重组改构人肿瘤坏死因子对人多药耐药SKOV3/DDP细胞的体外作用及其机制的研究
摘要
目的:卵巢癌患者对化疗产生耐药性是影响疗效的主要原因之一。传统的逆转剂因副作用大而使得临床应用受到限制,因此寻找高效、低毒的逆转剂是目前研究方向及热点之一。重组改构人肿瘤坏死因子(recombinant mutant human tumor necrosis factor, rmh-TNF)是由国内学者通过蛋白质工程技术改造天然TNF的结构而研制出的一种高效、低毒的TNF突变体。本研究通过体外实验观察rmh-TNF对人卵巢癌多药耐药细胞系SKOV3/DDP耐药性的影响,并对可能存在的机制进行初步的探讨,以期寻找一种新的药物帮助解决卵巢癌治疗中出现的多药耐药问题。
方法:体外培养人卵巢癌多药耐药细胞株SKOV3/DDP,采用四氮唑噻蓝盐(MTT)比色法检测rmh-TNF对SKOV3/DDP耐药株的细胞毒作用,选择杀伤率<10%的浓度作为该药物的无毒剂量,即本实验的逆转剂量。同法测定逆转剂量的rmh-TNF干预后细胞株SKOV3/DDP对顺铂耐药性的变化;应用流式细胞术(FCM)检测rmh-TNF干预不同时间段SKOV3/DDP细胞株中GST-π蛋白、BCL-2蛋白的表达,应用逆转录酶链反应(RT-PCR)分析rmh-TNF处理SKOV3/DDP细胞前后mdr1 mRNA、BCL-2 mRNA的表达水平。
结果:1 MTT结果显示: rmh-TNF在(50-3200)U/ml以内能抑制SKOV3/DDP细胞的体外增殖,且随着浓度增加抑制率相应上升。回归分析得出当rmh-TNF终浓度<122.34U/ml,其对细胞生长无明显抑制作用(细胞存活率均﹥90%)因此本实验选用100U/ml作为逆转剂量。100U/ml rmh-TNF作用SKOV3/DDP 24、48、72h的逆转倍数分别为1.19倍、2.20倍、2.64倍。2流式细胞术检测结果显示rmh-TNF处理前SKOV3、SKOV3/DDP两组细胞GST-π蛋白表达量的相对值(FI)分别是1、2.33±0.30,两株细胞GST-π蛋白的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后GST-π蛋白表达量的相对值随作用时间的延长而逐渐降低,FI值分别为1.82±0.13、1.73±0.28、1.31±0.17,作用前后比较均有显著性差异(P<0.05);rmh-TNF处理前SKOV3、SKOV3/DDP两株细胞BCL-2蛋白表达量的相对值(FI)分别是1、2.62±0.15,两株细胞BCL-2蛋白的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后BCL-2蛋白表达量的相对值随作用时间的延长而逐渐降低,FI值分别为2.24±0.20、1.71±0.19、1.30±0.15,作用前后比较均有显著性差异(P<0.05)。3 RT-PCR法检测结果显示rmh-TNF处理前SKOV3细胞株不表达mdr1 mRNA, SKOV3/DDP细胞株mdr1 mRNA表达量的相对值(mdr1/β-actin)是0.76±0.01。100U/ml rmh-TNF作用细胞株SKOV3/DDP 24、48、72,mdr1 mRNA表达量的相对值随作用时间的延长而逐渐降低,mdr1/β-actin值分别为0.66±0.05、0.52±0.04、0.45±0.03,作用前后差异均有显著性。(P<0.05);rmh-TNF处理前SKOV3、SKOV3/DDP两株细胞BCL-2 mRNA表达量的相对值(BCL-2/β-actin)分别是0.09±0.15、0.47±0.01,两株细胞BCL-2 mRNA的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后BCL-2 mRNA表达量的相对值随作用时间的延长而逐渐降低,BCL-2/β-actin值分别为0.45±0.01、0.31±0.01、0.27±0.01,作用前后比较均有极显著性差异(P<0.01)
结论:1重组改构人肿瘤坏死因子(rmh-TNF)在(50-3200)U/ml浓度范围内,对SKOV3/DDP细胞有生长抑制作用,且呈浓度依赖性。2逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)对SKOV3/DDP细胞耐顺铂效应有一定的逆转作用,且逆转效应存在时间-效应关系。3逆转机制可能为:①逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可能通过下调耐药相关基因mdr1 mRNA表达,进而降低SKOV3/DDP细胞的耐药性实现逆转耐药。②逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可在转录和蛋白翻译水平降低BCL-2表达,进而降低BCL-2抑制凋亡的作用,使耐药细胞株凋亡增加,发挥对细胞耐顺铂效应的逆转作用。③逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可能通过下调GSTπ表达,减少细胞毒性药物代谢产物的外排从而实现耐药性的逆转。
Object:Multidrug resistance (MDR) of Ovarian cancer cells is one of the major reasons that lead to chemothrapy falure, therefor how to overcome MDR to increase the curative effect of chemotherapy has become the focus. Rcombinant mutant human tumor necrosis factor(rmh-TNF) is a high- efficiency low-toxicity mutation developed by chinese scholar. This experiment aimed to investigate the reversing effect and its possible mechanism of rmh-TNF on Cisplatin-resistant Human Ovarian cell lines in vitro.
Methods:Choosing Human multidrug-resistant Ovarian cell lines SKOV3/DDP ,Detect rmh-TNF’s cell toxicity to SKOV3/DDP to select nontoxic dose and examine the vary of Cisplatin-resistant by MTT assay. The expression of BCL-2 protein and GST-πprotein were measured by flow cytometry. The expression of BCL-2 mRNA and mdr1 mRNA were studied by RT-PCR in SKOV3/DDP cells.
Results:1 MTT assay results: Within the concentration of ( 50-3200 ) U/ml, rmh-TNF can inhibit proliferation of SKOV3/DDP cells in vitro. The inhibition ration was stepping up as the concentration increasing, When rmh-TNF’s concentration was low than 122.34U/ml , rmh-TNF could not obviously proliferation inhibiting by regression analysis, and the experiment choose 100 U/ml for nontoxic dose. After being treated with 100U/ml rmh-TNF for 24、48、72h,the RF are 1.19、2.06 and 2.64 .2 FCM assay results: The FI-value of GST-πprotein expression in SKOV3 was 1,while 2.33±0.30 in SKOV3/DDP, it was significant difference in the expression of GST-πprotein (P<0.01).After being treated with rmh-TNF for 24、48、72h,GST-πprotein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 1.82±0.13 1.73±0.28 and 1.31±0.17 , There was significantly difference in the GST-πprotein expression between before and after rmh-TNF action. (P<0.05) ;The FI-value of BCL-2 protein expression in SKOV3 was 1,while 2.62±0.15 in SKOV3/DDP, their BCL-2 protein expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h、48h、72h,BCL-2 protein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 2.24±0.20、1.71±0.19 and 1.30±0.15, There was significant difference of BCL-2 protein expression between before and after rmh-TNF action. (P<0.05) 3 RT-PCR detection results: Before being treated with rmh-TNF , The gene mdr1 mRNA was expressed in SKOV3/DDP cell, and hardly espressed in SKOV3 cell.After being treated by rmh-TNF for 24、48、72h mdr1 mRNA expression in SKOV3/DDP cell became reduing as action time increasing, the weakest expression was in the group with 72h. The mdr1/β-actin value are 0.66±0.05、0.52±0.04 and 0.45±0.03,rmh-TNF could inhibiting the expression of mdr1 mRNA in a certain extent. There was significant difference of mdr1 mRNA expression between before and after rmh-TNF action. (P<0.05); The value of BCL-2 mRNA expression in SKOV3 was 0.09±0.15,while 0.47±0.01 in SKOV3/DDP, their BCL-2 mRNA expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h、48h、72h BCL-2 mRNA expression in SKOV3/DDP cell became reduing as action time increasing,the weakest expression was in the group with 72h. The mdr1/β-actin value are 2.24±0.20、1.71±0.19、1.30±0.15, rmh-TNF could inhibiting the expression of BCL-2 mRNA in a certain extent. There was significantly difference of BCL-2 mRNA expression between before and after rmh-TNF action.
Conclusion: 1 Within the concentration of (50-3200)U/ml, rmh-TNF might inhibit proliferation of SKOV3/DDP cells in vitro 2 Nontoxic doses rmh-TNF could partly reverse the Cisplatin-resistant of SKOV3/DDP cells. 3 The mechanisnm of reverse might conclude three sides:①Within a certain drug concentration , rmh-TNF might reverse multidrug-resistance by inhibiting expression of mdr1 mRNA.②rmh-TNF could reduce the apoptosis-inhibition action by inhibition the expression of BCL-2 mRNA and BCL-2 protein to promote cells apoptosis.③Within a certain drug concentration, rmh-TNF might reduce the drainage of celltoxicitive drug’s metabolizable producetion by inhibiting expression of GST-πprotein.
方法:体外培养人卵巢癌多药耐药细胞株SKOV3/DDP,采用四氮唑噻蓝盐(MTT)比色法检测rmh-TNF对SKOV3/DDP耐药株的细胞毒作用,选择杀伤率<10%的浓度作为该药物的无毒剂量,即本实验的逆转剂量。同法测定逆转剂量的rmh-TNF干预后细胞株SKOV3/DDP对顺铂耐药性的变化;应用流式细胞术(FCM)检测rmh-TNF干预不同时间段SKOV3/DDP细胞株中GST-π蛋白、BCL-2蛋白的表达,应用逆转录酶链反应(RT-PCR)分析rmh-TNF处理SKOV3/DDP细胞前后mdr1 mRNA、BCL-2 mRNA的表达水平。
结果:1 MTT结果显示: rmh-TNF在(50-3200)U/ml以内能抑制SKOV3/DDP细胞的体外增殖,且随着浓度增加抑制率相应上升。回归分析得出当rmh-TNF终浓度<122.34U/ml,其对细胞生长无明显抑制作用(细胞存活率均﹥90%)因此本实验选用100U/ml作为逆转剂量。100U/ml rmh-TNF作用SKOV3/DDP 24、48、72h的逆转倍数分别为1.19倍、2.20倍、2.64倍。2流式细胞术检测结果显示rmh-TNF处理前SKOV3、SKOV3/DDP两组细胞GST-π蛋白表达量的相对值(FI)分别是1、2.33±0.30,两株细胞GST-π蛋白的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后GST-π蛋白表达量的相对值随作用时间的延长而逐渐降低,FI值分别为1.82±0.13、1.73±0.28、1.31±0.17,作用前后比较均有显著性差异(P<0.05);rmh-TNF处理前SKOV3、SKOV3/DDP两株细胞BCL-2蛋白表达量的相对值(FI)分别是1、2.62±0.15,两株细胞BCL-2蛋白的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后BCL-2蛋白表达量的相对值随作用时间的延长而逐渐降低,FI值分别为2.24±0.20、1.71±0.19、1.30±0.15,作用前后比较均有显著性差异(P<0.05)。3 RT-PCR法检测结果显示rmh-TNF处理前SKOV3细胞株不表达mdr1 mRNA, SKOV3/DDP细胞株mdr1 mRNA表达量的相对值(mdr1/β-actin)是0.76±0.01。100U/ml rmh-TNF作用细胞株SKOV3/DDP 24、48、72,mdr1 mRNA表达量的相对值随作用时间的延长而逐渐降低,mdr1/β-actin值分别为0.66±0.05、0.52±0.04、0.45±0.03,作用前后差异均有显著性。(P<0.05);rmh-TNF处理前SKOV3、SKOV3/DDP两株细胞BCL-2 mRNA表达量的相对值(BCL-2/β-actin)分别是0.09±0.15、0.47±0.01,两株细胞BCL-2 mRNA的表达具有极显著性差异(P<0.01),100U/ml rmh-TNF作用细胞SKOV3/DDP 24、48、72h后BCL-2 mRNA表达量的相对值随作用时间的延长而逐渐降低,BCL-2/β-actin值分别为0.45±0.01、0.31±0.01、0.27±0.01,作用前后比较均有极显著性差异(P<0.01)
结论:1重组改构人肿瘤坏死因子(rmh-TNF)在(50-3200)U/ml浓度范围内,对SKOV3/DDP细胞有生长抑制作用,且呈浓度依赖性。2逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)对SKOV3/DDP细胞耐顺铂效应有一定的逆转作用,且逆转效应存在时间-效应关系。3逆转机制可能为:①逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可能通过下调耐药相关基因mdr1 mRNA表达,进而降低SKOV3/DDP细胞的耐药性实现逆转耐药。②逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可在转录和蛋白翻译水平降低BCL-2表达,进而降低BCL-2抑制凋亡的作用,使耐药细胞株凋亡增加,发挥对细胞耐顺铂效应的逆转作用。③逆转剂量的重组改构人肿瘤坏死因子(rmh-TNF)可能通过下调GSTπ表达,减少细胞毒性药物代谢产物的外排从而实现耐药性的逆转。
Object:Multidrug resistance (MDR) of Ovarian cancer cells is one of the major reasons that lead to chemothrapy falure, therefor how to overcome MDR to increase the curative effect of chemotherapy has become the focus. Rcombinant mutant human tumor necrosis factor(rmh-TNF) is a high- efficiency low-toxicity mutation developed by chinese scholar. This experiment aimed to investigate the reversing effect and its possible mechanism of rmh-TNF on Cisplatin-resistant Human Ovarian cell lines in vitro.
Methods:Choosing Human multidrug-resistant Ovarian cell lines SKOV3/DDP ,Detect rmh-TNF’s cell toxicity to SKOV3/DDP to select nontoxic dose and examine the vary of Cisplatin-resistant by MTT assay. The expression of BCL-2 protein and GST-πprotein were measured by flow cytometry. The expression of BCL-2 mRNA and mdr1 mRNA were studied by RT-PCR in SKOV3/DDP cells.
Results:1 MTT assay results: Within the concentration of ( 50-3200 ) U/ml, rmh-TNF can inhibit proliferation of SKOV3/DDP cells in vitro. The inhibition ration was stepping up as the concentration increasing, When rmh-TNF’s concentration was low than 122.34U/ml , rmh-TNF could not obviously proliferation inhibiting by regression analysis, and the experiment choose 100 U/ml for nontoxic dose. After being treated with 100U/ml rmh-TNF for 24、48、72h,the RF are 1.19、2.06 and 2.64 .2 FCM assay results: The FI-value of GST-πprotein expression in SKOV3 was 1,while 2.33±0.30 in SKOV3/DDP, it was significant difference in the expression of GST-πprotein (P<0.01).After being treated with rmh-TNF for 24、48、72h,GST-πprotein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 1.82±0.13 1.73±0.28 and 1.31±0.17 , There was significantly difference in the GST-πprotein expression between before and after rmh-TNF action. (P<0.05) ;The FI-value of BCL-2 protein expression in SKOV3 was 1,while 2.62±0.15 in SKOV3/DDP, their BCL-2 protein expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h、48h、72h,BCL-2 protein expression in SKOV3/DDP cell became reducing as action time increasing, and the FI-value are 2.24±0.20、1.71±0.19 and 1.30±0.15, There was significant difference of BCL-2 protein expression between before and after rmh-TNF action. (P<0.05) 3 RT-PCR detection results: Before being treated with rmh-TNF , The gene mdr1 mRNA was expressed in SKOV3/DDP cell, and hardly espressed in SKOV3 cell.After being treated by rmh-TNF for 24、48、72h mdr1 mRNA expression in SKOV3/DDP cell became reduing as action time increasing, the weakest expression was in the group with 72h. The mdr1/β-actin value are 0.66±0.05、0.52±0.04 and 0.45±0.03,rmh-TNF could inhibiting the expression of mdr1 mRNA in a certain extent. There was significant difference of mdr1 mRNA expression between before and after rmh-TNF action. (P<0.05); The value of BCL-2 mRNA expression in SKOV3 was 0.09±0.15,while 0.47±0.01 in SKOV3/DDP, their BCL-2 mRNA expression was significant difference (P<0.01). After being treated with rmh-TNF for 24h、48h、72h BCL-2 mRNA expression in SKOV3/DDP cell became reduing as action time increasing,the weakest expression was in the group with 72h. The mdr1/β-actin value are 2.24±0.20、1.71±0.19、1.30±0.15, rmh-TNF could inhibiting the expression of BCL-2 mRNA in a certain extent. There was significantly difference of BCL-2 mRNA expression between before and after rmh-TNF action.
Conclusion: 1 Within the concentration of (50-3200)U/ml, rmh-TNF might inhibit proliferation of SKOV3/DDP cells in vitro 2 Nontoxic doses rmh-TNF could partly reverse the Cisplatin-resistant of SKOV3/DDP cells. 3 The mechanisnm of reverse might conclude three sides:①Within a certain drug concentration , rmh-TNF might reverse multidrug-resistance by inhibiting expression of mdr1 mRNA.②rmh-TNF could reduce the apoptosis-inhibition action by inhibition the expression of BCL-2 mRNA and BCL-2 protein to promote cells apoptosis.③Within a certain drug concentration, rmh-TNF might reduce the drainage of celltoxicitive drug’s metabolizable producetion by inhibiting expression of GST-πprotein.
引文
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23 周清华,侯梅,李潞,等。新型重组人肿瘤坏死因子治疗非小细胞肺癌的多中心 II 期临床随机试验。中国肺癌杂志,2003,6(1)B:42-45
24 杨亚菁,袁志军,罗以,等.重组改构人肿瘤坏死因子治疗 47例恶性胸腹腔积液[J].中国癌症杂志,2004,14(4):396-398
25 周清华,鄢希,任莉,等.注射用重组改构人肿瘤坏死因子联合化疗药物治疗非小细胞肺癌的多中心III期临床试验[J].中国肺癌杂志,2003,6(4):264-267
26 彭宝岗,梁力键,何强,等.肿瘤坏死因子联合干扰素 C 治疗原发肝癌的实验研究.中华肝胆外科杂志,2003,9(1):25-25
27 Boumendjel A. Anticancer multidrug resistance mediated by MRP1:recent advances in the discovery of reversa lagents [J].Medicinal Research Reviews,2005,25(4): 453-472
28 Ludwig JA,Szakacs G,Martin SE, et al.Selective toxicity of NSE73306 in mdr1-positive cells a new strategy to circumvent multidrug resistance in cancer[J]. Cancer Res,2006,66 (9):4808-4815
29 刘景丰,陈孝平,肿瘤坏死因子α逆转人肝癌多药耐药性的实验研究[J].中华实验外科杂志,1999,16[3]:256-258
30 张珂.卵巢癌顺铂耐药靶向逆转的初步研究[D]. 山东大学,2005
31 Beeghly A, Katsaros D, Chen H, et al. Glutathione Stransferase polymorphisms and ovarian cancer trentment and surivival[J]. Gynecol Oncol,2006,100(2):330-337.
32 Boresellino N,Crescimanno M,flandina C,et,al.Combined activity of interleukin-1 alpha or TNF-alpha and doxorubicin on multidrug resistantcell lines:evidence that TNF and DXR gave synergistic antitumor and differertiation-inducing effects[J]. Anticancer Res,1994,14(6B):2643-2648
1 Carla Frova. Glutathione transferases in the genomicsera new insights and perspectives [J]. Biomol Eng, 2006,23(4):149-169
2 Mclwain CC,Townsend DM, Tew KD. et al .Glutathione Stransferase polymorphisms: cancer incidence and therapy[J]. Oncogene ,2006 ,25(11):1639-1648
3 Doherty M ,Michael M. Tumoral drug metabolism: perspectives and therapeutic implications [J].Curr Drug Metab,2003,4(2):131-149
4 Lin JH, Masay oY. Clinical relevance of P-glycoprotein in Drugtherapy [J]. Drug Metab Rev, 2003,35(4):417-454
5 Valkov NI,Sullivan DM, Tumor p53 statusand response to topoisomerase II inhibitors [J].Drug Resist Updatesx, :2003,6(1):27-39
6 汪毅,周理.多药耐药相关蛋白 1,谷胱甘肽 S 转移酶和DNA 拓 酶 在 骨 肉 瘤 中 的 表 达 [J]. 重 庆 医学,2006,35(14):1273-1275
7 Lorenzo PS,Dennis PA.Modulating protein kinaseC (PKC ) to increase the efficacy of chemotherapy stepping into darkness [J].Drug Resist Updates ,2003 ,(6):329-339
8 于韬,赵桂森,臧恒昌等 肿瘤多药耐药逆转剂研究进展[J]. 中国药物化学杂志,2003,13(53):172-178
9 冯凤芝,向阳,张卫光等 人肿瘤坏死因子α基因转导绒癌耐药细胞系体外耐药逆转的研究[J]。肿瘤,2003,23(4):283-286
10 Stein U ,Walther W, Shoemaker RH,et al. Modulation of mdr1 expression by cytokines in human colon carcinoma cells: an approach for reversal of multidrug resistance[J].Br J Cancer,1996,74(9):1384-1391
11 Walther W, Stein U,Pfeil D,et al.Gene transfer of human TNF alpha into glioblastoma cells permits of mdr1expression and potentiation of chemosensitivity[J]. Int J Cancer,1995,61(6):832-839
12 Stein U ,Walther W, Shoemaker RH,et al.Reversal of multidrug resistance by transduction of cytokine genes into human colon carcinoma cells[J].J Natl Cancer Inst,1996,88(19): 1383-1392
13 Boresellino N, Rainaldi G,Tritarell E,et al .Tumor necrosis factor is a powerful apoptotic inducer in lymphoid leukemic cells expressing the p-170 glycoprotein [J].Anticancer Res,1994,14(6B):2643-2648
14 庄静丽,徐建民,王宝珍等. TNF 诱导 K562/VCR 和 K562细胞凋亡及逆转其耐药作用的实验研究[J].实用肿瘤杂志,2004,19(4):296-300
15 Matarrese P,Testa U,cauda R,et al. Expression of P-170 glycoprotein sensitizes lymphoblastoid CEM cell tomitochondia-mediated apoptosis [J].Biochem J,2001,335 (pt3):587-595
16 Yin Y,ALLen PD,Jia L,et al.Constitutive levels of cAMP-dependent protein kinase activity determine sensitivity of human multidmg-resistant leukaemic cell lines to growth inhibition and apoptosis by forskolin and tumor necrosis factor alpha[J].Br J Haematol ,2000,108(3):565.-573
17 Stein U ,Walther W,Laurencot C M ,et al.Increased activityand sensitivity of the multidrug resistance associated genes LRP and MRP [J]. Natl Cancer Inst,1997,89(11):807-813
18 Cimoli G, Valenti M, Parodi S, et al. Reversal of "atypical"-multidrug resistance by recombinant human tumor necrosis factor in the human ovarian cancer cell line A2780-DX3.[J].Oncol Res, 1993,5(8): 311-323
19 杨亚菁,袁志军,罗以,等.重组改构人肿瘤坏死因子治疗
47 例 恶 性 胸 腹 腔 积 液 [J]. 中 国 癌 症 杂志,2004,14(4):396-398
20 周清华,鄢希,任莉,等.注射用重组改构人肿瘤坏死因子联合化疗药物治疗非小细胞肺癌的多中心 III 期临床试验[J].中国肺癌杂志,2003,6(4):264-267
21 彭宝岗,梁力键,何强,等.肿瘤坏死因子联合干扰素 C 治疗 原 发 肝 癌 的 实 验 研 究 . 中 华 肝 胆 外 科 杂志,2003,9(1):25-25
22 魏虎来,葛建国,赵怀顺等.干扰素和环孢素 A 协同逆转 K562/ADM 细胞对阿霉素的耐药性[J].肿瘤防治研究,2005,32(2):99-101
23 钱其军,严文伟, 杨纯正等 白血病细胞株对马法兰的耐药机制与α干扰素的逆转作用[J].中华医学杂志,1996,76(7):485-488
24 平宝红,周淑芸,刘启发等.干扰素逆转白血病细胞多药耐药性的研究及机制的探讨[J].中华血液学杂志,1996,17(1):13-15
25 Takeuchi A,kaneko S,Matsushhita E,et,al.Interferon-alphamodulates resistance to cisplatin in three human hepatoma cell lines[J]. J Gastroenterol, 1999,34(3): 351-358
26 张荣河,何三光等.肿瘤坏死因子、白介素-2、α-干扰素及合用异博定对乳腺癌细胞多药耐药性的逆转作用研究[J].中国肿瘤临床与康复,2001,8(2):51-53
27 丁泓文,何文芳,徐谊朝等.α-干扰素、异搏定协同逆转肾癌细胞药物耐受性[J].暨南大学学报,2000,21(6):5-8
28 Corrias MV,Basso S,Meazza R,et al. Characterization and tumorigenicity of human neuroblastoma cells transfected with the IL-2 gene [J]. Cancer Gene Ther,1998,5(1): 38-44
29 崔竹梅,杨秀玉,向阳等. IL2 基因转染逆转绒癌耐药细胞系多药耐药性[J]. 基础医学与临床,2001,21(6 )523-526
30 付劲蓉,李成荣,杨锡强等。 白介素-4 基因修饰对儿童肝母细胞瘤多药耐药的逆转及其机制的研究[J].中华儿科杂志,2001,39(11): 650-653
31 贾庆瑞,葛祥花,隋金财等.白细胞介素-2 逆转急性白血病细胞多药耐药的临床研究[J]. 白血病? 淋巴瘤,2003,12(1):28-30
32 沈文状,易继林,李兴睿等. IL-2 治疗对肝癌化疗敏感性影响的研究[J]. 中国已是杂志,2005,7(1):11-13
33 平宝红,周淑芸,杨纯正等.4 种细胞因子对 K562/S 及其耐药细胞株 K562/AO2 细胞毒性作用检测 [J].解放军医学杂志,1998,23(4):299-300
34 Spicka I,Jirasek A,Koubek K et al. Progression of multiple myeloma during treatment with recombinant G-CSF and absence of G-CSF and IL-6 cell surface receptors on malignant cells [J].Cas Lek Cesk, 1998,137(21): 660-663
35 List A F. Role of mulitidrug resistance and its pharam acological modulation in acute myoloid leukem ia.Leukem ia[J]. 1996,10(6): 937-942
2 程国钧,李亚里,田方,等.不同方式诱导人卵巢癌细胞系的比较.中华肿瘤杂志 , 2001,23: 305-308
3 李宏,赵丽嫣,张松平.卵巢癌顺铂耐药细胞系的建立及耐药机制的研究.中国实用妇科与产科杂志,2001,17:497-498
4 Han R,ShunY,Cancer chem Oprevention and drug therapy of the new millennium [M]. Beijing :Peoples Military Medical Press ,2005 ,860
5 TakanoM, KudoK, GotoT ,et al. Analysis by comparative genomic hybridization of genes relating with cisplatin resistance in ovarian cancer[J]. Hum Cell ,2001,14(4): 267-271
6 Gadducci A, Cosio S, Muraca S ,et al. Molecular mechanisms of apoptosis and chemosensitivity to platinum and paclitaxel in ovarian cancer :Biological data and clinical implications . Eur J gynaecol Oncol ,2002 ,23 (5 ):390 -396
7 Skirnisdottir I ,Seidal T, Gerdin E ,et al. The prognostic Importance of p53 ,bcl-2 ,and bax in early stage epithelial ovarian carcinoma treated with adjuvant chemotherapy. Int J Gynecol Cancer, 2002,12(3):265-276
8 Yang X ,Zheng F, Xing H et al .Resistance to chemotherapy-induced apoptosis via decreased caspase-3 activity and over expression of antiapoptotic proteins in ovarian cancer .J Cancer Res Clin Oncol, 2004,130(7):423-428
9 Strathdee G, MacKeancMJ,Illand M, et al. A role formethylation of the hMLH1 promoter in loss of hMHL1 expression and drug resistance in ovarian cancer [J].Oncogene, 1999, 18(14):2335-2341
10 Duam Z,Brakora KA,Seliden MV,et al.Inhibition of ABC1 ( mdr1) and ABC4(MDR3) expression by small interfering RNA and reversal of paclitaxel resistance in human ovarian cancer cells[J].Mol Cancer Ther,2004,3(7):833-838
11 Krasznai ZT,Frieflander E,Nagy A, et al.Quantitative and functional assay of mdr1/P170-mediated MDR inascites cells of patients with ovarian cancer[J].Anticancer Res,2005,25(2A):1187-1192
12 程国钧,祝 华,孙丽亚,等.耐药相关基因在卵巢组织中的表达及其临床意义[J].中华妇产科杂志,2000,35(2):87~90
13 Liu JR,OpipariAW, Tan L ,et al Dysfunctional apoptosome activation in ovarian cancer : implications for chemoresistance[J].Cancer Res.2002.62: 924-931
14 Raspollini MR,Amunni G, Villanucci A, et al. HER-2 /neu and bcl-2 in ovarian carcinoma clinicopathologic immunohistochemical and molecular study in patients with shorter and longer survival [J]. Appl immunohistochem MolMorphol 2006, 14(2):181-186
15 Daniel V, Glutathiones-transferases: gene structure and regula of expression[J].Crit Rev Biochem Mol Biol, 1993,28(3):173-270
16 冯凤芝,向阳,张卫光等 人肿瘤坏死因子α基因转导绒癌耐药细胞系体外耐药逆转的研究[J]。肿瘤,2003,23(4):283-286
17 Matarrese P,Testa U,cauda R,et al. Expression of P-170 glycoprotein sensitizes lymphoblastoid CEM cell tomitochondia-mediated apoptosis [J].Biochem J,2001,335 (pt3):587-595
18 Stein U ,Walther W,Laurencot C M ,et al.Increased activity and sensitivity of the multidrug resistance associated genes LRP and MRP [J]. Natl Cancer Inst,1997,89(11):807-813
19 Cimoli G, Valenti M, Parodi S, et al. Reversal of "atypical"-multidrug resistance by recombinant human tumor necrosis factor in the human ovarian cancer cell line A2780-DX3.[J].Oncol Res, 1993,5(8): 311-323
20 Lee KY,Walther W,Qiu D,et al.PF491 (triptolide) cooperates with tumor necrosis fator-alpha toinduce apoptosis in tumor cells. J Biol Chem,1999,274(19)B:1345-1345
21 Stein U,Walther W,Shoemaker RH,et,al.Reversal of multidrug resistance by transduction of cytokine genes into human colon carcinoma cells.J Natl Cancer rInst,1996,88(19)B:1383-1392
22 lase kW,Giermasz A,Kuc K,et al.potentiation of theanti-tumor effect of action mycin D by tumor necrosis fator-alpha in mice:correlation between in vitro and in vivo results.Int J Cancer,1996,66(3)B:374-379
23 周清华,侯梅,李潞,等。新型重组人肿瘤坏死因子治疗非小细胞肺癌的多中心 II 期临床随机试验。中国肺癌杂志,2003,6(1)B:42-45
24 杨亚菁,袁志军,罗以,等.重组改构人肿瘤坏死因子治疗 47例恶性胸腹腔积液[J].中国癌症杂志,2004,14(4):396-398
25 周清华,鄢希,任莉,等.注射用重组改构人肿瘤坏死因子联合化疗药物治疗非小细胞肺癌的多中心III期临床试验[J].中国肺癌杂志,2003,6(4):264-267
26 彭宝岗,梁力键,何强,等.肿瘤坏死因子联合干扰素 C 治疗原发肝癌的实验研究.中华肝胆外科杂志,2003,9(1):25-25
27 Boumendjel A. Anticancer multidrug resistance mediated by MRP1:recent advances in the discovery of reversa lagents [J].Medicinal Research Reviews,2005,25(4): 453-472
28 Ludwig JA,Szakacs G,Martin SE, et al.Selective toxicity of NSE73306 in mdr1-positive cells a new strategy to circumvent multidrug resistance in cancer[J]. Cancer Res,2006,66 (9):4808-4815
29 刘景丰,陈孝平,肿瘤坏死因子α逆转人肝癌多药耐药性的实验研究[J].中华实验外科杂志,1999,16[3]:256-258
30 张珂.卵巢癌顺铂耐药靶向逆转的初步研究[D]. 山东大学,2005
31 Beeghly A, Katsaros D, Chen H, et al. Glutathione Stransferase polymorphisms and ovarian cancer trentment and surivival[J]. Gynecol Oncol,2006,100(2):330-337.
32 Boresellino N,Crescimanno M,flandina C,et,al.Combined activity of interleukin-1 alpha or TNF-alpha and doxorubicin on multidrug resistantcell lines:evidence that TNF and DXR gave synergistic antitumor and differertiation-inducing effects[J]. Anticancer Res,1994,14(6B):2643-2648
1 Carla Frova. Glutathione transferases in the genomicsera new insights and perspectives [J]. Biomol Eng, 2006,23(4):149-169
2 Mclwain CC,Townsend DM, Tew KD. et al .Glutathione Stransferase polymorphisms: cancer incidence and therapy[J]. Oncogene ,2006 ,25(11):1639-1648
3 Doherty M ,Michael M. Tumoral drug metabolism: perspectives and therapeutic implications [J].Curr Drug Metab,2003,4(2):131-149
4 Lin JH, Masay oY. Clinical relevance of P-glycoprotein in Drugtherapy [J]. Drug Metab Rev, 2003,35(4):417-454
5 Valkov NI,Sullivan DM, Tumor p53 statusand response to topoisomerase II inhibitors [J].Drug Resist Updatesx, :2003,6(1):27-39
6 汪毅,周理.多药耐药相关蛋白 1,谷胱甘肽 S 转移酶和DNA 拓 酶 在 骨 肉 瘤 中 的 表 达 [J]. 重 庆 医学,2006,35(14):1273-1275
7 Lorenzo PS,Dennis PA.Modulating protein kinaseC (PKC ) to increase the efficacy of chemotherapy stepping into darkness [J].Drug Resist Updates ,2003 ,(6):329-339
8 于韬,赵桂森,臧恒昌等 肿瘤多药耐药逆转剂研究进展[J]. 中国药物化学杂志,2003,13(53):172-178
9 冯凤芝,向阳,张卫光等 人肿瘤坏死因子α基因转导绒癌耐药细胞系体外耐药逆转的研究[J]。肿瘤,2003,23(4):283-286
10 Stein U ,Walther W, Shoemaker RH,et al. Modulation of mdr1 expression by cytokines in human colon carcinoma cells: an approach for reversal of multidrug resistance[J].Br J Cancer,1996,74(9):1384-1391
11 Walther W, Stein U,Pfeil D,et al.Gene transfer of human TNF alpha into glioblastoma cells permits of mdr1expression and potentiation of chemosensitivity[J]. Int J Cancer,1995,61(6):832-839
12 Stein U ,Walther W, Shoemaker RH,et al.Reversal of multidrug resistance by transduction of cytokine genes into human colon carcinoma cells[J].J Natl Cancer Inst,1996,88(19): 1383-1392
13 Boresellino N, Rainaldi G,Tritarell E,et al .Tumor necrosis factor is a powerful apoptotic inducer in lymphoid leukemic cells expressing the p-170 glycoprotein [J].Anticancer Res,1994,14(6B):2643-2648
14 庄静丽,徐建民,王宝珍等. TNF 诱导 K562/VCR 和 K562细胞凋亡及逆转其耐药作用的实验研究[J].实用肿瘤杂志,2004,19(4):296-300
15 Matarrese P,Testa U,cauda R,et al. Expression of P-170 glycoprotein sensitizes lymphoblastoid CEM cell tomitochondia-mediated apoptosis [J].Biochem J,2001,335 (pt3):587-595
16 Yin Y,ALLen PD,Jia L,et al.Constitutive levels of cAMP-dependent protein kinase activity determine sensitivity of human multidmg-resistant leukaemic cell lines to growth inhibition and apoptosis by forskolin and tumor necrosis factor alpha[J].Br J Haematol ,2000,108(3):565.-573
17 Stein U ,Walther W,Laurencot C M ,et al.Increased activityand sensitivity of the multidrug resistance associated genes LRP and MRP [J]. Natl Cancer Inst,1997,89(11):807-813
18 Cimoli G, Valenti M, Parodi S, et al. Reversal of "atypical"-multidrug resistance by recombinant human tumor necrosis factor in the human ovarian cancer cell line A2780-DX3.[J].Oncol Res, 1993,5(8): 311-323
19 杨亚菁,袁志军,罗以,等.重组改构人肿瘤坏死因子治疗
47 例 恶 性 胸 腹 腔 积 液 [J]. 中 国 癌 症 杂志,2004,14(4):396-398
20 周清华,鄢希,任莉,等.注射用重组改构人肿瘤坏死因子联合化疗药物治疗非小细胞肺癌的多中心 III 期临床试验[J].中国肺癌杂志,2003,6(4):264-267
21 彭宝岗,梁力键,何强,等.肿瘤坏死因子联合干扰素 C 治疗 原 发 肝 癌 的 实 验 研 究 . 中 华 肝 胆 外 科 杂志,2003,9(1):25-25
22 魏虎来,葛建国,赵怀顺等.干扰素和环孢素 A 协同逆转 K562/ADM 细胞对阿霉素的耐药性[J].肿瘤防治研究,2005,32(2):99-101
23 钱其军,严文伟, 杨纯正等 白血病细胞株对马法兰的耐药机制与α干扰素的逆转作用[J].中华医学杂志,1996,76(7):485-488
24 平宝红,周淑芸,刘启发等.干扰素逆转白血病细胞多药耐药性的研究及机制的探讨[J].中华血液学杂志,1996,17(1):13-15
25 Takeuchi A,kaneko S,Matsushhita E,et,al.Interferon-alphamodulates resistance to cisplatin in three human hepatoma cell lines[J]. J Gastroenterol, 1999,34(3): 351-358
26 张荣河,何三光等.肿瘤坏死因子、白介素-2、α-干扰素及合用异博定对乳腺癌细胞多药耐药性的逆转作用研究[J].中国肿瘤临床与康复,2001,8(2):51-53
27 丁泓文,何文芳,徐谊朝等.α-干扰素、异搏定协同逆转肾癌细胞药物耐受性[J].暨南大学学报,2000,21(6):5-8
28 Corrias MV,Basso S,Meazza R,et al. Characterization and tumorigenicity of human neuroblastoma cells transfected with the IL-2 gene [J]. Cancer Gene Ther,1998,5(1): 38-44
29 崔竹梅,杨秀玉,向阳等. IL2 基因转染逆转绒癌耐药细胞系多药耐药性[J]. 基础医学与临床,2001,21(6 )523-526
30 付劲蓉,李成荣,杨锡强等。 白介素-4 基因修饰对儿童肝母细胞瘤多药耐药的逆转及其机制的研究[J].中华儿科杂志,2001,39(11): 650-653
31 贾庆瑞,葛祥花,隋金财等.白细胞介素-2 逆转急性白血病细胞多药耐药的临床研究[J]. 白血病? 淋巴瘤,2003,12(1):28-30
32 沈文状,易继林,李兴睿等. IL-2 治疗对肝癌化疗敏感性影响的研究[J]. 中国已是杂志,2005,7(1):11-13
33 平宝红,周淑芸,杨纯正等.4 种细胞因子对 K562/S 及其耐药细胞株 K562/AO2 细胞毒性作用检测 [J].解放军医学杂志,1998,23(4):299-300
34 Spicka I,Jirasek A,Koubek K et al. Progression of multiple myeloma during treatment with recombinant G-CSF and absence of G-CSF and IL-6 cell surface receptors on malignant cells [J].Cas Lek Cesk, 1998,137(21): 660-663
35 List A F. Role of mulitidrug resistance and its pharam acological modulation in acute myoloid leukem ia.Leukem ia[J]. 1996,10(6): 937-942