中文摘要：

目的研究生存素（survivin）抑制剂YMl55对滤泡淋巴瘤细胞的凋亡效应及作用机制，为应用YMl55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YMl55形成一系列浓度梯度：100、10…101、0rig／ml，培养24、48、72h后进行活细胞计数试剂盒（CCK）8实验，观察YMl55对其生长抑制效应，计算其细胞半数抑制药物浓度（IC50）。加入YMl55至培养瓶中使其药物浓度为1．g／ml，培养24、48、72h进行流式细胞术检测，膜联蛋白（Annexin）V一异硫氰酸荧光素（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YMl55后24、48h的实验组细胞抽提总RNA，然后进行反转录PCR（RT．PCR）检测48h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survivinmRNA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一3蛋白的表达量。结果YMl55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、72h的Ic，。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％4-2．1％、35．7％-4-3．3％、54．6％±4．3％比2．1％-t-O．3％，均P〈0．05）；PCR结果显示YMl55作用后24和48h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easpase-3表达逐渐增加。结论YMl55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easpase-3，从而切?

英文摘要：

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-$ 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 （ICs0） values calculated. SUDHL-4 cells were co- cultured with YM155 （1 ng/ml） for 0, 24, 48 and 72 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 timepointwith Western blot respectively. Results SUDHL-4 cell line showed significant growth inhibition effect depending on time and dose. And the 24, 48, 72 h ICs0 was 6. 1, 2. 7 and 1.2 ng/ml respectively. SUDHL-4 ceils 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% -＋ O. 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 c