耐药株,建立相应的细胞亚系并探讨其耐药机制.方法采用阿霉素(adriamycin,ADM)大剂量冲击法筛选原发性耐药细胞株,倒置显微镜下观察其形态学变化,细胞计数法绘制生长曲线,MTT法检测药物敏感性,流式细胞术分析细胞周期和检测耐药蛋白的表达.结果随冲击次数的增加,筛选出细胞的耐药指数(resistance index, RI)逐渐升高,筛选3次后对ADM的RI可达30.5;其形态学特点、生长特性及细胞周期分布与亲本细胞有明显差异,同时发现耐药蛋白P-gp、MRPt LRP表达增强.结论肝癌细胞系HepG2是个非均质的群体,其中存在着原发性耐药细胞株.     

结肠癌奥沙利铂耐药细胞系的建立及其生物学特性的研究

Background and Objective:Chemotherapy is the main treatment for colon cancer,while multidrugresistance is the main reason for chemotherapy failure and tumor relapse.This study was to establish two oxaliplatinresistant colon cancer cell lines and evaluate their biological characteristics.Met hods:Oxaliplatin-resistant colon cancer cell lines SW620/L-OHP and lovo/L-OHP were established in vitro by continuous exposure to oxaliplatin(L-OHP) of low and gradually increased concentration.Growth curve,cross-resista...     

MDR 逆转因子筛选技术P-gp 细胞系的建立及其生物学特性评价

 目的 通过基因转导和药物诱导，建立稳定表达P—gp的细胞系，用于筛选特异性逆转P-gp的药物。方法 经RT-PCR反应得到小鼠肿瘤细胞P-gp DNA，转化大肠杆茵，构建并将质粒DNA转导逆转录病毒载体中，进而感染B-MD-C1细胞。采用Northern印迹法和流式细胞技术检测细胞P-gp mRNA和P-gp分子的表达；以MTT法分析细胞的增殖活性；应用药物聚集和排放实验检测其生物学活性。结果 成功构建了稳定表达特异性P-gp基因和P-gP分子的B-MD-C1(ADR+／+)细胞系。经阿霉素诱导，B-MD-C1(ADR+／+)细胞P-gp的表达明显增高，在阿霉素增加至12000ng／mL时，仍有80％的细胞保持良好增殖状态，而B-MD-C1(wt)细胞在1000ng／mL时100％细胞死亡。B-MD-C1(ADR+／+)较(wt)细胞对MI>123有较低的聚集量和较高的排放量，加入P—gP逆转剂Verapamil后，B-MD-C1(ADR+／+)细胞MD-123聚集量增加，药物外排作用消失。结论 B-MD-C1(ADR+／+)细胞系具有较强的药物耐受性，可用于筛选特异性逆转P-gp的药物，Verapamil可逆转P—gP耐药性，具有Verapamil特性者可视为具有逆转P—gp耐药性。     

人骨肉瘤耐药细胞系MG-63/PTX的建立及多药耐药机制研究

目的 建立人骨肉瘤细胞多药耐药亚系,探讨骨肉瘤细胞多药耐药的发生机制.方法 采用紫杉醇大剂量间断冲击培养法,诱导建立紫杉醇耐药骨肉瘤细胞系(MG-63/PTX),并运用MTT法检测6种药物敏感性变化及细胞生长规律变化,流式细胞仪分析亲本细胞系和耐药细胞系的细胞膜表面P-gp蛋白表达率、罗丹明123排出情况,RT-PCR检测细胞内耐药基因的变化,免疫细胞化学法检测细胞内P-gp蛋白的表达,Westernblotting法检测细胞内P-gp蛋白的表达.结果 历时12个月建成人骨肉瘤耐药细胞系MG-63/PTX,能在含PTX1μg/mL的培养基中稳定生长并传代,其对PTX的耐药指数为69.8,并与卡铂、顺铂、表柔比星、甲氨蝶呤、阿霉素等多种化疗药物存在不同程度的交叉耐药性;罗丹明123排出实验显示MG-63/PTX内罗丹明含量明显低于亲本细胞;RT-PCR结果显示在MG-63/PTX细胞系中存在MDR1、MRP1、LRP、ABCG2基因的表达,而在MG-63细胞系中无MDR1基因的表达.免疫细胞化学、流式细胞仪及Western blotting实验均显示在MG-63/PTX中存在P-gp的表达.结论 MDR1/ P-gp在多药耐药的特性上起着至关重要的作用,而且骨肉瘤多药耐药细胞亚系为进一步研究骨肉瘤耐药特征及逆转方法打下基础.     

COX-2介导MDR1/P-gp调控人结肠癌细胞多药耐药的研究

背景与目的:肿瘤多药耐药(multidrug resistance,MDR)是导致临床上结直肠癌化疗失败的重要原因之一,环氧化酶-2(cyclooxygenase-2,COX-2)过表达与肿瘤多药耐药的发生密切相关,但其引起多药耐药的作用机制尚不清楚.本研究通过调节人结肠癌HCT8/V多药耐药细胞COX-2的表达,以探讨COX-2对人结肠癌细胞多药耐药性的影响及其对P-糖蛋白(P-glycoprotein,p-gp)的调控作用.方法:应用COX-2基因重组慢病毒载体和COX-2特异性抑制剂NS-398(20 μ mol/L)上调及抑制人结肠癌HCT8细胞和多药耐药细胞HCT8/V的COX-2的表达,MTT法检测COX-2对HCT8和HCT8/V细胞化疗药敏感性的影响,Real-time PCR(RFQ-PCR)、Westernblot等方法检测多药耐药基因MDR1和P-gp的表达.结果:人结肠癌耐药细胞HCT8/V对多种化疗药具有耐药性,其COX-2、MDR1 mRNA和P-gp的表达水平明显高于HCT8细胞(P<0.01).感染COX-2过表达慢病毒(COX-2-GFP-Lentivirus)后,HCT8细胞对长春新碱的半数抑制浓度(IC50)由(18.2±7.1)μg/mL上升至(122.5±13.4)μg/mL长春新碱(t=11.9,P<0.01),MDRI mRNA和P-gp的表达水平显著增加(P<0.01);抑制COX-2表达后,HCT8/V细胞对VCR的半数抑制浓度IC50由(191.1±18.2)μg/mL下降至(32.2±7.5)μg/mL(t=14.0,P<0.01),MDR1 mRNA和P-gp的表达水平分别下降了53.3%和42.5%(P<0.01).结论:COX-2通过介导MDR1/P-gp的表达调控人结肠癌细胞多药耐药,抑制COX-2过表达对于逆转结肠癌多药耐药具有重要意义.     

结肠癌耐药细胞株LoVo/L-OHP的建立及其耐药机制

目的:建立获得性奥沙利铂(L-OHP)耐药的结肠癌细胞模型LoVo/L-OHP,并初步研究其耐药机制。方法:采用L-OHP浓度递增法建立人结肠癌细胞耐药模型LoVo/L-OHP,观察其生长规律并绘制细胞生长曲线;用MTT法鉴定耐药细胞株耐药性并计算耐药指数(RI);用半定量RT-PCR方法对部分耐药相关基因在耐药细胞及其亲本细胞中的表达情况进行分析。结果:成功建立了耐药的结肠癌细胞模型LoVo/L-OHP,LoVo/L-OHP细胞与LoVo细胞相比,生长缓慢,触角增多。通过RT-PCR半定量分析,P-gp、bcl-2、ERCC-1在LoVo/L-OHP中的表达上调,而p53基因表达下调。结论:LoVo/L-OHP细胞株耐药性稳定,耐药机制可能与P-gp、bcl-2、ERCC-1基因上调、p53基因下调多因素有关。     

结肠癌耐药细胞株LoVo/L-OHP的建立及其耐药机制

目的：建立获得性奥沙利铂（L-OHP）耐药的结肠癌细胞模型LoVo/L-OHP,并初步研究其耐药机制。方法：采用L-OHP浓度递增法建立人结肠癌细胞耐药模型LoVo/L-OHP,观察其生长规律并绘制细胞生长曲线;用MTT法鉴定耐药细胞株耐药性并计算耐药指数（RI）;用半定量RT-PCR方法对部分耐药相关基因在耐药细胞及其亲本细胞中的表达情况进行分析。结果：成功建立了耐药的结肠癌细胞模型LoVo/L-OHP,LoVo/L-OHP细胞与LoVo细胞相比,生长缓慢,触角增多。通过RT-PCR半定量分析,P-gp、bcl-2、ERCC-1在LoVo/L-OHP中的表达上调,而p53基因表达下调。结论：LoVo/L-OHP细胞株耐药性稳定,耐药机制可能与P-gp、bcl-2、ERCC-1基因上调、p53基因下调多因素有关。     

人鼻咽癌紫杉醇耐药细胞系与其亲代细胞系比较基因组杂交研究

目的：比较基因组杂交（comparative genomic hybridization, CGH）是一种在荧光原位杂交（fluorescence in situ hybridiza?tion,FISH）技术上发展起来的用于检测两个基因组间相对DNA拷贝数的改变（缺失或扩增）, 并将这些变化在染色体上进行定位的分子细胞遗传学方法。为了解鼻咽癌紫杉醇耐药细胞 （CNE1/Taxol, HNE2/Taxol, 5-8F/Taxol）与亲代细胞（CNE1, HNE2, 5-8F）在基因组DNA水平上可能存在的差异, 以及这种差异在肿瘤获得性耐药产生中的意义, 采用CGH技术对鼻咽癌紫杉醇耐药细胞与亲本细胞的基因组DNA进行检测和分析。方法：三株鼻咽癌紫杉醇耐药细胞采用大剂量冲击与剂量逐渐递加相结合的方法诱导而成。采用集落形成实验测定药物的敏感性,利用比较基因组杂交技术 （CGH） 对耐药细胞和其亲本细胞的基因组DNA进行检测和分析, 比较耐药细胞与亲本细胞在染色体扩增与缺失上的共同点和差异。结果：三株鼻咽癌紫杉醇耐药细胞耐药指数分别为8.43、 8.27和5.26。鼻咽癌亲本细胞存在广泛的染色体改变,主要表现在3q21-qter, 5p13-pter, 12和20q11-qter的共同扩增以及10q11-qter, 18和X染色体的共同缺失,从亲本细胞系诱导的三株鼻咽癌紫杉醇耐药细胞系表现为3q21-qter, 5p13-pter, 12,20q11-qter和8q21-qter的共同扩增, 无明显共同缺失区域。与亲本细胞共同扩增区域相比, 其中最有意义的是8q21-qter区域在三株耐药细胞中出现了新的共同扩增。结论： 3q21-qter, 5p13-pter, 12和20q11-qter的共同扩增以及10q11-qter,18和X染色体的共同缺失可能与鼻咽癌的发生有关, 而8q21-qter染色体扩增可能与鼻咽癌获得性紫杉醇耐药相关,对这些区域的进一步研究, 有可能为肿瘤发生及耐药机制研究提供新的线索。     

卵巢癌细胞株 SKOV3/ADM的多药耐药性逆转研究

目的：研究糖脂合成酶抑制剂杠基棕榈酰胺吗啡丙醇（DL－threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol,PPMP)对卵巢癌多药耐药细胞株SKOV3／ADM的中性鞘糖脂成分－单己糖神经酰胺（monohexosylceramide,CMH)含量的影响,探讨PPMP对SKOV3／ADM多药耐药性的逆转作用。方法：收集SKOV3细胞和PPMP处理前后的SKOV3／ADM细胞,采用改良的Hakamort's方法提取中性鞘糖脂,用高效薄层层析技术（HPTLC）分析CMH含量变化,用体外细胞毒实验技术（MTT）检测PPMP对SKOV3／ADM耐药性的逆转作用。结果：耐药株SKOV3／ADM与其敏感株SKOV3相比,CMH含量明显增高,25μmol/L PPMP作用SKOV3／ADM细胞48h可完全抑制其CMH的合成;PPMP可增加SKOV3／ADM细胞对阿霉素、长春新碱的敏感性,但PPMP浓度不同时这种作用没有显著性差异。结论：糖脂合成酶抑制剂PPMP可有效抑制多药耐药卵巢癌细胞的中性鞘糖脂成分CMH的合成,并对卵巢癌细胞多药耐药性有逆转作用。     

Reversal of Cisplatin Resistance with Amphotericin B in a Non-small Cell Lung Cancer Cell Line

The potentiation of anticancer agents by non-anticancer drugs is one of the possible strategies for overcoming cellular resistance to chemotherapy. In order to overcome cis -diamminedichloroplatinum-(II) (CDDP) resistance, we evaluated the sensitizing effect on CDDP-induced cytotoxicity of various non-anticancer agents which might alter membrane transport, by means of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyItetrazolium bromide] (MTT) assay. Drugs which have previously been demonstrated to modify multidrug resistance did not show a sensitizing effect to cisplatin. Only amphotericin B (AmB) selectively conquered CDDP resistance in the CDDP-resistant cell line. A drug accumulation study done by the atomic absorption method demonstrated that the accumulation of CDDP in the resistant cell line recovered to the level of the parental cell line after treatment with AmB. Thus, AmB might overcome CDDP resistance by increasing the accumulation of CDDP.     

The Mechanism of Acquired Resistance to Cisplatin by a Human Ovarian Cancer Cell Line

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G₁-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 μg/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.     

Non-P-glycoprotein-mediated Atypical Multidrug Resistance in a Human Bladder Cancer Cell Line

A human bladder cancer cell line resistant to adriamycin (ADM), T24/ADM9 has been established in vitro by exposing T24 parent cells to progressively higher concentrations of the drug over a period of 12 months. The T24/ADM9 cells were found to be 9 times more resistant to ADM than the T24 parent, and showed various degrees of cross-resistance to an ADM derivative, vinca alkaloids and a DNA topoisomerase II (Topo ID-targeting agent, etoposide. No significant difference was observed in the cellular accumulation of ADM between the T24/ADM9 and T24 parent cells. A Northern blot analysis showed an overexpression of multidrug resistance-associated protein (MRP) mRNA, but no overexpression of multidrug resistance-1 (MDR1) mRNA was observed in the T24/ADM9 cells. A flow cytometric analysis showed that the MDR1 gene product, P-glycoprotein (Pgp), is not expressed on the T24/ADM9 cells. T24/ADM9 showed approximately the parental level of DNA Topo II catalytic activity. In Western blot and Northern blot analyses, however, the cellular level of DNA topo II was apparently much lower in T24/ADM9 than in the T24 parent. Thus, these results suggest that a decreased cellular level of DNA Topo II and an overexpression of MRP gene may be responsible for the expression of an MDR phenotype in the T24/ADM9 cells and that such non-Pgp-mediated, atypical MDR may develop in bladder cancer treated with chemotherapy including ADM.     

甲基莲心碱对乳腺癌MCF-7/Adr 细胞MDR逆转的研究

 目的 探讨甲基莲心碱（Neferine，Nef）对耐药人乳腺癌细胞增殖，细胞内ADM积聚浓度及mdr-1／P-gp表达的影响。方法 采用MTT法测定细胞毒作用，高效液相色谱法测定细胞内ADM积聚浓度，RT—PCR技术及蛋白质印迹技术检测mdr-1／P-gp表达。结果 10μg／ml Nef＋ADM组细胞的抑制率及细胞内ADM积聚浓度比ADM组高（P〈0.01）；20μg／ml Nef＋ADM组细胞抑制率及细胞内ADM积聚浓度较5μg／ml异博定＋ADM组及10μg／ml Nef＋ADM组均高（P〈0．01）。10μg／ml Nef＋ADM组MCF-7／Adr细胞mdr-1 mRNA及P-gp表达比ADM组明显下降（P〈0．01）。20μg／ml Nef＋ADM组细胞mdr-1mRNA及P-gp表达较10μg／ml Nef＋ADM组明显下调（P〈0．01）。结论 Nef能抑制耐药人乳腺癌细胞增殖。Nef能增加MCF-7／Adr细胞内ADM积聚浓度。Nef通过降低耐药人乳腺癌细胞mdr-1 mRNA及P-gp的表达逆转MDR。     

人胃癌耐药细胞株SGC-7901／HCPT的耐药机制初探

目的:探讨人胃癌耐羟基喜树碱细胞株即SGC-7901/HCPT的耐药机制。方法:通过光镜和电镜观测SGC-7901/HCPT与人胃癌细胞株即SGC-7901形态和超微结构差异和免疫细胞化学的方法检测SGC-7901/HCPT与SGC-7901之间的TOPO-Ⅰ、TOPO-Ⅱ、P-gp、GST-π、BCL-2表达异同来探讨SGC-7901/HCPT的可能耐药机制。结果:(1)光镜下SGC-7901/HCPT细胞大小不一致,形态不规则,胞浆较丰富,核小而不规则,可见巨核现象;而SGC-7901细胞呈圆梭形,大小一致,核圆形;电镜下SGC-7901/HCPT表面指状突起明显,胞浆内有较多的核糖体及粗面内质网,线粒体较多。(2)SGC-7901/HCPT的TOPO-Ⅰ表达较SGC-7901明显增加,差异具有显著性;而TOPO-Ⅱ、P-gp、GST-π、BCL-2的表达差异无显著性。结论:SGC-7901/HCPT趋向细胞成熟分化,这可能是其产生耐药的形态学表现。TOPO-Ⅰ的表达下降则可能是SGC-7901/HCPT耐HCPT形成的分子基础。     

异博定等药物对人卵巢癌耐药细胞系多药耐受的逆转作用

用已建立的耐长春新碱人卵巢癌的耐药细胞做实验,应用ＭＴＴ法证实：异博定、潘生丁、黄体酮、环孢菌素Ａ和他莫西芬等药,能不同程度的逆转耐药细胞对长春新碱的耐受;高效液相色谱仪测定进一步证实了上述逆转药可保持或增加耐药细胞内长春新碱的药物浓度。该作用系由于逆转药使耐药细胞表达的大量Ｐ糖蛋白功能失活,通过增加细胞摄取药物量或延长药物在细胞内滞留时间形成的。     

Determinants of Drug Response in a Cisplatin-resistant Human Lung Cancer Cell Line

To elucidate the mechanism(s) of cisplatin resistance, we have characterized a human non-small cell lung cancer cell line (PC-9/CDDP) selected from the wild type (PC-9) for acquired resistance to cisplatin. PC-9/CDDP demonstrated 28-fold resistance to cisplatin, with cross resistance to other chemotherapeutic drugs including chlorambucil (× 6.3), melphalan (× 3.7) and 3-[(4-amino-2-methyl-5-pyrimidinyl)]methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU) (× 3.9). There was no expression of mdr-1 mRNA in either wild-type or resistant cells. The mRNA and protein levels of glutathione S -transferase (GST) × were similar in the two lines. A GST-μ isozyme was present in equal amounts and the activities of selenium-dependent and independent glutathione peroxidase and glutathione reductase were unchanged. The mRNA level of human metallothionein IIA and the total intracellular metallothionein levels were reduced in the resistant cells. Significantly increased intracellular glutathione (GSH) levels were found in the resistant cells (20.0 vs. 63.5 nmol/mg protein) and manipulation of these levels with buthionine sulfoximine produced a partial sensitization to either cisplatin or chlorambucil. Increased GSH probably also played a role in determining cadmium chloride resistance of the PC-9/CDDP, even though this cell line had a reduced metallothionein level. Also contributing to the cisplatin resistance phenotype was a reduced intracellular level of platinum in the PC-9/CDDP. Thus, at least two distinct mechanisms have been selected in the resistant cells which confer the phenotype and allow degrees of cross resistance to other electrophilic drugs.     

结肠癌TRAIL耐药细胞株的建立及其耐药机制的初步研究

背景与目的以人结肠癌DLD1细胞株为载体,探讨恶性肿瘤获得性TRAIL基因耐药的可能机制。材料与方法用重组腺病毒介导的TRAIL基因(Ad/gTRAIL)反复处理人结肠癌DLD1细胞,得到耐药细胞群DLD1-TRAIL/R。通过MTT比色法和蛋白电泳,检测耐药细胞对Ad/gTRAIL杀伤作用的敏感性及其凋亡通路中信号分子的表达情况,分析其获得性耐药的可能机制。结果DLD1-TRAIL/R细胞对Ad/gTRAIL和重组TRAIL蛋白处理耐药,但对腺病毒介导的Bax基因处理仍然敏感。耐药细胞内Bcl-XL的表达明显升高,caspase-8的表达显著下降,未见明显的caspase-8活性裂解形式。结论人结肠癌DLD1细胞株经Ad/gTRAIL反复处理后产生特异性针对TRAIL基因的耐药,其发生机制可能与Bcl-XL表达上调和caspase-8表达下调有关。