CPP32在阿霉素诱导的胃癌耐药细胞的凋亡的作用
1第一军医大学南方医院消化内科 广东省广州市 510515
2第四军医大学唐都医院骨科医院 陕西省西安市 710038
王少东，女，1968-02-15生，江苏省无锡市人，汉族.1998年第四军 医大学消化专业硕士毕业，在读博士生，主治医师，主要从事肿瘤耐药机理的研究.发表论 著10篇.
项目负责人 王少东
收稿日期 2001-03-28 接受日期 2001-05-20
The function of CPP32 in apoptosis of multidrug-resistance gastric ca ncer cells
Shao-Dong Wang, Zheng-Shu Zhang, Xing-Quan Zhang, and Dian-Yuan Zhou
Correspondence to: Shao-Dong Wang
Received 2001-03-28 Accepted 2001-05-20
Abstract
AIM To identify the effect and the activity of CPP32 in multidrug resistance cell by comparing the gene expression, protein expression, and the relationship between them. We try to illuminate the effect and the significa nce of caspase-3 in the apoptosis induced by cheomotherapeutic agents in multi drug resistance cells.
METHODS Utilizing the established multidrug resistance cell lin e, we investigated the mRNA expression of caspase-3 in multidrug resistance ce lls at 0h,24h; and in drug sensitive cells at 0h, 6h by RT-PCR. We determined the protein expression of in multidrug resistance cells at 0h,6h, 24h, 30h; and in drug sensitive cells at 0h, 6h, 24h by western blot. DNA ladder was used to examine the characteristic changes of apoptosis, MTT was performed to get surviv al rate of cells.
RESULTS there are no significant difference between the mRNA ex pression of the resistant cells at 0h, 24h and that of the drug sensitive cells at 0h, 6h. Analysis of protein expression showed that there is trace expression in resistant cells at 6h; the small degradation products appears at 30h; the lit tle degradation products appears at 6h in sensitive cells, the caspase-3 prote in is splitted completely at 24h. DNA analysis show that the typical apoptotic l adder at 30h in resistant cells; the apoptosis of resistant cells was delayed af ter the inhibitor was applied. The same was occurred at 6h in sensitive cells.
CONCLUSIONS Our results imply that execution of apoptosis is th e same in mdr cells and sensitive cells,the only difference between mdr cell and sensitive cell is the concentration of the drug and the time of caspase-3 wh en they have their effect.
Subject headings stomach neoplasms/pathology; caspases/ metabolism; drug resistance, multiple; drug resistance, neoplasm; apoptosis
Wang SD, Zhang ZS, ZhangXQ, Zhou DY. The function of CPP32 in apoptosis of multidrug-resistance gastric cancer cells.Shijie Huaren Xiaohua Zazhi,2001; 9(10):1152-1156
摘 要
目的 探讨耐药细胞和药敏细胞中caspase-3 mRNA 、蛋白的表达以 及他们相互之间的关系，试图阐明caspase-3在诱导耐药细胞凋亡中的作用及意义.
方法 利用已有的胃癌耐药细胞系SGC7901/08，通过RT-PCR研究caspase -3的mRNA在细胞毒药物处理耐药细胞0h, 24h及药敏细胞0h, 6h时的表达，用Western-blot 的方法讨论其蛋白在耐药细胞中0h, 6h, 24h, 30h的表达和药敏细胞中0h, 6h, 24h的表达 ，DNA ladder 检测其特征性凋亡变化，MTT法检测细胞的存活率.
结果 耐药细胞在0h, 24h和药敏细胞在0h, 6h表达的caspase-3mRNA无 显著性差异，蛋白表达在耐药细胞中在6h出现微量的表达，在30h时出现更小片断；药敏细 胞在6h时出现小片断，在24h出现完全裂解.细胞DNA分析表明耐药细胞在30h出现典型的凋亡 谱带；应用了caspase-3抑制剂后细胞的凋亡延迟.药敏细胞在6h出现典型的凋亡谱带，应 用了抑制剂后也出现凋亡的延迟.
结论 提高耐药细胞的用药量仍然可以使耐药细胞出现凋亡，而且其凋亡 的过程与耐药细胞相同，均有caspase-3的激活，而抑制了caspase-3的活性，凋亡的发生可被抑制或是延迟，其差别就在于耐药细胞需要较高的药物诱导浓度.耐药细胞与药敏细胞 的凋亡机制从本质上并无显著的不同，其根本的差别可能在于凋亡发生前的诸多因素产生的 差异.
主题词 胃肿瘤/病理学；caspase类/代谢；抗药性，多药；抗药性，肿瘤；凋亡
王少东，张振书，张杏泉，周殿元.CPP32在阿霉素诱导的胃癌耐药细胞的凋亡的作用.世界华人消化杂志，2001；9(10)：1152-1156
0 引言胃癌在我国十分常见[1-16]，同时细胞凋亡的研究也很活跃[17-20].CPP32是ICE/CED-3半胱氨酸蛋白 酶家族成员, 自1994年Fernandes- Alnemri首次报道CPP32的发现以来, 已有大量的研究证 明了其在细胞死亡途径中的关键性作用.[21-55].CPP32，也称YAMA(印度神话中 的死亡之神)最为引人注目，他的过量表达可诱发细胞凋亡，他是迄今发现的caspase家族 成员中与ced-3同源性最高的，被认为是哺乳动物中ced-3的对应者，同时，他是caspase系列蛋白酶解级联反应中的下游分子[26-34].我们成功地诱导了一株胃癌的多药 耐药细胞株SGC7901/VCR08(前文已发表), 对胃癌耐药细胞及药敏细胞中的CPP32的基因型和 表达型进行了初步的研究, 为了进一步了解CPP32及其在胃癌耐药细胞中的作用, 我们对CPP 32在耐药细胞及药敏细胞中的表达及调控进行了研究, 这些研究为肿瘤耐药细胞中的抗凋亡 的研究、探索新的逆转耐药机制奠定了基础.
1 材料和方法
1.1 材料 测序载体pGEM-T VECTOR SYSTEM Ⅱ购自 美国promega 公司, 人Jurkat T淋巴瘤细胞系，由本校免疫教研室金伯泉教授惠赠. CPP32专 一性引物由日本宝生物(大连)公司合成.RNA提取试剂为美国GIBCOBRL公司的TRIzol reagent ，PCR kit 为上海生工产品， 反转录系统系日本宝生物(大连)公司的BcaBest kit， 电泳 凝胶seakem GTG agarose、Nusiene agarose 均购自FMC，CPP32抗体购自美国Santa公司，A C-DEVD-CHO购自SIGMA公司，其他药品及试剂均为美国SIGMA 公司产品或国产AR级试剂.PC R仪为MJC PTC-100，垂直电泳仪为美国HOFER AE-265.
1.2 方法1.2.1 胃癌耐药细胞系SGC7901/V08和药敏细胞系SG C7901采用RPMI1640培养基， 加100mL·L-1牛血清于37℃、5 0mL·L-1CO 2培养，SGC7901/V08加用长春新碱维持培养.RT-PCR：用酸性异硫氰酸胍法参照TRIzol reagent 的说明书提取耐药细胞及药敏细胞的总 RNA，并用宝生物公司的反转录系统，将他们反转录成cDNA作为模板，进行PCR反应. PCR法 扩增DNA及其产物鉴定：PCR反应条件：94℃，30S；56℃，30S；72℃， 1min， 共进行35个 循环，PCR产物进行30 g·L-1琼脂糖凝胶电泳鉴定.
1.2.2 噻唑蓝还原法(MTT法) 参照文献[35-37 ]进行.取对数生长期的SGC7901/WT细胞及耐药细胞分别接种于96孔板，培养24h后，每孔 中加入或不加入caspase-3的特异性抑制剂AC-DEVD-CO使其终水平为20μmol/ L-1 ，在上述各孔中加入5mg·L-1或30 mg·L-1的阿霉素(adr)，培养不同的 时间段后换无药培养液，继续培养2d，随后做MTT实验.SGC7901/WT细胞及耐药细胞均同时设 实验组及空白组，空白组即只加入培养液不加入细胞的空白对照孔，其他实验步骤一致，实 验组即加入不同浓度的adr的各实验孔，其余步骤同前.每组各设5孔，共做3次，取平均值.
1.2.3 DNA ladder 90mm平皿中生长的2×107个细胞，用PBS洗一次，用600μL Tris-NaCl-EDTA裂解缓冲液裂解细胞，刮取收集上述混合液 于eppendrof管中，随后加入20μL 3 g·L-1的proteinase K，55℃，overnight， 不时旋动，待裂解液清亮后，在冰上放置10min；加入同体积的酚：氯仿，来回颠摇，vorte x充分混合10min，冰浴低温10min，离心13000r·min-1×20min，取上清，加入1/10 体积的3 mol·L-1 NaOAc，2体积的无水乙醇，加入MgCl2 使其终水平为0.01 mol ·L-1，-20℃放置至少1h，13000 r·min-1×15min离心， 弃上清，收集 沉淀团块，加入70 mL·L-1乙醇500μL，13000 r·min-1×4min，弃上清， 倒置留有沉淀块的eppendrof管，在37℃孵箱中孵育10min，取出eppendrof管后，加入20μL TE(pH 8.0)溶解沉淀块， 充分冲洗管壁，3 0g·L-1 FMC 凝胶电泳鉴定.-80℃冰 箱保存.
1.2.4 Western-blot 2×105个细胞用50μl的冰 冷的Triton X-100裂解缓冲液(含有Leupeptin、pepstetin和aprotinin等蛋白酶抑制剂) 于冰上作用60min，12500 r·min-1，4℃，离心15min，取上清， 用分光光度计测 定其浓度.以每泳道100μg总蛋白的量用SDS-150 g·L-1的聚丙烯酰胺凝胶进行电 泳，丽春红染色标定marker，用Bio-Rad 半干式转移仪将蛋白条带转移到硝酸纤维膜上(mi llipore)，用Santa的抗人caspase-3 mAb进行共孵育，用NBT/BCIP呈色，保存.
2 结果
2.1 阿霉素(adr)诱发细胞凋亡及caspase-3的参与 在耐药细胞中加入终浓度为30 mg·L-1的Adr作用30h，耐药细胞出现DNA梯状谱(DNA ladder)；药敏细胞中加入终浓度为5μg/ml的Adr作用6h，药敏细胞出现DNA梯状谱(DNA ladder).而用caspase-3的特异性抑制剂AC-DEVD-CHO四肽衍生物于加入Adr前加入细胞 共培养，则于加Adr处理相同的时间段后 几乎不出现DNA梯状谱.揭示Adr诱发细胞凋亡依赖c aspase-3的激活，抑制caspase-3的活性，可以导致凋亡的延迟发生(图1).
2.2 细胞存活率实验 Adr对耐药细胞的IC50为18.4 mg ·L-1,而在其对照组药敏细胞中，该值为4 mg·L-1，即Adr而言，耐药细胞 的抗药性是药敏细胞的4.6倍(图2).加用了caspase-3的特异性抑制剂AC-DEVD-CHO四肽衍生物后，在30μg/ml的Adr的作用下，出现凋亡的时间被延迟.
图1 用阿霉素处理的耐药细胞和药敏细胞的DNA ladder电泳：1-3泳 道：用30 mg·L-1阿霉素处理的耐药细胞的DNA提取物的琼脂糖凝胶电泳，4-6泳道 ：用5 mg·L-1阿霉素处理的药敏细胞的DNA提取物的琼脂糖凝胶电泳.1：耐药细胞 对照组，2：阿霉素处理30h后，3：用特异性抑制剂和阿霉素处理30h的耐药细胞，4：7901对照组，5：7901用阿霉素处理6h后，6：用特异性抑制剂和阿霉素处理6h后的药敏细胞.
图2 细胞存活曲线
2.3 caspase-3 mRNA表达 Adr在诱发细胞凋亡过程中 ，可见明显的caspase-3的激活，耐药细胞与药敏细胞的表现是一致的，其差别仅在于Adr 用量的多少； 即耐药细胞出现的凋亡现象，也是caspase-3的激活所导致的.这种激活是来 源于Adr所导致的转录的升高还是翻译后修饰?为此，我们以加药后不同时间点提取细胞，进 行RT-PCR，观察Adr诱导细胞凋亡过程中，耐药细胞及药敏细胞caspase-3基因转录的情况 .结果如图3所示：加药与未加药相比，耐药细胞与药敏细胞相比，caspase-3 mRNA水平无 明显改变.提示：Adr所诱导的肿瘤耐药细胞及药敏细胞的凋亡中caspase-3的活性增高并非 源于其转录的提高，而可能是翻译后酶前体的活化.
图3 用RT-PCR的方法分析caspase-3的基因表达，用EB将PCR扩增产物 的琼脂糖电泳染色显示.β-actin 的基因表达作内参照(本图中未显示).1：marker，2：未 用adr的耐药细胞，3：用30mg·L-1 adr处理了24h的耐药细胞，4：未用adr的药敏细胞，5：用5mg·L-1 adr 处理了6h的药敏细胞.
2.4 凋亡过程中caspase-3的活性分析 已知caspase 家族的蛋白酶在细胞中均以非活性的前体形式(pro-caspase)出现，要经过活化形成(p20+ p10)2四聚体才能发挥酶切活性.而RT-PCR结果已显示Adr诱导的细胞凋亡中caspase-3 mR NA水平没有改变，肿瘤耐药细胞与药敏细胞中的caspase-3 mRNA的含量没有显著的差别.显 然，观察到的caspase-3在耐药细胞及药敏细胞中的活性的提高应该源于其前体的活化，we stern-blot的结果充分证实了这一推论.图4显示了加药前及加药后不同时间点用caspase- 3 mAb检测其活性亚基的结果.未加药细胞显示的是pro-caspase-3，但随着凋亡的进展， 活性亚基p20的产生增多，而且pro-caspase-3的量在6h后出现减少或消失.
图4 用western-blot方法分析caspase-3表达.我们用的二抗可以识别 p11,p20亚单位和caspase-3的酶原.1：未用adr的耐药细胞，2：用30mg·L-1adr处 理6h的耐药细胞，3：用30mg·L-1adr处理24h的耐药细胞，4：用30mg·L-1 adr作用30h的耐药细胞，5：未用adr的药敏细胞，6：用5mg·L-1adr作用6h的药敏 细胞，7：用30mg·L-1adr作用24h的药敏赴?
3 讨论ICE 是线虫发育过程中细胞凋亡基因ced-3产 物在哺乳动物中的同源体，至今共发现了14个CED-3和ICE的同源体，1996年后这些家族成 员统称为caspase.Caspase-3/CPP32/YAMA由2.6Kb mRNA编码，在多种组织细胞中有表达.正 常情况下，caspase-3以无活性的caspase-3酶原形式存在于哺乳动物的多种组织和细胞内 .目前普遍认为在ICE家族中，caspase-3在哺乳动物细胞凋亡中的关键酶[41-46] .目前的研究揭示：caspase-3可被各种各样的凋亡信号所激活，在哺乳动物中目前存在两 个主要的caspase级联反应.第一路径，caspase-8和caspase-10被Fas、TNFR1和DR3通过接 头分子FADD和死亡受体相连接而活化[47].第二路径，各种细胞内和细胞外的死亡 刺激促使线粒体释放细胞色素C，胞质内细胞色素C与Apaf-1相结合，Apaf-1激活caspase -9酶原[48-50].活化的caspase-8和caspase-9激活效应caspase:caspase-3, -6,-7[58].作为效应分子的caspase(如caspase-3,-6,-7)介导了一个共同的 细胞凋亡通路.他们降解细胞基因组稳定的蛋白质如PARP、调控细胞周期的蛋白质如Rb、或 是结构蛋白质如Gas2、以及DNA片断产生因子(DFF)等，导致阻抑细胞进程[51-54] ，丧失维持基因组稳定及DNA损伤修复机制，促使凋亡细胞同周围组织分离，破坏维系细胞 形态的结构蛋白，显现凋亡特征等等.[55-57] 肿瘤的耐药问题一直是肿瘤研究的热点问题，从第一个耐药分子发现至今， 已经过了1/4个 世纪，目前已有5种机理认为与肿瘤的耐药有关：(1)P-糖蛋白的表达[59-64](2) 耐药相关蛋白(MRP)的表达[65-67](3)拓扑异构酶的减低[68-69](4)谷 胱苷肽转移酶的改变(5)其他，包括蛋白激酶C(PKC)[70]，肺相关蛋白(LRP)等.随 着对化疗药物导致凋亡的认识， 使人们意识到肿瘤的耐药是由于不能启动凋亡程序.Zhang Y等的研究认为由喜树碱诱导而来的多药耐药细胞其抗拒肿瘤药物而导致的凋亡的原因在于c aspase-3类的蛋白酶的活性显著下降，导致这一原因的机制在于bcl-2、p21 Wafl/Cip1的 高表达[71].我们的研究结果显示：提高了耐药细胞的用药量仍然可以出现耐药细 胞的凋亡，而且其凋亡的过程与耐药细胞相同，均有caspase-3的激活，而抑制了caspase -3的活性，凋亡的发生可被抑制或是延迟；其差别就在于耐药细胞需要较高的药物诱寂?度，可见从本质上来讲耐药细胞与药敏细胞的凋亡机制并无显著的不同，其根本的差别可能 在于凋亡发生前的诸多因素产生的差异.从这个角度来讲，研究肿瘤耐药细胞的凋亡具有 重要的意义.我们的结果显示，caspase-3在Adr诱导的肿瘤耐药细胞凋亡中激活，并依赖其 前体的活化而发挥作用，据此我们有理由相信，以caspase-3为靶标的新的抗癌药物的设计 ，有望为肿瘤耐药的克服提供新的思路.
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selected human lung-cancer cell line. Int J Cancer,1999 Dec 10;83:790-7, http://www.100md.com(王少东1 张振书1 张杏泉2 周殿元1)
2第四军医大学唐都医院骨科医院 陕西省西安市 710038
王少东，女，1968-02-15生，江苏省无锡市人，汉族.1998年第四军 医大学消化专业硕士毕业，在读博士生，主治医师，主要从事肿瘤耐药机理的研究.发表论 著10篇.
项目负责人 王少东
收稿日期 2001-03-28 接受日期 2001-05-20
The function of CPP32 in apoptosis of multidrug-resistance gastric ca ncer cells
Shao-Dong Wang, Zheng-Shu Zhang, Xing-Quan Zhang, and Dian-Yuan Zhou
Correspondence to: Shao-Dong Wang
Received 2001-03-28 Accepted 2001-05-20
Abstract
AIM To identify the effect and the activity of CPP32 in multidrug resistance cell by comparing the gene expression, protein expression, and the relationship between them. We try to illuminate the effect and the significa nce of caspase-3 in the apoptosis induced by cheomotherapeutic agents in multi drug resistance cells.
METHODS Utilizing the established multidrug resistance cell lin e, we investigated the mRNA expression of caspase-3 in multidrug resistance ce lls at 0h,24h; and in drug sensitive cells at 0h, 6h by RT-PCR. We determined the protein expression of in multidrug resistance cells at 0h,6h, 24h, 30h; and in drug sensitive cells at 0h, 6h, 24h by western blot. DNA ladder was used to examine the characteristic changes of apoptosis, MTT was performed to get surviv al rate of cells.
RESULTS there are no significant difference between the mRNA ex pression of the resistant cells at 0h, 24h and that of the drug sensitive cells at 0h, 6h. Analysis of protein expression showed that there is trace expression in resistant cells at 6h; the small degradation products appears at 30h; the lit tle degradation products appears at 6h in sensitive cells, the caspase-3 prote in is splitted completely at 24h. DNA analysis show that the typical apoptotic l adder at 30h in resistant cells; the apoptosis of resistant cells was delayed af ter the inhibitor was applied. The same was occurred at 6h in sensitive cells.
CONCLUSIONS Our results imply that execution of apoptosis is th e same in mdr cells and sensitive cells,the only difference between mdr cell and sensitive cell is the concentration of the drug and the time of caspase-3 wh en they have their effect.
Subject headings stomach neoplasms/pathology; caspases/ metabolism; drug resistance, multiple; drug resistance, neoplasm; apoptosis
Wang SD, Zhang ZS, ZhangXQ, Zhou DY. The function of CPP32 in apoptosis of multidrug-resistance gastric cancer cells.Shijie Huaren Xiaohua Zazhi,2001; 9(10):1152-1156
摘 要
目的 探讨耐药细胞和药敏细胞中caspase-3 mRNA 、蛋白的表达以 及他们相互之间的关系，试图阐明caspase-3在诱导耐药细胞凋亡中的作用及意义.
方法 利用已有的胃癌耐药细胞系SGC7901/08，通过RT-PCR研究caspase -3的mRNA在细胞毒药物处理耐药细胞0h, 24h及药敏细胞0h, 6h时的表达，用Western-blot 的方法讨论其蛋白在耐药细胞中0h, 6h, 24h, 30h的表达和药敏细胞中0h, 6h, 24h的表达 ，DNA ladder 检测其特征性凋亡变化，MTT法检测细胞的存活率.
结果 耐药细胞在0h, 24h和药敏细胞在0h, 6h表达的caspase-3mRNA无 显著性差异，蛋白表达在耐药细胞中在6h出现微量的表达，在30h时出现更小片断；药敏细 胞在6h时出现小片断，在24h出现完全裂解.细胞DNA分析表明耐药细胞在30h出现典型的凋亡 谱带；应用了caspase-3抑制剂后细胞的凋亡延迟.药敏细胞在6h出现典型的凋亡谱带，应 用了抑制剂后也出现凋亡的延迟.
结论 提高耐药细胞的用药量仍然可以使耐药细胞出现凋亡，而且其凋亡 的过程与耐药细胞相同，均有caspase-3的激活，而抑制了caspase-3的活性，凋亡的发生可被抑制或是延迟，其差别就在于耐药细胞需要较高的药物诱导浓度.耐药细胞与药敏细胞 的凋亡机制从本质上并无显著的不同，其根本的差别可能在于凋亡发生前的诸多因素产生的 差异.
主题词 胃肿瘤/病理学；caspase类/代谢；抗药性，多药；抗药性，肿瘤；凋亡
王少东，张振书，张杏泉，周殿元.CPP32在阿霉素诱导的胃癌耐药细胞的凋亡的作用.世界华人消化杂志，2001；9(10)：1152-1156
0 引言胃癌在我国十分常见[1-16]，同时细胞凋亡的研究也很活跃[17-20].CPP32是ICE/CED-3半胱氨酸蛋白 酶家族成员, 自1994年Fernandes- Alnemri首次报道CPP32的发现以来, 已有大量的研究证 明了其在细胞死亡途径中的关键性作用.[21-55].CPP32，也称YAMA(印度神话中 的死亡之神)最为引人注目，他的过量表达可诱发细胞凋亡，他是迄今发现的caspase家族 成员中与ced-3同源性最高的，被认为是哺乳动物中ced-3的对应者，同时，他是caspase系列蛋白酶解级联反应中的下游分子[26-34].我们成功地诱导了一株胃癌的多药 耐药细胞株SGC7901/VCR08(前文已发表), 对胃癌耐药细胞及药敏细胞中的CPP32的基因型和 表达型进行了初步的研究, 为了进一步了解CPP32及其在胃癌耐药细胞中的作用, 我们对CPP 32在耐药细胞及药敏细胞中的表达及调控进行了研究, 这些研究为肿瘤耐药细胞中的抗凋亡 的研究、探索新的逆转耐药机制奠定了基础.
1 材料和方法
1.1 材料 测序载体pGEM-T VECTOR SYSTEM Ⅱ购自 美国promega 公司, 人Jurkat T淋巴瘤细胞系，由本校免疫教研室金伯泉教授惠赠. CPP32专 一性引物由日本宝生物(大连)公司合成.RNA提取试剂为美国GIBCOBRL公司的TRIzol reagent ，PCR kit 为上海生工产品， 反转录系统系日本宝生物(大连)公司的BcaBest kit， 电泳 凝胶seakem GTG agarose、Nusiene agarose 均购自FMC，CPP32抗体购自美国Santa公司，A C-DEVD-CHO购自SIGMA公司，其他药品及试剂均为美国SIGMA 公司产品或国产AR级试剂.PC R仪为MJC PTC-100，垂直电泳仪为美国HOFER AE-265.
1.2 方法1.2.1 胃癌耐药细胞系SGC7901/V08和药敏细胞系SG C7901采用RPMI1640培养基， 加100mL·L-1牛血清于37℃、5 0mL·L-1CO 2培养，SGC7901/V08加用长春新碱维持培养.RT-PCR：用酸性异硫氰酸胍法参照TRIzol reagent 的说明书提取耐药细胞及药敏细胞的总 RNA，并用宝生物公司的反转录系统，将他们反转录成cDNA作为模板，进行PCR反应. PCR法 扩增DNA及其产物鉴定：PCR反应条件：94℃，30S；56℃，30S；72℃， 1min， 共进行35个 循环，PCR产物进行30 g·L-1琼脂糖凝胶电泳鉴定.
1.2.2 噻唑蓝还原法(MTT法) 参照文献[35-37 ]进行.取对数生长期的SGC7901/WT细胞及耐药细胞分别接种于96孔板，培养24h后，每孔 中加入或不加入caspase-3的特异性抑制剂AC-DEVD-CO使其终水平为20μmol/ L-1 ，在上述各孔中加入5mg·L-1或30 mg·L-1的阿霉素(adr)，培养不同的 时间段后换无药培养液，继续培养2d，随后做MTT实验.SGC7901/WT细胞及耐药细胞均同时设 实验组及空白组，空白组即只加入培养液不加入细胞的空白对照孔，其他实验步骤一致，实 验组即加入不同浓度的adr的各实验孔，其余步骤同前.每组各设5孔，共做3次，取平均值.
1.2.3 DNA ladder 90mm平皿中生长的2×107个细胞，用PBS洗一次，用600μL Tris-NaCl-EDTA裂解缓冲液裂解细胞，刮取收集上述混合液 于eppendrof管中，随后加入20μL 3 g·L-1的proteinase K，55℃，overnight， 不时旋动，待裂解液清亮后，在冰上放置10min；加入同体积的酚：氯仿，来回颠摇，vorte x充分混合10min，冰浴低温10min，离心13000r·min-1×20min，取上清，加入1/10 体积的3 mol·L-1 NaOAc，2体积的无水乙醇，加入MgCl2 使其终水平为0.01 mol ·L-1，-20℃放置至少1h，13000 r·min-1×15min离心， 弃上清，收集 沉淀团块，加入70 mL·L-1乙醇500μL，13000 r·min-1×4min，弃上清， 倒置留有沉淀块的eppendrof管，在37℃孵箱中孵育10min，取出eppendrof管后，加入20μL TE(pH 8.0)溶解沉淀块， 充分冲洗管壁，3 0g·L-1 FMC 凝胶电泳鉴定.-80℃冰 箱保存.
1.2.4 Western-blot 2×105个细胞用50μl的冰 冷的Triton X-100裂解缓冲液(含有Leupeptin、pepstetin和aprotinin等蛋白酶抑制剂) 于冰上作用60min，12500 r·min-1，4℃，离心15min，取上清， 用分光光度计测 定其浓度.以每泳道100μg总蛋白的量用SDS-150 g·L-1的聚丙烯酰胺凝胶进行电 泳，丽春红染色标定marker，用Bio-Rad 半干式转移仪将蛋白条带转移到硝酸纤维膜上(mi llipore)，用Santa的抗人caspase-3 mAb进行共孵育，用NBT/BCIP呈色，保存.
2 结果
2.1 阿霉素(adr)诱发细胞凋亡及caspase-3的参与 在耐药细胞中加入终浓度为30 mg·L-1的Adr作用30h，耐药细胞出现DNA梯状谱(DNA ladder)；药敏细胞中加入终浓度为5μg/ml的Adr作用6h，药敏细胞出现DNA梯状谱(DNA ladder).而用caspase-3的特异性抑制剂AC-DEVD-CHO四肽衍生物于加入Adr前加入细胞 共培养，则于加Adr处理相同的时间段后 几乎不出现DNA梯状谱.揭示Adr诱发细胞凋亡依赖c aspase-3的激活，抑制caspase-3的活性，可以导致凋亡的延迟发生(图1).
2.2 细胞存活率实验 Adr对耐药细胞的IC50为18.4 mg ·L-1,而在其对照组药敏细胞中，该值为4 mg·L-1，即Adr而言，耐药细胞 的抗药性是药敏细胞的4.6倍(图2).加用了caspase-3的特异性抑制剂AC-DEVD-CHO四肽衍生物后，在30μg/ml的Adr的作用下，出现凋亡的时间被延迟.
图1 用阿霉素处理的耐药细胞和药敏细胞的DNA ladder电泳：1-3泳 道：用30 mg·L-1阿霉素处理的耐药细胞的DNA提取物的琼脂糖凝胶电泳，4-6泳道 ：用5 mg·L-1阿霉素处理的药敏细胞的DNA提取物的琼脂糖凝胶电泳.1：耐药细胞 对照组，2：阿霉素处理30h后，3：用特异性抑制剂和阿霉素处理30h的耐药细胞，4：7901对照组，5：7901用阿霉素处理6h后，6：用特异性抑制剂和阿霉素处理6h后的药敏细胞.
图2 细胞存活曲线
2.3 caspase-3 mRNA表达 Adr在诱发细胞凋亡过程中 ，可见明显的caspase-3的激活，耐药细胞与药敏细胞的表现是一致的，其差别仅在于Adr 用量的多少； 即耐药细胞出现的凋亡现象，也是caspase-3的激活所导致的.这种激活是来 源于Adr所导致的转录的升高还是翻译后修饰?为此，我们以加药后不同时间点提取细胞，进 行RT-PCR，观察Adr诱导细胞凋亡过程中，耐药细胞及药敏细胞caspase-3基因转录的情况 .结果如图3所示：加药与未加药相比，耐药细胞与药敏细胞相比，caspase-3 mRNA水平无 明显改变.提示：Adr所诱导的肿瘤耐药细胞及药敏细胞的凋亡中caspase-3的活性增高并非 源于其转录的提高，而可能是翻译后酶前体的活化.
图3 用RT-PCR的方法分析caspase-3的基因表达，用EB将PCR扩增产物 的琼脂糖电泳染色显示.β-actin 的基因表达作内参照(本图中未显示).1：marker，2：未 用adr的耐药细胞，3：用30mg·L-1 adr处理了24h的耐药细胞，4：未用adr的药敏细胞，5：用5mg·L-1 adr 处理了6h的药敏细胞.
2.4 凋亡过程中caspase-3的活性分析 已知caspase 家族的蛋白酶在细胞中均以非活性的前体形式(pro-caspase)出现，要经过活化形成(p20+ p10)2四聚体才能发挥酶切活性.而RT-PCR结果已显示Adr诱导的细胞凋亡中caspase-3 mR NA水平没有改变，肿瘤耐药细胞与药敏细胞中的caspase-3 mRNA的含量没有显著的差别.显 然，观察到的caspase-3在耐药细胞及药敏细胞中的活性的提高应该源于其前体的活化，we stern-blot的结果充分证实了这一推论.图4显示了加药前及加药后不同时间点用caspase- 3 mAb检测其活性亚基的结果.未加药细胞显示的是pro-caspase-3，但随着凋亡的进展， 活性亚基p20的产生增多，而且pro-caspase-3的量在6h后出现减少或消失.
图4 用western-blot方法分析caspase-3表达.我们用的二抗可以识别 p11,p20亚单位和caspase-3的酶原.1：未用adr的耐药细胞，2：用30mg·L-1adr处 理6h的耐药细胞，3：用30mg·L-1adr处理24h的耐药细胞，4：用30mg·L-1 adr作用30h的耐药细胞，5：未用adr的药敏细胞，6：用5mg·L-1adr作用6h的药敏 细胞，7：用30mg·L-1adr作用24h的药敏赴?
3 讨论ICE 是线虫发育过程中细胞凋亡基因ced-3产 物在哺乳动物中的同源体，至今共发现了14个CED-3和ICE的同源体，1996年后这些家族成 员统称为caspase.Caspase-3/CPP32/YAMA由2.6Kb mRNA编码，在多种组织细胞中有表达.正 常情况下，caspase-3以无活性的caspase-3酶原形式存在于哺乳动物的多种组织和细胞内 .目前普遍认为在ICE家族中，caspase-3在哺乳动物细胞凋亡中的关键酶[41-46] .目前的研究揭示：caspase-3可被各种各样的凋亡信号所激活，在哺乳动物中目前存在两 个主要的caspase级联反应.第一路径，caspase-8和caspase-10被Fas、TNFR1和DR3通过接 头分子FADD和死亡受体相连接而活化[47].第二路径，各种细胞内和细胞外的死亡 刺激促使线粒体释放细胞色素C，胞质内细胞色素C与Apaf-1相结合，Apaf-1激活caspase -9酶原[48-50].活化的caspase-8和caspase-9激活效应caspase:caspase-3, -6,-7[58].作为效应分子的caspase(如caspase-3,-6,-7)介导了一个共同的 细胞凋亡通路.他们降解细胞基因组稳定的蛋白质如PARP、调控细胞周期的蛋白质如Rb、或 是结构蛋白质如Gas2、以及DNA片断产生因子(DFF)等，导致阻抑细胞进程[51-54] ，丧失维持基因组稳定及DNA损伤修复机制，促使凋亡细胞同周围组织分离，破坏维系细胞 形态的结构蛋白，显现凋亡特征等等.[55-57] 肿瘤的耐药问题一直是肿瘤研究的热点问题，从第一个耐药分子发现至今， 已经过了1/4个 世纪，目前已有5种机理认为与肿瘤的耐药有关：(1)P-糖蛋白的表达[59-64](2) 耐药相关蛋白(MRP)的表达[65-67](3)拓扑异构酶的减低[68-69](4)谷 胱苷肽转移酶的改变(5)其他，包括蛋白激酶C(PKC)[70]，肺相关蛋白(LRP)等.随 着对化疗药物导致凋亡的认识， 使人们意识到肿瘤的耐药是由于不能启动凋亡程序.Zhang Y等的研究认为由喜树碱诱导而来的多药耐药细胞其抗拒肿瘤药物而导致的凋亡的原因在于c aspase-3类的蛋白酶的活性显著下降，导致这一原因的机制在于bcl-2、p21 Wafl/Cip1的 高表达[71].我们的研究结果显示：提高了耐药细胞的用药量仍然可以出现耐药细 胞的凋亡，而且其凋亡的过程与耐药细胞相同，均有caspase-3的激活，而抑制了caspase -3的活性，凋亡的发生可被抑制或是延迟；其差别就在于耐药细胞需要较高的药物诱寂?度，可见从本质上来讲耐药细胞与药敏细胞的凋亡机制并无显著的不同，其根本的差别可能 在于凋亡发生前的诸多因素产生的差异.从这个角度来讲，研究肿瘤耐药细胞的凋亡具有 重要的意义.我们的结果显示，caspase-3在Adr诱导的肿瘤耐药细胞凋亡中激活，并依赖其 前体的活化而发挥作用，据此我们有理由相信，以caspase-3为靶标的新的抗癌药物的设计 ，有望为肿瘤耐药的克服提供新的思路.
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