CAJ | 학술논문

目的研究生存素(survivin)抑制剂YM155对滤泡淋巴瘤细胞的凋亡效应及作用机制,为应用YM155治疗淋巴瘤提供实验依据.方法培养滤泡淋巴瘤细胞株SUDHL-4至对数生长期,加入YM155形成一系列浓度梯度:100、10、1、0.1、0 ng/ml,培养24、48、72 h后进行活细胞计数试剂盒(CCK)8实验,观察YM155对其生长抑制效应,计算其细胞半数抑制药物浓度(IC50).加入YM155至培养瓶中使其药物浓度为1 ng/ml,培养24、48、72 h进行流式细胞术检测,膜联蛋白(Annexin)V-异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染法观察细胞凋亡情况.选取不加药的对照组和加YM155后24、48 h的实验组细胞抽提总RNA,然后进行反转录PCR(RT-PCR)检测48 h时抗凋亡基因bcl-2、bcl-xl、survivin和促凋亡基因bid、bax的mRNA的相对表达量,检测24h时survivin mRNA的相对表达量.同时抽提各个时间点的总蛋白用Western印迹技术检测survivin、半胱氨酸蛋白酶(caspase)9、活化的caspase-9、caspase-3和活化的caspase-3蛋白的表达量.结果 YM155对SUDHL-4具有明显的生长抑制效应且呈剂量和时间依赖性,24、48、72 h的IC50分别为6.1、2.7、1.2ng/ml.用流式细胞仪分析发现细胞凋亡比例随时间的推移逐步增加(17.3％±2.1％、35.7％±3.3％、54.6％±4.3％比2.1％±0.3％,均P＜0.05)；PCR结果显示YM155作用后24和48 h下调survivin基因mRNA的表达(0.72±0.02、0.56±0.01比1.00,均P＜0.05),但bcl-2家族基因表达变化不显著(P＞0.05).Western印迹检测发现随着时间的推移,survivin、caspase-3蛋白的表达逐渐减低,caspase-9和活化的caspase-9蛋白表达没有明显变化,而活化的caspase-3表达逐渐增加.结论 YM155能够有效促进淋巴瘤SUDHL-4细胞株凋亡,其机制可能是由于survivin蛋白下调绕过caspase-9直接激活下游caspase-3,从而切割DNA引发凋亡；bcl-2家族基因可能并未参与此过程.
목적 연구생존소(survivin)억제제YM155대려포림파류세포적조망효응급작용궤제,위응용YM155치료림파류제공실험의거.방법 배양려포림파류세포주SUDHL-4지대수생장기,가입YM155형성일계렬농도제도:100、10、1、0.1、0 ng/ml,배양24、48、72 h후진행활세포계수시제합(CCK)8실험,관찰YM155대기생장억제효응,계산기세포반수억제약물농도(IC50).가입YM155지배양병중사기약물농도위1 ng/ml,배양24、48、72 h진행류식세포술검측,막련단백(Annexin)V-이류청산형광소(FITC)/전화병정(PI)쌍염법관찰세포조망정황.선취불가약적대조조화가YM155후24、48 h적실험조세포추제총RNA,연후진행반전록PCR(RT-PCR)검측48 h시항조망기인bcl-2、bcl-xl、survivin화촉조망기인bid、bax적mRNA적상대표체량,검측24h시survivin mRNA적상대표체량.동시추제각개시간점적총단백용Western인적기술검측survivin、반광안산단백매(caspase)9、활화적caspase-9、caspase-3화활화적caspase-3단백적표체량.결과 YM155대SUDHL-4구유명현적생장억제효응차정제량화시간의뢰성,24、48、72 h적IC50분별위6.1、2.7、1.2ng/ml.용류식세포의분석발현세포조망비례수시간적추이축보증가(17.3％±2.1％、35.7％±3.3％、54.6％±4.3％비2.1％±0.3％,균P＜0.05)；PCR결과현시YM155작용후24화48 h하조survivin기인mRNA적표체(0.72±0.02、0.56±0.01비1.00,균P＜0.05),단bcl-2가족기인표체변화불현저(P＞0.05).Western인적검측발현수착시간적추이,survivin、caspase-3단백적표체축점감저,caspase-9화활화적caspase-9단백표체몰유명현변화,이활화적caspase-3표체축점증가.결론 YM155능구유효촉진림파류SUDHL-4세포주조망,기궤제가능시유우survivin단백하조요과caspase-9직접격활하유caspase-3,종이절할DNA인발조망；bcl-2가족기인가능병미삼여차과정.
Objective To explore the apoptotic effect of follicular lymphoma and related mechanism induced by YM155 in vitro and provide laboratory rationales for the clinical treatment of follicular of lymphoma with YM155 in the future.Methods SUDHL-4 cells were cultured to logarithmic phase and transferred to 96-well plates.There were a series of YM155 concentration gradients:100,10,1,0.1 and 0 ng/ml and cultured for 24,48 and 72 h.After the addition of CCK-8 reagent for 2 h at each time point,optical density values were obtained from the cell growth inhibition curves depending on time and drug concentration and the half growth inhibition concentration ( IC50 ) values calculated.SUDHL-4 cells were cocultured with YM155 (1 ng/ml) for0,24,48 and72 h respectively.Then flow cytometry (FCM) was used to detect apoptosis.SUDHL-4 cell line was treated with YM155 for 24 and 48 h to extract the total RNA.The mRNA expressions of bcl-2,bcl-xl,bid,bax and survivin gene at the timepoint of 48 h and the survivin mRNA expression at 24 h were detected by reverse transcription-PCR(RT-PCR).The protein expressions of survivin,caspase-9,cleaved caspase-9,caspase-3 and cleaved caspase-3 were detected at each timepoint with Western blot respectively.Results SUDHL-4 cell line showed significant growth inhibition effect depending on time and dose.And the 24,48,72 h IC50 was 6.1,2.7 and 1.2 ng/ml respectively.SUDHL-4 cells stained AnnexinV-FITC and PI examined by FCM demonstrated that the proportion of AnnexinV-FITC positive cells gradually increased with time(17.3％ ±2.1％,35.7％ ±3.3％,54.6％ ±4.3％ vs 2.1％ ± 0.3％,all P ＜0.05). And the results of real-time fluorescent PCR proved that YM155 decreased the expression of survivin gene obviously(24 h:0.72 ± 0.02,48 h:0.56 ± 0.01 vs 1.00,both P ＜ 0.05 ) but had little effects on the gene expressions of bax,bid,bcl-2 and bcl-xl. The Western blot results further confirmed that the protein expressions of survivin and caspase-3 decreased with time while caspase-9 and cleaved caspase-9 showed no obvious changes. But cleaved caspase-3 increased significantly.Conclusions YM155 displays significant apoptotic effects in SUDHL-4 cell lines. The mechanism may be the direct activation of caspase-3 through the down-regulation of survivin.And the apoptotic pathway is probably not regulated by bcl-2 family.