视网膜母细胞瘤细胞株SO-Rb50及SO-Rb70对六种抗癌药物敏感性测定

目的 利用体外培养的视网膜母细胞瘤（retinoblastoma,Rb）细胞株筛选对Rb敏感的化疗药物。方法 采用噻唑蓝（3,-4,5 Dimethyliazol tetrazolium bromide,MTT）法对SO-Rb50和SO-Rb70瘤细胞株进行更生霉素、长春新碱、鬼臼噻吩甙、柔红霉素、顺氯氧铂、平阳霉素6种药物体外敏感实验．结果 6种药物作用于SO-Rb5048小时50％抑制浓度（50％ inhibitory concenrtation,IC50）值（μg／ml）分别为0.0004、0.0016、0.0389、0.047、0.29、0.44．72小时分别为0.00025、0.00081、0.0151、0.0192、0.097、0.11,作用于SO-R5048小时IC50值分别为0.00065、0.00149、0.0282、0.043、0.37、0.215,72小时分别为0.00042、0.00082、0.0146、0.0176、0.035、0.084．结论 SO-Rb50和SO-RS70瘤细胞对于上述6种化疗药物均敏感,根据其IC50值．药物敏感性顺序依次为更生霉素、长春新碱、鬼臼噻吩甙、柔红霉素、顺氯氨铂、平阳霉素。     

The long-term effects of 5-fluorouracil and sodium butyrate on human Tenon's fibroblasts.

The use of subconjunctival 5-fluorouracil (5-FU) in the first weeks after filtration surgery may ensure long-term bleb survival despite a continuing proliferative stimulus such as in eyes with neovascular glaucoma. In addition, long-term side effects may occur, such as increasing bleb thinning. To ascertain the long-term effects of 5-FU and sodium butyrate, an agent with differentiating and antiproliferative properties, we exposed proliferating human Tenon's capsule fibroblasts to different concentrations of the drugs. The cells were exposed to 5-FU for 1-12 d. The cells were subsequently observed for up to 30 d. Cell proliferation was assessed using cell counting and bromodeoxyuridine uptake, and cell viability was assessed with trypan blue uptake. 5-FU and sodium butyrate inhibited fibroblast proliferation during the treatment period. Higher concentrations of 5-FU (100 and 1000 micrograms/ml) for as little as 1 d resulted in no significant increase in the number of fibroblasts for at least 29 d after treatment was stopped, despite continued stimulation with serum. When treatment with sodium butyrate was stopped, there was greater recovery of proliferation. At a constant concentration of 1000 micrograms/ml of 5-FU for 3 or more days, or a concentration of 100 mmol/l sodium butyrate for 12 d, the entire fibroblast population gradually died over the 30 d period. Thus, short-term treatment with 5-FU may result in long-term inhibition of proliferation of fibroblasts. Long-term inhibition depends on the duration of treatment or on the concentration of 5-FU. Short-term treatment may be affecting the ability of the tissues at the bleb site to heal in the long term. Different dosage regimens may have advantages and are discussed.     

视网膜母细胞瘤细胞系SO—Rb50瘤细胞Rb基因突变动态变化研究

目的：研究SO－Rb50细胞系瘤细胞Rb基因突变的动态变化。方法：1．用Southern blot杂交法分析SO－Rb50细胞系第327代瘤细胞DNA。2．用PCR－SSCP法对SO－Rb50细胞系第415代及第713代瘤细胞的Rb基因27个外显子和1个启动子逐个筛查。3．将SO－Rb50细胞系的第775代克隆出3个细胞株：MC2、MC3及MC4。用PCR－SSCP－HA法对MC2、MC3及MC4第11代细胞和MC3第138代细胞的Rb基因的27个外显子逐个筛查。结果：SO－Rb50细胞系第327代细胞DNA缺失3．5Kb、2.9Kb及1.0Kb的三条带,证明SO－Rb50细胞系瘤细胞Rb基因缺失。第415代瘤细胞Rb基因第23外显子少一条单链;第713代第25外显子发生新的突变。SO－Rb50细胞系第775代的3个克隆株MC2、MC3及MC4的第11代细胞,只有MC4第24外显子突变;而MC3第138代第24外显子突变,提示MC3经多次传代后第24外显子发生新突变。结论：SO－Rb50细胞系在长期培养传代过程中,瘤细胞的Rb基因突变有动态变化。     

人视网膜母细胞瘤SO-Rb_(50)细胞系单克隆方法探索

目的:为了使培养细胞的遗传性状、生物学特性相似,利于多种实验研究,而对视网膜母细胞瘤SO-Rb_(50)细胞系进行克隆。方法:用多孔塑料板克隆细胞法,对SO-Rb_(50)细胞进行克隆及分离培养。结果:没有菊花团形成的Rb细胞克隆成功,而有菊花团的Rb细胞、连续三次克隆均不能成功。结论:提示分化好的Rb细胞难以克隆成功。眼科学报 1996;12:79—82。     

The effect of 5-fluorouracil and cytarabine on human fibroblasts from Tenon's capsule

The in vitro cellular inhibitory effects of two pyrimidine antimetabolites, 5-fluorouracil (5-FU) and cytarabine (ara-C), on the attachment and proliferation of human Tenon's capsule fibroblasts after 1, 2, 3, 5, 7, and 10 days of growth were measured with a Coulter counter, a colorimetric method using the endogenous enzyme hexosaminidase, and 3H-thymidine uptake. Neither 5-FU nor ara-C affected cell attachment. The 50% inhibitory dose (ID50) for 5-FU, as measured by the Coulter counter and hexosaminidase assay, was 0.2 and 0.4 micrograms/ml, respectively, at day 5 and decreased to 0.01 and 0.10 micrograms/ml, respectively, on later days. The ID50 for ara-C as measured by the Coulter counter and hexosaminidase assay was 0.01 and 0.1 micrograms/ml at day 3 and remained constant over time. Much lower ID50s were measured by thymidine uptake for both drugs. These findings may indicate that 5-FU has a delayed effect on cellular proliferation due to conversion into more active metabolites. The ara-C has a direct and constant inhibitory effect on cellular proliferation and is ten times more potent than 5-FU as an antiproliferative drug. Thus ara-C may have clinical utility in preventing failure of glaucoma filtering surgery.     

SO-Rb 50克隆株生物学及分子生物学特性动态观察

目的 研究SO-Rb50细胞系三个克隆株MC 2、MC 3、MC 4第11代和MC 3 -138代染色体畸变的差异及Rb-1基因突变的情况。 方法 对SO-Rb50建立的三个克隆细胞株第11代进行G-显带核型分析；用常规方法抽提三个细胞株的第11代和MC 138代基因组DNA，用PCR联合SSCP法，逐个外显子筛查其Rb1基因编码区及启动子。 结果 1. 三个克隆株细胞的染色体数目和结构均有异常，出现不同类型的染色体畸变，13号染色体畸变仅见于个别核型且在三个克隆株中畸变情况不同。发现5个标记染色体M1，M2，M3，M4和M5，其中标记染色体M1出现频率最高，是由1号染色体长臂部分重复构成［t(1;１)qter-p35∷124-ter］。M4和M5为2号染色体的变化，是在该细胞系染色体变化动态研究中首次发现。2. 外显子24的SSCP电泳结果显示，MC4-11代细胞第2条单链泳动率比正常对照标本增快，单链数相同；MC4-138代细胞比对照多一条链。 结论 SO-Rb50细胞系至少存在两个不同克隆株，同一克隆株不同代数的细胞生物学特性有动态变化。13号染色体的畸变不是RB发生的唯一原因；1号染色体的变化与本细胞系的永生关系密切，其与RB发生、发展的关系值得深入研究。 (中华眼底病杂志, 1999, 15: 146-148)     

小干扰RNA技术下调视网膜母细胞瘤细胞株SO-Rb50中Survivin基因表达的研究

目的 应用RNA干扰(RNAi)技术沉默视网膜母细胞瘤细胞株SO-Rb50中凋亡抑制因子Survivin的表达,观察其干扰效果及对细胞的影响.方法 实验研究.体外化学法合成针对人Survivin基因的特异性小干扰RNA(siRNA)和对Survivin基因无沉默效果的阴性对照siRNA,利用转染试剂分别将siRNA转入SO-Rb50细胞株,通过半定量RT-PCR和Western blot检测其对SO-Rb50细胞中Survivin基因和蛋白表达的抑制作用,四甲基偶氮唑盐(MTT)法检测不同浓度siRNA对细胞增殖抑制率,流式细胞仪检测转染后细胞凋亡率和细胞周期改变,荧光显微镜下观察细胞凋亡形态.对多组间数据比较采用单因素方差分析,处理组与对照组比较采用Dunnett-t检验.结果 Survivin特异性siRNA转染细胞24 h后Survivin mRNA和蛋白表达水平明显下调,对照组中其表达则无明显改变.MTT法结果显示Survivin特异性siRNA浓度为35、70、100 nmol/L时均对SO-Rb50细胞增殖具有抑制作用(P＜0.05),且具有剂茸依赖性.流式细胞仪检测发现Survivin特异性siRNA组出现凋亡亚二倍体峰,细胞被阻滞于G0/G1期,较对照组增加了8.11%,G2/M期细胞比例减少了5.75%,S期细胞仅减少了2.26%.荧光显微镜下可见部分细胞出现染色质浓缩和凋亡小体等典型的凋亡形态学变化.结论 针对Survivin的RNA干扰技术可有效地下调SO-Rb50细胞中Survivin基因表达,进而抑制SO-Rb50细胞增殖和诱导其凋亡,为视网膜母细胞瘤的基因治疗提供了重要的途径.     

三氧化二砷诱导的视网膜母细胞瘤SO-Rb50细胞白噬的研究

背景细胞的自噬是肿瘤细胞非凋亡形式的死亡过程,研究证实三氧化二砷（As2O3）可诱导肿瘤细胞凋亡,但As2O3是否可致SO—Rb50细胞发生自噬的研究鲜有报道。目的探讨As2O3体外诱导SO—Rb50细胞自噬的作用。方法用浓度为0．5、1．0、2．0、4．0μmol／L的As2O3培养液及不含As2O3的未处理组处理SO—Rb50细胞系48h,采用MTT法测定各As2O3,浓度组SO—Rb50细胞的吸光度（As2O3）值。构建自噬标志物磷酸化绿色荧光蛋白（pGFP）-LC3体外转染SO—Rb50细胞,并分为新鲜RPMI一1640培养组（未处理组）、As2O3,RPMI一1640培养组（As2O3处理组）和rapamycin培养组（阳性对照组）,于48h后行激光共焦显微镜检测细胞自噬;用自噬特异性染料单丹磺酰戊二胺（MDC）行荧光染色检测SO—Rb50细胞的自噬,并用透射电子显微镜检测SO—Rb50细胞的自噬表现,采用流式细胞仪定量检测不同浓度As2O3,处理组自噬泡阳性细胞百分比。结果未处理组和0．5、1．0、2．0、4．0txmolAs2O3,作用48h后SO—Rb50细胞的A570值分别为2．194＋0．066、1．841±0．213、1．035±0．046、0．374±0．042和0．167±0．019,总体比较差异有统计学意义（F=547．636,P〈0．05）,其中各浓度As2O3组A。值均明显低于未处理组,差异均有统计学意义（均P=0．000）。As2O3处理组和阳性对照组可见GFP—LC3融合蛋白呈点状聚集在相应的自噬泡,未处理组GFP—LC3呈弥散分布。透射电子显微镜检查可见未处理组SO—Rb50细胞超微结构正常,As2O3处理组和阳性对照组见大量双层膜状结构及自噬溶酶体。未处理组48h见极少量MDC阳性荧光颗粒,As2O3处理组及阳性对照组48h可见明显的MDC阳性荧光颗粒,聚集在细胞质相应的自噬发生区。流式细胞术检测发现未处理组和0．5、1．0、2．0、4．0μmol／LAs2O3及rapamycin作用48h后自噬泡阳性的SO—Rb50细胞百分比分别为0、15．6％、42．7％、57．9％、79．5％和89．0％,随As2O3,浓度的增加MDC阳性细胞百分比递增。结论As2O3,抑制SO—Rb50细胞的生长和增生,并致SO—Rb50细胞发生自噬;SO—Rb50细胞经As2O3处理后,自噬的发生早于细胞核的改变,早中期自噬程度呈明显的As2O3浓度依赖性。     

The effects of 5-fluorouracil and mitomycin C on the corneal endothelium.

5-Fluorouracil (5-FU) and Mitomycin C (MMC) are used as adjunct chemotherapy during glaucoma filtering surgery to suppress conjunctival fibroblast proliferation. Since part of these agents may gain access to the anterior chamber and cause cytotoxicity to the corneal endothelium we set up an in vitro system to establish a dose-response effect. Cytotoxicity of MMC and 5-FU was quantified using Mosmann's colorimetric assay in a bovine endothelial cell culture system. In this assay the respiratory activity of the cells is used as a marker for cell viability. After incubation for 5 minutes the 3.0 mg/ml concentration of MMC showed endothelial cytotoxicity, whereas no endothelial toxicity of 5-FU was noted in concentrations up to 50 mg/ml. After incubation for 30 minutes endothelial cytotoxicity was demonstrated for 50 mg/ml of 5-FU and 1 mg/ml of MMC. After an exposure-time of 60 minutes the toxicity level remained 50 mg/ml for 5-FU but decreased to 0.5 mg/ml for MMC. We conclude that with respect to the clinically used concentrations and methods of application of 5-FU and MMC in vivo endothelial toxicity is not to be expected. However, in cases of accidental access of MMC to the anterior eye chamber and following a reduction of aqueous turnover rate the safety of MMC is unwarranted.