CAJ | 학술논문

目的观察野甘草醇(SDC)在人端粒酶逆转录酶(hTERT)启动子调控腺病毒介导的单纯疱疹病毒胸腺激酶基因/丙氧鸟苷(HSV-TK/GCV)自杀基因系统对人膀胱癌细胞的体外杀伤中的增效作用.方法测定胸苷激酶(TK)活性,应用高效液相色谱(HPLC)法测定三磷酸丙氧鸟苷(GCV-TP)在肿瘤细胞中的浓度,按转染倍数(MOI)=100加入Ad-hTERT-HSV-TK,24 h后分别加入5％血清培养液含GCV(200、100、50、10、5、l、0 mg/L),含SDC(0、0.04或0.20 μmol/L),应用于膀胱癌253J细胞,观察对膀胱癌细胞的杀伤作用.旁杀伤效应观察:将Ad-hTERT-HSV-TK按MOI=100转染253J细胞,24 h后将转染后的253J细胞和未转染的253J细胞以不同比例混合培养,共分11组(1∶1、1∶2、1∶3、1∶4、2∶1、2∶3、3∶1、3∶2、3∶4、4∶1、4∶3),在11组253J肿瘤细胞培养液中分别加入GCV使其浓度为100μmoL/L或浓度为100 μmol/L的GCV联合应用SDC 0.04μmoL/L,噻唑蓝(MTT)测定细胞存活率,观察旁杀伤作用.结果 SDC对转染Ad-hTERT-HSV-TK的膀胱癌253J细胞的TK酶活性影响不明显.应用高效液相色谱法分析,以单加GCV组的GCV-TP浓度记作100％.加入0.04 μmol/L SDC后,GCV-TP浓度变化差异无统计学意义(P＞0.05)；加入5倍治疗剂量0.20 μmol/L SDC后,GCV-TP浓度提高到(105.0±5.0)％,差异有统计学意义(P＜0.05).进一步细胞实验证实,SDC对Ad-hTERT-HSV-TK/GCV系统杀伤膀胱癌253J细胞具有增效作用,旁杀伤作用增强明显.结论 SDC对胸腺激酶依赖的GCV阻断膀胱癌进展有一定增效作用,将天然药物SDC与构建的具有肿瘤特异性的重组腺病毒Ad-hTERT-HSV-TK/GCV系统联合应用,可以达到减毒增效的目的.
목적 관찰야감초순(SDC)재인단립매역전록매(hTERT)계동자조공선병독개도적단순포진병독흉선격매기인/병양조감(HSV-TK/GCV)자살기인계통대인방광암세포적체외살상중적증효작용.방법 측정흉감격매(TK)활성,응용고효액상색보(HPLC)법측정삼린산병양조감(GCV-TP)재종류세포중적농도,안전염배수(MOI)=100가입Ad-hTERT-HSV-TK,24 h후분별가입5％혈청배양액함GCV(200、100、50、10、5、l、0 mg/L),함SDC(0、0.04혹0.20 μmol/L),응용우방광암253J세포,관찰대방광암세포적살상작용.방살상효응관찰:장Ad-hTERT-HSV-TK안MOI=100전염253J세포,24 h후장전염후적253J세포화미전염적253J세포이불동비례혼합배양,공분11조(1∶1、1∶2、1∶3、1∶4、2∶1、2∶3、3∶1、3∶2、3∶4、4∶1、4∶3),재11조253J종류세포배양액중분별가입GCV사기농도위100μmoL/L혹농도위100 μmol/L적GCV연합응용SDC 0.04μmoL/L,새서람(MTT)측정세포존활솔,관찰방살상작용.결과 SDC대전염Ad-hTERT-HSV-TK적방광암253J세포적TK매활성영향불명현.응용고효액상색보법분석,이단가GCV조적GCV-TP농도기작100％.가입0.04 μmol/L SDC후,GCV-TP농도변화차이무통계학의의(P＞0.05)；가입5배치료제량0.20 μmol/L SDC후,GCV-TP농도제고도(105.0±5.0)％,차이유통계학의의(P＜0.05).진일보세포실험증실,SDC대Ad-hTERT-HSV-TK/GCV계통살상방광암253J세포구유증효작용,방살상작용증강명현.결론 SDC대흉선격매의뢰적GCV조단방광암진전유일정증효작용,장천연약물SDC여구건적구유종류특이성적중조선병독Ad-hTERT-HSV-TK/GCV계통연합응용,가이체도감독증효적목적.
Objective To investigate the enhanced effect of dulciol (SDC) on the killing effect on antitumor actions by Ad-human telomerase reverse transcriptase (hTERT)-herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir(GCV) suicide gene system on bladder cancer cells in vitro.Methods To clarigy the mechanism of enhanced effect of SDC in the gene therapy of human bladder cancer cell line 253J by thymidine kinase (TK)-denendent GCV in molecular level,the TK enzyme activity and the concentration of GCV-TP in the tumor cells were measured by using high performance liquid chromatography (HPLC).The bladder cancer cells,respectively,were treated with different doses and different concentrations Ad-hTERT-HSV-TK [multiplicity of infection (MOI) =100],GCV (200,100,50,10,5,1,0 mg/L) and SDC (0,0.04 or 0.20 μmol/L) with The killing effect.The bystander effect of these drugs in therapy of human bladder cancer cells were observed by treated with different mixing proportion of TK + cells andTK-cells (1∶1,1∶2,1∶3,1∶4,2∶1,2∶ 3,3∶1,3∶2,3∶4,4∶1,4∶3).Results SDC had slightly effect on the TK enzyme activity of bladder cancer cells that were transduced by Ad-hTERT-HSV-TK.The GCV-TP concentration in the GCV treatment group was 100％.There was no significant difference when 0.04 μmoL/L SDC was added,The GCV-TP concentration was up to (105.0 ± 5.0) ％ when adding 0.20 μmol/L SDC,and there was significant difference campared to the GCV treatment group (P ＜0.05).It was confirmed that SDC had strong killing effect on the bladder cancer cells by combination of Ad-hTERT-HSV-TK and GCV,the bystander effect was also significantly augmented by the combined treatment of GCV and SDC.Conclusion The synergistic effect of SDC is significantly shown in the therapy of human bladder cancer cells by TK-dependent GCV.SDC is effective in the Ad-hTERT-HSV-TK/GCV administration system in killing bladder cancer cells.