CAJ | 학술논문

目的：探讨水飞蓟宾对胰腺癌 AsPC －1细胞的增殖抑制作用及其作用机制．方法：MTT 法和克隆形成抑制实验观察水飞蓟宾对人胰腺癌 AsPC －1细胞的增殖抑制作用，碘化丙锭（PI）单染色检测细胞周期改变，An-nexin V －FITC ／PI 双染流式细胞术检测细胞凋亡水平，Western blotting 检测细胞周期及细胞凋亡相关蛋白的表达．结果：不同浓度的水飞蓟宾对胰腺癌 AsPC －1细胞的生长均有抑制作用，且呈剂量－效应和时间－效应关系（P ＜0.05），水飞蓟宾作用于 AsPC －1细胞48、72 h 的 IC50浓度分别为224.20、87.25μmol／L；克隆形成抑制实验显示，随着水飞蓟宾浓度增加，AsPC －1细胞克隆形成逐渐减少．细胞周期检测结果显示，随着水飞蓟宾浓度的增加，胰腺癌 AsPC －1细胞出现明显 G1期阻滞；水飞蓟宾处理组细胞的周期蛋白 CyclinD1、CyclinE2、CyclinA、CyclinB1表达下降，细胞周期蛋白激酶 CDK4、CDK6表达不变，细胞周期素依赖性蛋白激酶抑制蛋白 P15 INK4B、P21 WAF1／CIP1表达升高，与流式检测的结果相一致．不同浓度水飞蓟宾作用48 h 后，出现明显的凋亡细胞群；同时发现 Caspase －9、Caspase －3活化降解，Caspase3下游效应蛋白 PARP 出现切割条带．JNK 蛋白表达增加并磷酸化活化，Bcl －2蛋白家族中抗凋亡蛋白 Bcl －2、Bcl －xL、Mcl －1表达明显降低，促凋亡蛋白 Bax 表达基本不变，BH3－only 蛋白 Bcl －xs、Bid、Bim 表达增加．结论：水飞蓟宾明显抑制胰腺癌细胞增殖，通过诱导 P15 INK4B、P21 WAF1／CIP1表达阻滞细胞周期在 G1期，并通过诱导 JNK 活化激活线粒体细胞凋亡途径，进而诱导胰腺癌 AsPC －1细胞凋亡．
목적：탐토수비계빈대이선암 AsPC －1세포적증식억제작용급기작용궤제．방법：MTT 법화극륭형성억제실험관찰수비계빈대인이선암 AsPC －1세포적증식억제작용，전화병정（PI）단염색검측세포주기개변，An-nexin V －FITC ／PI 쌍염류식세포술검측세포조망수평，Western blotting 검측세포주기급세포조망상관단백적표체．결과：불동농도적수비계빈대이선암 AsPC －1세포적생장균유억제작용，차정제량－효응화시간－효응관계（P ＜0.05），수비계빈작용우 AsPC －1세포48、72 h 적 IC50농도분별위224.20、87.25μmol／L；극륭형성억제실험현시，수착수비계빈농도증가，AsPC －1세포극륭형성축점감소．세포주기검측결과현시，수착수비계빈농도적증가，이선암 AsPC －1세포출현명현 G1기조체；수비계빈처리조세포적주기단백 CyclinD1、CyclinE2、CyclinA、CyclinB1표체하강，세포주기단백격매 CDK4、CDK6표체불변，세포주기소의뢰성단백격매억제단백 P15 INK4B、P21 WAF1／CIP1표체승고，여류식검측적결과상일치．불동농도수비계빈작용48 h 후，출현명현적조망세포군；동시발현 Caspase －9、Caspase －3활화강해，Caspase3하유효응단백 PARP 출현절할조대．JNK 단백표체증가병린산화활화，Bcl －2단백가족중항조망단백 Bcl －2、Bcl －xL、Mcl －1표체명현강저，촉조망단백 Bax 표체기본불변，BH3－only 단백 Bcl －xs、Bid、Bim 표체증가．결론：수비계빈명현억제이선암세포증식，통과유도 P15 INK4B、P21 WAF1／CIP1표체조체세포주기재 G1기，병통과유도 JNK 활화격활선립체세포조망도경，진이유도이선암 AsPC －1세포조망．
Aim:To investigate the inhibitory effects of Silibinin on pancreatic cancer AsPC-1 cells and the possible mechanism.Methods:MTT and cell clone formation inhibitory assay were used to in- <br> vestigate the inhibitory effects of Silibinin on human pancreatic cancer AsPC-1 cells.Cell cycle distribu-tion and cell apoptosis were determined by flow cytometry staining with propidium iodide (PI)or AnnexinⅤ-FITC and PI respectively.The expressions of cell cycle and apoptosis related proteins were analyzed by Western blotting.Results:MTT results showed that Silibinin inhibited pancreatic cancer AsPC-1 cells′proliferation in dose and time-dependent manner(P <0.05).The IC50 of Silibinin against pancreat-ic cancer AsPC-1 cells for 48 hours and 72 hours was 224.20 μmol /L and 87.25 μmol /L,respectively. Cell colony formation inhibition assay showed that the number of cell clones were decreased with the in-creasing of Silibinin′s concentration.PI staining analysis showed that cell cycle was arrested at G1 phase. Western blotting assay showed that the levels of Cyclins (D1,E2,A,B1)decreased,without changes in cyclin-dependent kinases (CDK4 and CDK6 ),but cyclin-dependent kinase inhibitors (P15 INK4B , P21 WAF1 /CIP1 )increased,which was in consistence with the results of flow cytometric analysis.Annexin V-FITC /PI staining analysis also showed a dose-dependent effect on apoptotic cells when the pancreatic cancer cells were incubated with Silibinin for 48 hrs.Caspase-9,Caspase-3 were cleaved,and the cleaved bands of PARP were detected.The expression of JNK and phospho-JNK were found to be in-creased,while the anti-apoptotic proteins of Bcl-2,Bcl-xL and Mcl-1 were decreased,and pro-apoptotic proteins Bax was not changed.The BH-3 only proteins,Bcl-xs,Bid and Bim,were found to be in-creased by Western blotting.Conclusion:Silibinin inhibits proliferation of pancreatic cancer AsPC-1 cells,induces G1 phase arrest via upregulating P15 INK4B and P21 WAF1 /CIP1 ,and induces cell apoptosis through the mitochondrial pathway with activation of JNK.