背景与目的:维甲酸诱导甲状腺癌分化的有效率约30%,但其毒副作用限制了临床应用.两种以上诱导剂联合应用于肿瘤的分化,临床已开展了Ⅰ～Ⅲ期试验.但对维甲酸和组蛋白脱乙酰基酶的抑制剂的联合应用,未有报道.本试验初步研究全反式维甲酸(retinoic acid,RA)联合曲古抑素(trichostain A,TSA)诱导人乳头状甲状腺癌细胞株(K1)和人滤泡状甲状腺癌细胞株(FTC-133)的分化,增强诱导作用的效果,同时降低单一药物的毒副作用,为临床前期试验提供理论依据.方法:RA联合TSA,分为4种浓度,分别为1组RA1×10-4mol/L+TSA 1.65×10-7mol/L、2组RA1×10-4mol/L+TSA 3.31×10-7mol/L、3组RA1×10-5mol/L+TSA 1.65 ×10-7mol/L、4组RA 1×10-5mol/L+TSA 3.31×10-7mol/L,在3个时间点(12、24、48 h)处理两种细胞后.采用苏木精-伊红染色后,观察细胞生长的数量、形态;采用氮蓝四唑盐法(methythiazolyltetrazolium,MTT)计算细胞生存率(survival rate,SR),研究联合药物对细胞的增殖、抑制和毒性作用;采用电化学发光法测定两种细胞株培养液中,甲状腺球蛋白(thyroglobulin,Tg)的水平,研究联合药物对肿瘤细胞的诱导分化作用.结果:联合药物作用K1和FFC-133后,细胞形态趋于规则,细胞间隔增大,细胞核明显缩小.4种浓度、3个时间点SR的方差分析,K1的F值分别为:23.52、170.14,FTC-133的F值分别为:57.09、224.35(P=0.000,均<0.01),有统计学意义.SNK分析,SR由低到高的排列分别为:2组<1组<4组<3组.4种浓度、3个时间点Tg方差分析,K1的F值分别为:69.63、101.07,FTC-133的F值分别为:79.77、81.72(P=0.000,均<0.01),差异有统计学意义.LSD两两比较:K1和FTC-133的1组与3组比较,P分别为:0.06、0.2,>0.05,两者之间差异无统计学意义,其他组均有统计学意义.结论:低浓度RA联合低浓度TSA,既可以抑制K1和FFC-133增殖,降低药物毒性作用.又可以增强对肿瘤细胞的诱导分化.其可能的机制是,TSA作用于转录步骤的DNA前调节,RA可能作用于转录步骤的信号后续调节,两种作用可能形成增强作用.通过转录后的传导系统,达到了抑制肿瘤细胞增殖和增强诱导分化的作用.
揹景與目的:維甲痠誘導甲狀腺癌分化的有效率約30%,但其毒副作用限製瞭臨床應用.兩種以上誘導劑聯閤應用于腫瘤的分化,臨床已開展瞭Ⅰ～Ⅲ期試驗.但對維甲痠和組蛋白脫乙酰基酶的抑製劑的聯閤應用,未有報道.本試驗初步研究全反式維甲痠(retinoic acid,RA)聯閤麯古抑素(trichostain A,TSA)誘導人乳頭狀甲狀腺癌細胞株(K1)和人濾泡狀甲狀腺癌細胞株(FTC-133)的分化,增彊誘導作用的效果,同時降低單一藥物的毒副作用,為臨床前期試驗提供理論依據.方法:RA聯閤TSA,分為4種濃度,分彆為1組RA1×10-4mol/L+TSA 1.65×10-7mol/L、2組RA1×10-4mol/L+TSA 3.31×10-7mol/L、3組RA1×10-5mol/L+TSA 1.65 ×10-7mol/L、4組RA 1×10-5mol/L+TSA 3.31×10-7mol/L,在3箇時間點(12、24、48 h)處理兩種細胞後.採用囌木精-伊紅染色後,觀察細胞生長的數量、形態;採用氮藍四唑鹽法(methythiazolyltetrazolium,MTT)計算細胞生存率(survival rate,SR),研究聯閤藥物對細胞的增殖、抑製和毒性作用;採用電化學髮光法測定兩種細胞株培養液中,甲狀腺毬蛋白(thyroglobulin,Tg)的水平,研究聯閤藥物對腫瘤細胞的誘導分化作用.結果:聯閤藥物作用K1和FFC-133後,細胞形態趨于規則,細胞間隔增大,細胞覈明顯縮小.4種濃度、3箇時間點SR的方差分析,K1的F值分彆為:23.52、170.14,FTC-133的F值分彆為:57.09、224.35(P=0.000,均<0.01),有統計學意義.SNK分析,SR由低到高的排列分彆為:2組<1組<4組<3組.4種濃度、3箇時間點Tg方差分析,K1的F值分彆為:69.63、101.07,FTC-133的F值分彆為:79.77、81.72(P=0.000,均<0.01),差異有統計學意義.LSD兩兩比較:K1和FTC-133的1組與3組比較,P分彆為:0.06、0.2,>0.05,兩者之間差異無統計學意義,其他組均有統計學意義.結論:低濃度RA聯閤低濃度TSA,既可以抑製K1和FFC-133增殖,降低藥物毒性作用.又可以增彊對腫瘤細胞的誘導分化.其可能的機製是,TSA作用于轉錄步驟的DNA前調節,RA可能作用于轉錄步驟的信號後續調節,兩種作用可能形成增彊作用.通過轉錄後的傳導繫統,達到瞭抑製腫瘤細胞增殖和增彊誘導分化的作用.
배경여목적:유갑산유도갑상선암분화적유효솔약30%,단기독부작용한제료림상응용.량충이상유도제연합응용우종류적분화,림상이개전료Ⅰ～Ⅲ기시험.단대유갑산화조단백탈을선기매적억제제적연합응용,미유보도.본시험초보연구전반식유갑산(retinoic acid,RA)연합곡고억소(trichostain A,TSA)유도인유두상갑상선암세포주(K1)화인려포상갑상선암세포주(FTC-133)적분화,증강유도작용적효과,동시강저단일약물적독부작용,위림상전기시험제공이론의거.방법:RA연합TSA,분위4충농도,분별위1조RA1×10-4mol/L+TSA 1.65×10-7mol/L、2조RA1×10-4mol/L+TSA 3.31×10-7mol/L、3조RA1×10-5mol/L+TSA 1.65 ×10-7mol/L、4조RA 1×10-5mol/L+TSA 3.31×10-7mol/L,재3개시간점(12、24、48 h)처리량충세포후.채용소목정-이홍염색후,관찰세포생장적수량、형태;채용담람사서염법(methythiazolyltetrazolium,MTT)계산세포생존솔(survival rate,SR),연구연합약물대세포적증식、억제화독성작용;채용전화학발광법측정량충세포주배양액중,갑상선구단백(thyroglobulin,Tg)적수평,연구연합약물대종류세포적유도분화작용.결과:연합약물작용K1화FFC-133후,세포형태추우규칙,세포간격증대,세포핵명현축소.4충농도、3개시간점SR적방차분석,K1적F치분별위:23.52、170.14,FTC-133적F치분별위:57.09、224.35(P=0.000,균<0.01),유통계학의의.SNK분석,SR유저도고적배렬분별위:2조<1조<4조<3조.4충농도、3개시간점Tg방차분석,K1적F치분별위:69.63、101.07,FTC-133적F치분별위:79.77、81.72(P=0.000,균<0.01),차이유통계학의의.LSD량량비교:K1화FTC-133적1조여3조비교,P분별위:0.06、0.2,>0.05,량자지간차이무통계학의의,기타조균유통계학의의.결론:저농도RA연합저농도TSA,기가이억제K1화FFC-133증식,강저약물독성작용.우가이증강대종류세포적유도분화.기가능적궤제시,TSA작용우전록보취적DNA전조절,RA가능작용우전록보취적신호후속조절,량충작용가능형성증강작용.통과전록후적전도계통,체도료억제종류세포증식화증강유도분화적작용.
Background and Objective: The effectiveness rate of all-trans-retinoic acid(RA)is only about 30% in the clinical application of inducing thyroid carcinoma differentiation.In addition,there are severe toxic side effects,which limit its clinical application.Phase Ⅰ-Ⅲ clinical studies have been conducted on the combined application of two or more kinds of inductors in tumors.Nevertheless,the combination of RA with histone deacetylase inhibitors is rarely reported.This article studied the effects of differentiation for papillary thyroid carcinoma and follicular thyroid carcinoma cell lines induced by RA combined with trichostatin A(TSA),enhancing the effect of induction,while reducing the toxic side effects of a single drug,to provide a theoretical basis for preclinical trials.Methods: After incubation with RA combined with TSA,K1 and FTC-133 were grouped into Group 1(RA 1×10-4 mol/L plus TSA 1.65×10-7 mol/L),Group 2(RA 1×10-4 mol/L plus TSA 3.31×10-7 mol/L),Group 3(RA 1×10-5 mol/L plus TSA 1.65×10-7mol/L),Group 4(RA 1×10-5 mol/L plus TSA 3.31×10-7 mol/L)by four varied concentrations and three time points(12,24,and 48 h).The cell proliferation,conformation,toxic effect,and induced differentiation on K1 and FTC-133 cell lines were studied microscopically with hematoxylin-eosin(HE)to observe cell quantity and morphology,methyl-thiazolyl-tetrazolium(MTT)to calculate cell survival rates,and electrochemiluminescence analysis to measure in vitro thyroglobulin(Tg)levels.Results: The research showed that K1 and FTC-133 cells had cell spacing increases,with an outer edge of smooth,nuclear chromatin condensation after RA combined TSA.Survival rates were assessed by an analysis of variance(ANOVA)by concentration and time point,F values of K1 were 23.52 and 170.14,and F values of FTC-133 were 57.09 and 224.35,respectively.There were significant differences for both cells(P <0.01).The SNK analysis indicated that survival rates were in the order of Group 2<Group1<Group4<Group 3.Tg was also assessed by ANOVA,F values of K1 were 69.63 and 101.07,and F values of FTC-133were 79.77 and 81.72(P<0.01).Group 1 was compared with Group 3 of K1 and FTC-133 by the least significant difference(LSD)method,and there was no statistical difference between the two groups(P=0.06,0.20,respectively,P>0.05),yet a significant difference was seen between the other groups.Conclusions: Lower concentrations of RA combined with lower concentrations of TSA have both inhibited cell proliferation,decreased toxicity of the drugs,and increased the effect of K1 and FTC-133 cell differentiation.The mechanism of action may be that TSA has pretranscription DNA regulation and that RA has posttranscriptional signal regulation to enhance the effects ofinhibited proliferation and differentiation of cells by transcription systems.
