三七总皂甙体外逆转K562/VCR细胞多药耐药的实验性研究
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摘要
目的 从中药中寻找安全有效的多药耐药逆转剂，并初步研究三七总皂甙(PNS)对人白血病细胞K562(敏感细胞)及K562／VCR(耐药细胞)的影响。
方法 以MTT法测定三七总皂甙注射液对K562及K562／VCR的直接细胞毒作用；测定阿霉素(DOX)对两种细胞的毒性作用并计算出耐药倍数；以无细胞毒性的三七总皂甙注射液作为耐药逆转剂用MTT法体外药敏感实验观察其对多药耐药细胞株K562／VCR的逆转作用；用流式细胞仪测定PNS作用后敏感细胞和耐药细胞内阿霉素的浓度；以流式细胞仪测定敏感细胞、用和未用三七总皂甙作用的耐药细胞表达多耐耐药糖蛋白P-gp(P-170)的阳性率。以上实验均用维拉帕米作为阳性对照。
结果 ①三七总皂甙在300μg／ml浓度以下时对两细胞基本无毒性，为安全有效的逆转剂量。②阿霉素对敏感细胞和耐药细胞的半数抑制浓度(IC_(50))分别为28.7ng／ml和1141.8ng／ml，耐药倍数为39.7倍。③三七总皂甙能够增强阿霉素(DOX)对K562／VCR的细胞毒作用，K562／VCR耐药
细胞在50 pg/ml、100 pg/ml、200 pg/m1PNS作用24小时
后IC:(，分别下降至392.8士58.Ing/ml、282.6士53.sng/ml、
203.6士19.75ng/ml，与对照组相t匕均P(0.01逆转倍数分别
为2.9倍、4.0倍5.6倍。④三七总皂试能够增加耐药细胞
K562/VCR细胞内的阿霉素的蓄积浓度，200 pg/m1的三七总
皂贰作用于K562/VCR耐药细胞后，流式细胞仪检测耐药细
胞内的阿霉素平均荧光强度由用药前的108.36士n.73上升
至145.76士4.51(二者t匕较只0.01)。⑤三七总皂贰(200。
g/m1)可下调多药耐药基因mdr一1表达的P一gP糖蛋白的量，
而未发现维拉帕米的类似作用。三七总皂贰和用带荧光标记
的P一170单克隆抗体IgGZa作用后的耐药细胞，用流式细胞
仪测定其平均荧光强度由用药前的163.32士7.23下降到
101.60士4. 60(二者比较只0.01)。
结论①高浓度的三七总皂贰具有一定的抗肿瘤作用;
②三七总皂贰可增加K562/VCR细胞内阿霉素药物浓度;③
三七总皂贰能够下调K562/VCR细胞表达P一gP的量;④三七
总皂贰可部分逆转耐药细胞系K562/VCR的多药耐药性;⑥
是一种有效安全的多药耐药逆转剂。
Objective To search a sort of safe and effective reversal agent for multidrug resistantce from Chinese traditional medicine. And study the impact of the panaxnotoginseng saponins(PNS) to human leukemia clone K562 and multidrug resistance clone K562/VCR in vitro. Method to measure direct cell toxicant effect of PNS to K562(senstive strain) and K562/VCR(multidrug-resistant strain) and measure toxicant effect of dox to both strains for resistant index by MTT; To observe the reverse effect of PNS which has screened for nontoxicant concentration to K562/VCR by MTT; to measure concentration of dox in both sort of cell after applied PNS (200ug/ml). To measure expressional positve rate of the both cell after applied PNS (200ug/ml). Applied Veraparmil as a positive control to experiment above
Result (1) there is no obvious cell toxicity of PNS to K562 and K562/VCR cell, when the concentration of PNS is 300ug/ml below. (2) Inhibitive concentration 50 of Dox to K562 and k562/VCR are 28.7ng/ml and 1141.8ng/ml respectively. Drug-Resistant index of K562/VCR is 39.7. (3) PNS can increased cell toxic effect of dox , after applied PNS of 50ug/mk 100ug/mk 200ug/ml concentration respectively to K562/VCR for 24 hours, inhibitive concentration 50 respectively decline to 392.8 ± 58.1ng/mK 282.6±53.8ng/ml, 203.6 ±19.75ng/ml(P<0.01): (4) PNS can increased K562/VCR's accumulated concentration of dox ,after applied PNS of 200ug/ml to K562/VCR for 3hours, find that fluorescence
intension increased from 108.36+11.73 tol45.76?.51 on average by flow cytometry(P<0.01). (5) measuring P-gp expression of mdr-1 of drug-resista-nt cell K562/VCR by flow cytometry after applied PNS and Ig-a(monoclony antibody of P-170) to K562/VCR for 3 hours ,indicate that average fluroescence intension of P-gp of expression decline from 138 to 65 .in contrast find no significant effect with verapamil. Conclusion (1) PNS of high concentration has certain tumour-resistant effect;.(2) PNS can increased K562/VCR's accumulated concentration of dox;(3) PNS can reduced K562/VCR's P-gp expression of mdr-1;(4) PNS can partly revese multidrug-resistance of K562/VCR in vitro;(5) PNS may be a kind of safe and efficient reversal agent for clinic.
方法 以MTT法测定三七总皂甙注射液对K562及K562／VCR的直接细胞毒作用；测定阿霉素(DOX)对两种细胞的毒性作用并计算出耐药倍数；以无细胞毒性的三七总皂甙注射液作为耐药逆转剂用MTT法体外药敏感实验观察其对多药耐药细胞株K562／VCR的逆转作用；用流式细胞仪测定PNS作用后敏感细胞和耐药细胞内阿霉素的浓度；以流式细胞仪测定敏感细胞、用和未用三七总皂甙作用的耐药细胞表达多耐耐药糖蛋白P-gp(P-170)的阳性率。以上实验均用维拉帕米作为阳性对照。
结果 ①三七总皂甙在300μg／ml浓度以下时对两细胞基本无毒性，为安全有效的逆转剂量。②阿霉素对敏感细胞和耐药细胞的半数抑制浓度(IC_(50))分别为28.7ng／ml和1141.8ng／ml，耐药倍数为39.7倍。③三七总皂甙能够增强阿霉素(DOX)对K562／VCR的细胞毒作用，K562／VCR耐药
细胞在50 pg/ml、100 pg/ml、200 pg/m1PNS作用24小时
后IC:(，分别下降至392.8士58.Ing/ml、282.6士53.sng/ml、
203.6士19.75ng/ml，与对照组相t匕均P(0.01逆转倍数分别
为2.9倍、4.0倍5.6倍。④三七总皂试能够增加耐药细胞
K562/VCR细胞内的阿霉素的蓄积浓度，200 pg/m1的三七总
皂贰作用于K562/VCR耐药细胞后，流式细胞仪检测耐药细
胞内的阿霉素平均荧光强度由用药前的108.36士n.73上升
至145.76士4.51(二者t匕较只0.01)。⑤三七总皂贰(200。
g/m1)可下调多药耐药基因mdr一1表达的P一gP糖蛋白的量，
而未发现维拉帕米的类似作用。三七总皂贰和用带荧光标记
的P一170单克隆抗体IgGZa作用后的耐药细胞，用流式细胞
仪测定其平均荧光强度由用药前的163.32士7.23下降到
101.60士4. 60(二者比较只0.01)。
结论①高浓度的三七总皂贰具有一定的抗肿瘤作用;
②三七总皂贰可增加K562/VCR细胞内阿霉素药物浓度;③
三七总皂贰能够下调K562/VCR细胞表达P一gP的量;④三七
总皂贰可部分逆转耐药细胞系K562/VCR的多药耐药性;⑥
是一种有效安全的多药耐药逆转剂。
Objective To search a sort of safe and effective reversal agent for multidrug resistantce from Chinese traditional medicine. And study the impact of the panaxnotoginseng saponins(PNS) to human leukemia clone K562 and multidrug resistance clone K562/VCR in vitro. Method to measure direct cell toxicant effect of PNS to K562(senstive strain) and K562/VCR(multidrug-resistant strain) and measure toxicant effect of dox to both strains for resistant index by MTT; To observe the reverse effect of PNS which has screened for nontoxicant concentration to K562/VCR by MTT; to measure concentration of dox in both sort of cell after applied PNS (200ug/ml). To measure expressional positve rate of the both cell after applied PNS (200ug/ml). Applied Veraparmil as a positive control to experiment above
Result (1) there is no obvious cell toxicity of PNS to K562 and K562/VCR cell, when the concentration of PNS is 300ug/ml below. (2) Inhibitive concentration 50 of Dox to K562 and k562/VCR are 28.7ng/ml and 1141.8ng/ml respectively. Drug-Resistant index of K562/VCR is 39.7. (3) PNS can increased cell toxic effect of dox , after applied PNS of 50ug/mk 100ug/mk 200ug/ml concentration respectively to K562/VCR for 24 hours, inhibitive concentration 50 respectively decline to 392.8 ± 58.1ng/mK 282.6±53.8ng/ml, 203.6 ±19.75ng/ml(P<0.01): (4) PNS can increased K562/VCR's accumulated concentration of dox ,after applied PNS of 200ug/ml to K562/VCR for 3hours, find that fluorescence
intension increased from 108.36+11.73 tol45.76?.51 on average by flow cytometry(P<0.01). (5) measuring P-gp expression of mdr-1 of drug-resista-nt cell K562/VCR by flow cytometry after applied PNS and Ig-a(monoclony antibody of P-170) to K562/VCR for 3 hours ,indicate that average fluroescence intension of P-gp of expression decline from 138 to 65 .in contrast find no significant effect with verapamil. Conclusion (1) PNS of high concentration has certain tumour-resistant effect;.(2) PNS can increased K562/VCR's accumulated concentration of dox;(3) PNS can reduced K562/VCR's P-gp expression of mdr-1;(4) PNS can partly revese multidrug-resistance of K562/VCR in vitro;(5) PNS may be a kind of safe and efficient reversal agent for clinic.
引文
1 俞恩珠．三七总皂甙对心脑血管的药理作用．浙江中西医结合杂志，2000，4(10)：25～26．
2 彭宝岗，吕明德，肖定璋，等．三七总皂甙对大鼠钙内流的阻断作用．中国药理学通报，1997，13(1)：70～73．
3 Hamada H,Hagiwara KI,Nakafima T, et al. Phosphorization of the Mdr 17000 to 18000glycoprotien specific to multidrug resistance tumor cells.Eftects of verapamil,trifluoperazine,and phorbl esters. Cancer Res ,1987,47:2860.
4 胡振，张惠斌．肿瘤多药耐药逆转剂的研究进展．药学进展，2002，26(3)：129～133．
5 梁蓉，杨平地，陈协群．川芎嗪和异博定联合逆转HL60／HT细胞的多药耐药．第四军医大学学报．1998，19(4)：385～387．
6 许文林，钱军，费霞，等．汉防己甲素逆转血液系统肿瘤细胞多药耐药的临床观察．中华血液学杂志，1999，20(7)：383～384．
7 石淑文，郭淑芬．多药耐药细胞系K562／HHT和K562／VCR的建立及耐药性逆转的研究．中华儿科杂志，1998，36(1)：15～18．
8 陈泽涛，董倩．肿瘤细胞多药耐药及其中医药研究．山东中医药大学学报,1999，5(3)：12～13．
9 李云凤，张永恒，于桂兰等．十全大补冲剂对肿瘤化疗药物增效减毒作用的研究．肿瘤杂志，1996，1(16)：38～39．
10 张代钊，于尔辛，余桂清等．中医药对肿瘤放化疗的增敏减毒作用
中国中西医结合杂志，1992，3(2)：135～137．
11 孙华丽，余桂清．扶正增效方对恶性肿瘤放射增敏作用临床和实验研究．中医杂志，1990，(6)：25．
12 谢兆霞，谭达人，谢俊明，等．“拮新康”的逆转耐药与抗肿瘤作用的研究．中国现代医学杂志，1996，6(4)：18～19．
13 梁蓉，杨平地，陈协群．川芎嗪逆转HL-60／VCR细胞多药耐药的体外研究．第六届全国实验血液学会议论文摘要(97年11月)：196～197．
14 史曦凯，张翼军，赵春景，等．人参皂甙单体Rb1对多药耐药细胞系K562／HHT的耐药逆转作用．第三军医大学学报1999，21(11)：825～827．
2 彭宝岗，吕明德，肖定璋，等．三七总皂甙对大鼠钙内流的阻断作用．中国药理学通报，1997，13(1)：70～73．
3 Hamada H,Hagiwara KI,Nakafima T, et al. Phosphorization of the Mdr 17000 to 18000glycoprotien specific to multidrug resistance tumor cells.Eftects of verapamil,trifluoperazine,and phorbl esters. Cancer Res ,1987,47:2860.
4 胡振，张惠斌．肿瘤多药耐药逆转剂的研究进展．药学进展，2002，26(3)：129～133．
5 梁蓉，杨平地，陈协群．川芎嗪和异博定联合逆转HL60／HT细胞的多药耐药．第四军医大学学报．1998，19(4)：385～387．
6 许文林，钱军，费霞，等．汉防己甲素逆转血液系统肿瘤细胞多药耐药的临床观察．中华血液学杂志，1999，20(7)：383～384．
7 石淑文，郭淑芬．多药耐药细胞系K562／HHT和K562／VCR的建立及耐药性逆转的研究．中华儿科杂志，1998，36(1)：15～18．
8 陈泽涛，董倩．肿瘤细胞多药耐药及其中医药研究．山东中医药大学学报,1999，5(3)：12～13．
9 李云凤，张永恒，于桂兰等．十全大补冲剂对肿瘤化疗药物增效减毒作用的研究．肿瘤杂志，1996，1(16)：38～39．
10 张代钊，于尔辛，余桂清等．中医药对肿瘤放化疗的增敏减毒作用
中国中西医结合杂志，1992，3(2)：135～137．
11 孙华丽，余桂清．扶正增效方对恶性肿瘤放射增敏作用临床和实验研究．中医杂志，1990，(6)：25．
12 谢兆霞，谭达人，谢俊明，等．“拮新康”的逆转耐药与抗肿瘤作用的研究．中国现代医学杂志，1996，6(4)：18～19．
13 梁蓉，杨平地，陈协群．川芎嗪逆转HL-60／VCR细胞多药耐药的体外研究．第六届全国实验血液学会议论文摘要(97年11月)：196～197．
14 史曦凯，张翼军，赵春景，等．人参皂甙单体Rb1对多药耐药细胞系K562／HHT的耐药逆转作用．第三军医大学学报1999，21(11)：825～827．