全反式维甲酸和5-Fu对裸鼠胃癌移植瘤生长和端粒酶活性的影响
中山医科大学孙逸仙纪念医院消化内科 广东省广州市 510120
夏忠胜，男，1971-10-08生，湖北省嘉鱼县人，汉族. 1994年湖北咸宁医学院临床医疗系毕业，1999年中山医科大学医学硕士毕业，住院医师，主要从事消化道疾病的研究.
项目负责人 夏忠胜，510120, 广东省广州市，中山医科大学孙逸仙纪念医院消化内科.
Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University of Medical Sciences, Guangzhou 510120, Guangdong Province, China
Correspondence to Zhong Sheng Xia, Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University of Medical Sciences, Guangzhou 510120, Guangdong Province, China
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Tel. 0086-20-81332489
收稿日期 2000-01-10 接收日期 2000-03-02
Effects of ATRA and 5-Fu on growth and telomerase activity of xenografts of gastric cancer in nude mice
Zhong-Sheng Xia, Zhao-Hua Zhu and Shou-Gao He
Abstract
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AIM To investigate the influence of ATRA and/or 5-Fu on growth and telomerase activity of xenografts of gastric cancer.
METHODS Thirty-four female Balb/c nude mice were randomly divided into control group, solvent control group, ATRA group, 5-Fu group and AF group. The volumes of xenografts were measured at the 1st and the 11th day of experiment. The telomerase activity of xenografts was measured by telomere repeat amplification protocol (TRAP).
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RESULTS At the 11th day of experiment, the volumes of tumors in ATRA group (9.26mm3±0.84mm3) and AF group (5.86mm3±0.87mm3) were significantly smaller than those in solvent control group (13.41mm3±3.12mm3), the volumes of tumors in 5-Fu group (5.92mm3±1.25mm3) were significantly smaller than those in control group (13.19mm3±2.60mm3). Meanwhile, the telomerase activity of xenogrfts in ATRA group, 5-Fu group and AF group was 61% (versus solvent control group, P<0.01), 100% and 63% (versus solvent control group, P<0.01).
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CONCLUSION In vivo, both ATRA and 5-Fu inhibited the growth of subcutaneous xenografts of gastric cancer in nude mice. ATRA inhibited the telomerase activity of xenografts of gastric cancer, but 5-Fu did not. No synergistic inhibitory effect on the tumor growth and telomerase activity of xenografts of gastric cancer was found when ATRA combined with 5-Fu were given in vivo on the conditions of this study. The inhibition of telomerase activity of gastric cancer cells is one of the possible mechanisms by which ATRA inhibits tumor growth.
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Subject headings tretinoin; fluorouracil; stomach neoplasms; telomerase; transplantation; drug therapy; mice, nude
Xia ZS, Zhu ZH, He SG. Effects of ATRA and 5-Fu on growth and telomerase activity of xenografts of gastric cancer in nude mice.
Shijie Huaren Xiaohua Zazhi, 2000;8(6):674-677
摘要
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目的 观察ATRA，5-Fu单独和联合应用对裸鼠胃癌移植瘤生长和端粒酶活性的影响.
方法 34只裸鼠随机分成对照组、溶剂对照组、ATRA组、5-Fu组和AF组，分别于给药的d1，d11测量瘤体积，并采用TRAP法测定端粒酶活性.
结果 用药后ATRA组(9.26±0.84)mm3、AF组(5.86.87)mm3瘤体积显著小于溶剂对照组(13.41±3.12)mm3(P<0.01)，5-Fu组(5.92±1.25)mm3瘤体积显著小于对照组(13.19±2.60)mm3(P<0.01)；用药后ATRA组、5-Fu组、AF组端粒酶活性分别为61%(与溶剂对照组比较，P<0.01)、100%，63%(与溶剂对照组比较，P<0.01).
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结论 ATRA，5-Fu均能显著抑制裸鼠胃癌移植瘤的生长，ATRA与5-Fu合用在本体内实验条件下未发现有协同抗肿瘤作用，ATRA能抑制胃癌移植瘤端粒酶活性，而5-Fu对其活性无抑制作用，二者合用对胃癌移植瘤端粒酶活性无协同抑制作用. 抑制端粒酶活性可能是ATRA抗癌机制之一.
主题词 维甲酸；氟脲嘧啶；胃肿瘤；端粒酶；移植；药物疗法；小鼠，裸
夏忠胜,朱兆华,何守搞. 全反式维甲酸和5-Fu对裸鼠胃癌移植瘤生长和端粒酶活性的影响. 世界华人消化杂志，2000；8(6)：674-677
0 引言
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端粒酶与肿瘤之间关系密切，已发现90%的肿瘤细胞端粒酶阳性，而正常的体细胞几乎阴性[1]. 端粒酶的激活可能是各种基因改变导致肿瘤细胞获得“永生化”的最后共同通路，因此，通过抑制端粒酶活性阻断肿瘤细胞“永生化”的通路，可达到治疗肿瘤的目的. 本研究在体外实验发现ATRA,5-Fu合用对胃癌细胞具有协同抗肿瘤作用的基础上，进一步探讨ATRA和5-Fu单独和联合应用对胃癌裸鼠移植瘤的生长及端粒酶活性的影响，并从端粒酶角度探讨ATRA和5-Fu的抗肿瘤作用机制.
1 材料和方法
1.1 材料 ①细胞：人低分化胃粘液腺癌MGC-803细胞株由中科院生物物理研究所提供. ②动物：SPF级♀Balb/c裸小鼠(6～8周龄)由中山医科大学实验动物中心提供. ③主要药品及试剂：全反式维甲酸(上海第六制药厂)，5-氟脲嘧啶(上海旭东海普药业有限公司)，RPMI-1640培养基(Gibco公司)，DMSO(二甲基亚砜)、MTT(噻唑蓝)、CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)均为Sigma公司产品，Taq酶(Gene公司)，TS，ACX引物由加拿大Gender公司合成.
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1.2 方法
1.2.1 药物的配制 5-Fu用生理盐水配制成3mg/mL；ATRA先用DMSO溶解，再加溶剂配制成4mg/mL. 该溶剂[2]内含20mmol/L NaOH, 0.5% Tween-80，3%乙醇，pH值为7.4. 溶剂与DMSO按9∶1体积比混合即为溶剂对照.
1.2.2 荷瘤裸鼠的制备 胃癌MGC-803细胞培养于含150mL/L%胎牛血清的RPMI-1640培养液中，50mL/L CO2，37℃培养，2.5g/L胰蛋白酶和0.2g/L EDTA复合消化液消化传代. 制备细胞悬液，镜下调整细胞浓度至300万/mL，将细胞悬液0.3mL(约100万个细胞)注入裸鼠颈背皮下，无菌喂养4wk，制备成瘤源裸鼠. 处死瘤源裸鼠，取出移植瘤剪碎成2mm3大小的瘤块，接种至各实验鼠. 无菌喂养1wk制备成荷瘤裸鼠.
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1.2.3 实验分组及给药 实验设对照组、溶剂对照组、ATRA组、5-Fu组及合用组(AF组)，分别用生理盐水、溶剂、ATRA，5-Fu，ATRA+5-Fu腹腔给药，每次以0.1mL/10g裸鼠重量的剂量腹腔注射(5-Fu每日用量为30mg/kg鼠重[3]；ATRA每日用量为40mg/kg鼠重[2])，1次/d，共10d. 停药24h后处死裸鼠. 用药d1及停药24h测量肿瘤长短径，按公式T=a2b/2(a为短径，b为长径)计算瘤体积[4].
1.2.4 移植瘤的处理 无菌取出移植瘤，部分用100mL/L缓冲甲醛固定，用于常规病理学HE染色检查，确证为胃癌组织；部分装入无菌Eppendoff管中，液氮速冻后放入-85℃超低温冰箱保存待测端粒酶.
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1.2.5 端粒酶定量检测 采用Kim et al[1]创建的TRAP法，引物采用Kim et al[5]报道的序列，TS引物序列为5＇-AATCCGTCGAGCAGAGTT-3＇，ACX引物序列为5＇-GCGCGG(CTTACC)3CTAACC-3＇. 电泳凝胶银染显色后用CS-930薄层扫描仪(日本岛津)测定最大吸收峰面积值. 以对照组端粒酶活性为100%，按公式计算各组端粒酶活性. 端粒酶活性(%)＝(药物组最大吸收峰面积值÷对照组最大吸收峰面积值)×100%.
统计学处理 两组资料的均数比较用t检验，多组资料的均数比较用方差分析.
2 结果
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2.1 实验处理前后各组裸鼠移植瘤瘤体积情况 用药前各组之间裸鼠移植瘤体积无差别(P>0.05，表1)，用药后ATRA组、AF组裸鼠移植瘤体积明显小于溶剂对照组，5-Fu组裸鼠移植瘤体积明显小于对照组，用药后5-Fu组、AF组裸鼠移植瘤体积明显小于用药后ATRA组. 用药后对照组、溶剂对照组、ATRA组与用药前自身比较裸鼠移植瘤体积明显增加，而用药后5-Fu组、AF组裸鼠移植瘤体积与用药前自身比较无显著差别(P>0.05).
表1 用药前后不同处理组移植瘤瘤体积
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aP<0.01, vs 用药前自身；bP<0.01, vs 用药后溶剂对照组；cP<0.01, vs 用药后对照组； dP<0.01, vs 用药后ATRA组.
2.2 ATRA，5-Fu及二者合用(AF)对移植瘤端粒酶活性的影响
图1显示的是各处理组电泳凝胶银染条带，将电泳条带进行薄层色谱扫描得各组最大吸收峰面积值(表2). ATRA能显著抑制体内移植瘤胃癌细胞端粒酶活性，而5-Fu对裸鼠移植瘤胃癌细胞端粒酶活性无影响. ATRA与5-Fu合用对裸鼠胃癌移植瘤细胞端粒酶活性无协同抑制作用.
图1 各处理组移植瘤胃癌细胞端粒酶活性检测电泳条带.M：DNA marker； 1：阴性对照； 2：对照组(自身阳性对照)3：溶剂对照组； 4：ATRA组； 5：5-Fu组； 6：AF组
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表2 不同处理组最大吸收峰面积值(A值)和端粒酶活性及其抑制率
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aP<0.01, vs 溶剂对照组.
3 讨论
端粒是染色体自然末端的保护性结构，对于维持染色体的稳定性至关重要，缺少端粒的染色体将导致染色体的不稳定，染色体互相融合、细胞死亡. 端粒的维持通过三种途径，即端粒酶的激活、染色体的重排和转位[6]. 国内外作者[7-15]报道，约90%的肿瘤组织端粒酶阳性，90%的肿瘤细胞都依赖端粒酶途径来维持端粒长度，也就是说，端粒酶是90%的肿瘤细胞“永生化”所必须的，抑制端粒酶活性可导致端粒的净丢失、细胞死亡. 因此，我们可以通过抑制端粒酶活性来治疗肿瘤. 到目前为止，已发现的端粒酶抑制剂包括：①反义核酸[16,17]；②反义肽核酸[18]；③核苷酸类似物[19-21]；④核酶[22]；⑤叠氮胸甙[23,24]；⑥细胞分化诱导剂[25,26]；⑦某些化疗药[27]. 目前检测端粒酶活性方法包括TRAP-银染法[28]、TRAP-放射自显影法[5]、TRAP-闪烁计数法[29]、TRAP-ELISA法[30].
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本研究发现ATRA，5-Fu均能抑制胃癌裸鼠皮下移植瘤的生长，但ATRA对胃癌移植瘤生长的抑制作用不如5-Fu强，ATRA与5-Fu合用对胃癌移植瘤生长的抑制作用与单用5-Fu无差别，说明在本体内实验条件下未发现ATRA与5-Fu合用具有协同抗肿瘤作用，这与我们在体外实验中发现二者合用具有协同抗肿瘤作用不同. 从各处理组治疗前后移植瘤体积的变化，我们可以看到，经处理10d后，对照组、溶剂对照组、ATRA组瘤体积均显著增加(P<0.01)，5-Fu组瘤体积轻度增加(P>0.05)，而AF组治疗后瘤体积小于治疗前，尽管没有统计学的显著性，但可能提示ATRA与5-Fu联用对肿瘤生长有更强的抑制作用.
1996年，Xu et al[31]用ATRA诱导白血病HL-60细胞分化时，发现HL-60细胞端粒酶活性显著下降. 1997年杨骅et al[32]报道用ATRA诱导分化大肠癌时，大肠癌细胞端粒酶活性下降. 但这些都是在体外实验取得的结果. 目前尚未见ATRA对胃癌细胞端粒酶活性的影响，特别是在体内实验条件下ATRA对裸鼠胃癌移植瘤端粒酶活性影响的报道. 本研究发现ATRA在抑制胃癌移植瘤生长的同时伴有端粒酶活性的降低. 1997年Ku et al[33]报道5-Fu在抑制鼻咽癌细胞生长时未发现其端粒酶活性下降. 1998年Asai et al[34]在比较食管癌细胞对5-Fu化疗敏感性、食管癌细胞的增殖、分化与端粒长度及端粒酶活性的关系时发现，5-Fu不影响端粒酶活性. 本研究在体内实验条件下亦证实上述实验结果. 5-Fu能明显抑制胃癌细胞生长，但对其端粒酶活性无影响，ATRA与5-Fu二者合用与ATRA单独应用对端粒酶活性的影响相比较，二者无差别，说明二者合用既无协同作用，也无拮抗作用.
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国内外多位作者报道ATRA对多种肿瘤细胞均有诱导分化、抑制生长的作用[35-43]. 近10+a来人们一直在探讨ATRA的作用机制. 关于ATRA的作用机制，近几年研究最为活跃的是受体与基因调节[44-47]. 关于ATRA的作用机制，近几年研究最为活跃的是受体与基因调节. 现已发现二大类维甲酸特异性受体即维甲酸受体(RARs)和视黄醇受体RXRs，RARs又包括RARα，RARβ，RARγ. Naka et al[48]发现ATRA能在转录水平调节对其敏感的胃癌细胞RARs及RXR-α mRNA的生成. Liu et al[49]发现ATRA能在转录水平调节对其敏感的细胞产生RAR-β2 mRNA. 也有作者发现，ATRA可直接调控三磷酸肌醇(IP3)受体基因转录，使IP3受体数目增加，功能表达增强，细胞内钙动员增加，提示ATRA可通过下调节蛋白激酶C(PKC)信息途径发挥作用[50]. 本研究发现ATRA在抑制胃癌移植瘤生长的同时，移植瘤胃癌细胞端粒酶活性下降. 端粒酶作为肿瘤细胞特异性标志，而ATRA能作用于该靶点，为阐明ATRA的作用机制提供了一条新的思路. 根据以往的研究成果和本实验的研究结果，我们推测ATRA的作用机制可能是：ATRA在转录水平调节维甲酸受体mRNA的生成，之后翻译成维甲酸受体，ATRA与维甲酸受体结合通过IP3途径下调节PKC，PKC又进一步调节端粒酶，使端粒酶活性下降，抑制肿瘤细胞生长. 我们发现5-Fu能明显抑制胃癌细胞生长，但对胃癌细胞端粒酶活性无影响. 该实验结果与Ku[33]、Asai[34]的报道一致，虽然所采用的肿瘤细胞株不同，但同样能说明问题. 5-Fu作为消化道肿瘤的首选化疗药物，能特异地作用于细胞周期中的S期，通过抑制胸腺嘧啶合成酶阻止DNA复制，阻止细胞分裂，抑制细胞生长，导致细胞死亡，这是5-Fu的经典作用机制. 本研究发现5-Fu对移植瘤胃癌细胞端粒酶活性无影响，但能显著抑制胃癌移植瘤的生长，说明5-Fu抑制胃癌细胞生长不是通过抑制端粒酶途径来实现的. 我们目前仍认为5-Fu是通过经典的作用机制来实现其抗肿瘤作用. 我们先前在体外实验中发现ATRA与5-Fu合用具有协同抗肿瘤效应，但在体内实验中却未发现二者的协同抗肿瘤作用. 分析可能的原因是：①在体内实验条件下，药物经过体内代谢过程并受到体内各种药物外因素的影响，其作用方式、效应都可能与体外实验不同，因而也有可能ATRA与5-Fu联用在体内实验条件下确实没有协同抗肿瘤效应. ②用药剂量的因素：用药剂量较大，本实验所采用的ATRA的剂量是参考Jiang et al[2]报道的剂量，5-Fu的剂量是参考Kubota et al[3]报道的剂量. 而该二位作者研究的都是某一种药物对裸鼠移植瘤生长的抑制作用，采用的是能产生比较明显作用的较大剂量，我们知道在某种药物已达其最大效应时是观察不出它和其他药物合用的协同作用的. ③用药时间的因素：用药时间过长、过短均不利于观察药物的协同作用,用药时间过短，可能因最大疗效未出现而观察不到协同作用，而当用药时间过长时又可因无论单用还是合用药物的作用已达极限而掩盖了曾经存在的协同作用. 本实验因条件的限制，只选用了一种剂量和一个观察时间终点，如果实施一项采用分级剂量和不同用药时间的研究，将有可能解决这一问题.
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48 Naka K, Yokozaki H, Domen T, Tayashi K, Kuniyasu H, Yasui W, Lotan R, Tahara E. Growth inhibition of cultured human gastric
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muscle development and adult chemosensory functions. Dev Biol, 1995;171:564-577, http://www.100md.com(夏忠胜 朱兆华 何守搞)
夏忠胜，男，1971-10-08生，湖北省嘉鱼县人，汉族. 1994年湖北咸宁医学院临床医疗系毕业，1999年中山医科大学医学硕士毕业，住院医师，主要从事消化道疾病的研究.
项目负责人 夏忠胜，510120, 广东省广州市，中山医科大学孙逸仙纪念医院消化内科.
Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University of Medical Sciences, Guangzhou 510120, Guangdong Province, China
Correspondence to Zhong Sheng Xia, Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University of Medical Sciences, Guangzhou 510120, Guangdong Province, China
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Tel. 0086-20-81332489
收稿日期 2000-01-10 接收日期 2000-03-02
Effects of ATRA and 5-Fu on growth and telomerase activity of xenografts of gastric cancer in nude mice
Zhong-Sheng Xia, Zhao-Hua Zhu and Shou-Gao He
Abstract
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AIM To investigate the influence of ATRA and/or 5-Fu on growth and telomerase activity of xenografts of gastric cancer.
METHODS Thirty-four female Balb/c nude mice were randomly divided into control group, solvent control group, ATRA group, 5-Fu group and AF group. The volumes of xenografts were measured at the 1st and the 11th day of experiment. The telomerase activity of xenografts was measured by telomere repeat amplification protocol (TRAP).
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RESULTS At the 11th day of experiment, the volumes of tumors in ATRA group (9.26mm3±0.84mm3) and AF group (5.86mm3±0.87mm3) were significantly smaller than those in solvent control group (13.41mm3±3.12mm3), the volumes of tumors in 5-Fu group (5.92mm3±1.25mm3) were significantly smaller than those in control group (13.19mm3±2.60mm3). Meanwhile, the telomerase activity of xenogrfts in ATRA group, 5-Fu group and AF group was 61% (versus solvent control group, P<0.01), 100% and 63% (versus solvent control group, P<0.01).
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CONCLUSION In vivo, both ATRA and 5-Fu inhibited the growth of subcutaneous xenografts of gastric cancer in nude mice. ATRA inhibited the telomerase activity of xenografts of gastric cancer, but 5-Fu did not. No synergistic inhibitory effect on the tumor growth and telomerase activity of xenografts of gastric cancer was found when ATRA combined with 5-Fu were given in vivo on the conditions of this study. The inhibition of telomerase activity of gastric cancer cells is one of the possible mechanisms by which ATRA inhibits tumor growth.
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Subject headings tretinoin; fluorouracil; stomach neoplasms; telomerase; transplantation; drug therapy; mice, nude
Xia ZS, Zhu ZH, He SG. Effects of ATRA and 5-Fu on growth and telomerase activity of xenografts of gastric cancer in nude mice.
Shijie Huaren Xiaohua Zazhi, 2000;8(6):674-677
摘要
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目的 观察ATRA，5-Fu单独和联合应用对裸鼠胃癌移植瘤生长和端粒酶活性的影响.
方法 34只裸鼠随机分成对照组、溶剂对照组、ATRA组、5-Fu组和AF组，分别于给药的d1，d11测量瘤体积，并采用TRAP法测定端粒酶活性.
结果 用药后ATRA组(9.26±0.84)mm3、AF组(5.86.87)mm3瘤体积显著小于溶剂对照组(13.41±3.12)mm3(P<0.01)，5-Fu组(5.92±1.25)mm3瘤体积显著小于对照组(13.19±2.60)mm3(P<0.01)；用药后ATRA组、5-Fu组、AF组端粒酶活性分别为61%(与溶剂对照组比较，P<0.01)、100%，63%(与溶剂对照组比较，P<0.01).
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结论 ATRA，5-Fu均能显著抑制裸鼠胃癌移植瘤的生长，ATRA与5-Fu合用在本体内实验条件下未发现有协同抗肿瘤作用，ATRA能抑制胃癌移植瘤端粒酶活性，而5-Fu对其活性无抑制作用，二者合用对胃癌移植瘤端粒酶活性无协同抑制作用. 抑制端粒酶活性可能是ATRA抗癌机制之一.
主题词 维甲酸；氟脲嘧啶；胃肿瘤；端粒酶；移植；药物疗法；小鼠，裸
夏忠胜,朱兆华,何守搞. 全反式维甲酸和5-Fu对裸鼠胃癌移植瘤生长和端粒酶活性的影响. 世界华人消化杂志，2000；8(6)：674-677
0 引言
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端粒酶与肿瘤之间关系密切，已发现90%的肿瘤细胞端粒酶阳性，而正常的体细胞几乎阴性[1]. 端粒酶的激活可能是各种基因改变导致肿瘤细胞获得“永生化”的最后共同通路，因此，通过抑制端粒酶活性阻断肿瘤细胞“永生化”的通路，可达到治疗肿瘤的目的. 本研究在体外实验发现ATRA,5-Fu合用对胃癌细胞具有协同抗肿瘤作用的基础上，进一步探讨ATRA和5-Fu单独和联合应用对胃癌裸鼠移植瘤的生长及端粒酶活性的影响，并从端粒酶角度探讨ATRA和5-Fu的抗肿瘤作用机制.
1 材料和方法
1.1 材料 ①细胞：人低分化胃粘液腺癌MGC-803细胞株由中科院生物物理研究所提供. ②动物：SPF级♀Balb/c裸小鼠(6～8周龄)由中山医科大学实验动物中心提供. ③主要药品及试剂：全反式维甲酸(上海第六制药厂)，5-氟脲嘧啶(上海旭东海普药业有限公司)，RPMI-1640培养基(Gibco公司)，DMSO(二甲基亚砜)、MTT(噻唑蓝)、CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)均为Sigma公司产品，Taq酶(Gene公司)，TS，ACX引物由加拿大Gender公司合成.
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1.2 方法
1.2.1 药物的配制 5-Fu用生理盐水配制成3mg/mL；ATRA先用DMSO溶解，再加溶剂配制成4mg/mL. 该溶剂[2]内含20mmol/L NaOH, 0.5% Tween-80，3%乙醇，pH值为7.4. 溶剂与DMSO按9∶1体积比混合即为溶剂对照.
1.2.2 荷瘤裸鼠的制备 胃癌MGC-803细胞培养于含150mL/L%胎牛血清的RPMI-1640培养液中，50mL/L CO2，37℃培养，2.5g/L胰蛋白酶和0.2g/L EDTA复合消化液消化传代. 制备细胞悬液，镜下调整细胞浓度至300万/mL，将细胞悬液0.3mL(约100万个细胞)注入裸鼠颈背皮下，无菌喂养4wk，制备成瘤源裸鼠. 处死瘤源裸鼠，取出移植瘤剪碎成2mm3大小的瘤块，接种至各实验鼠. 无菌喂养1wk制备成荷瘤裸鼠.
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1.2.3 实验分组及给药 实验设对照组、溶剂对照组、ATRA组、5-Fu组及合用组(AF组)，分别用生理盐水、溶剂、ATRA，5-Fu，ATRA+5-Fu腹腔给药，每次以0.1mL/10g裸鼠重量的剂量腹腔注射(5-Fu每日用量为30mg/kg鼠重[3]；ATRA每日用量为40mg/kg鼠重[2])，1次/d，共10d. 停药24h后处死裸鼠. 用药d1及停药24h测量肿瘤长短径，按公式T=a2b/2(a为短径，b为长径)计算瘤体积[4].
1.2.4 移植瘤的处理 无菌取出移植瘤，部分用100mL/L缓冲甲醛固定，用于常规病理学HE染色检查，确证为胃癌组织；部分装入无菌Eppendoff管中，液氮速冻后放入-85℃超低温冰箱保存待测端粒酶.
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1.2.5 端粒酶定量检测 采用Kim et al[1]创建的TRAP法，引物采用Kim et al[5]报道的序列，TS引物序列为5＇-AATCCGTCGAGCAGAGTT-3＇，ACX引物序列为5＇-GCGCGG(CTTACC)3CTAACC-3＇. 电泳凝胶银染显色后用CS-930薄层扫描仪(日本岛津)测定最大吸收峰面积值. 以对照组端粒酶活性为100%，按公式计算各组端粒酶活性. 端粒酶活性(%)＝(药物组最大吸收峰面积值÷对照组最大吸收峰面积值)×100%.
统计学处理 两组资料的均数比较用t检验，多组资料的均数比较用方差分析.
2 结果
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2.1 实验处理前后各组裸鼠移植瘤瘤体积情况 用药前各组之间裸鼠移植瘤体积无差别(P>0.05，表1)，用药后ATRA组、AF组裸鼠移植瘤体积明显小于溶剂对照组，5-Fu组裸鼠移植瘤体积明显小于对照组，用药后5-Fu组、AF组裸鼠移植瘤体积明显小于用药后ATRA组. 用药后对照组、溶剂对照组、ATRA组与用药前自身比较裸鼠移植瘤体积明显增加，而用药后5-Fu组、AF组裸鼠移植瘤体积与用药前自身比较无显著差别(P>0.05).
表1 用药前后不同处理组移植瘤瘤体积
| 组别 | 用药前 | 用药后 | ||
| n | 瘤体积(mm3) | n | 瘤体积(mm3) | |
| 对照组 | 5 | 7.80±3.00 | 5 | 13.19±2.60a |
| 溶剂对照组 | 5 | 6.40±4.17 | 5 | 13.41±3.12a |
| ATRA组 | 8 | 5.48±1.45 | 8 | 9.26±0.84a,b |
| 5-Fu组 | 8 | 5.58±1.83 | 8 | 5.92±1.25c,d |
| AF组 | 8 | 6.76±2.29 | 8 | 5.86±0.87b,d |
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aP<0.01, vs 用药前自身；bP<0.01, vs 用药后溶剂对照组；cP<0.01, vs 用药后对照组； dP<0.01, vs 用药后ATRA组.
2.2 ATRA，5-Fu及二者合用(AF)对移植瘤端粒酶活性的影响
图1显示的是各处理组电泳凝胶银染条带，将电泳条带进行薄层色谱扫描得各组最大吸收峰面积值(表2). ATRA能显著抑制体内移植瘤胃癌细胞端粒酶活性，而5-Fu对裸鼠移植瘤胃癌细胞端粒酶活性无影响. ATRA与5-Fu合用对裸鼠胃癌移植瘤细胞端粒酶活性无协同抑制作用.
图1 各处理组移植瘤胃癌细胞端粒酶活性检测电泳条带.M：DNA marker； 1：阴性对照； 2：对照组(自身阳性对照)3：溶剂对照组； 4：ATRA组； 5：5-Fu组； 6：AF组
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表2 不同处理组最大吸收峰面积值(A值)和端粒酶活性及其抑制率
| 组别 | n | A值 | 端粒酶活性(%) | 端粒酶抑制率(%) |
| 对照组 | 5 | 45490.25±9714.66 | 100.00 | 0.00 |
| 溶剂对照组 | 5 | 45742.53±7028.91 | 100.00 | 0.00 |
| ATRA组 | 8 | 27922.13±5278.48a | 61.04a | 38.96a |
| 5-Fu组 | 8 | 46243.34±5820.48 | 100.00 | 0.00 |
| AF组 | 8 | 28813.68±5265.62a | 62.99a | 37.10a |
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aP<0.01, vs 溶剂对照组.
3 讨论
端粒是染色体自然末端的保护性结构，对于维持染色体的稳定性至关重要，缺少端粒的染色体将导致染色体的不稳定，染色体互相融合、细胞死亡. 端粒的维持通过三种途径，即端粒酶的激活、染色体的重排和转位[6]. 国内外作者[7-15]报道，约90%的肿瘤组织端粒酶阳性，90%的肿瘤细胞都依赖端粒酶途径来维持端粒长度，也就是说，端粒酶是90%的肿瘤细胞“永生化”所必须的，抑制端粒酶活性可导致端粒的净丢失、细胞死亡. 因此，我们可以通过抑制端粒酶活性来治疗肿瘤. 到目前为止，已发现的端粒酶抑制剂包括：①反义核酸[16,17]；②反义肽核酸[18]；③核苷酸类似物[19-21]；④核酶[22]；⑤叠氮胸甙[23,24]；⑥细胞分化诱导剂[25,26]；⑦某些化疗药[27]. 目前检测端粒酶活性方法包括TRAP-银染法[28]、TRAP-放射自显影法[5]、TRAP-闪烁计数法[29]、TRAP-ELISA法[30].
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本研究发现ATRA，5-Fu均能抑制胃癌裸鼠皮下移植瘤的生长，但ATRA对胃癌移植瘤生长的抑制作用不如5-Fu强，ATRA与5-Fu合用对胃癌移植瘤生长的抑制作用与单用5-Fu无差别，说明在本体内实验条件下未发现ATRA与5-Fu合用具有协同抗肿瘤作用，这与我们在体外实验中发现二者合用具有协同抗肿瘤作用不同. 从各处理组治疗前后移植瘤体积的变化，我们可以看到，经处理10d后，对照组、溶剂对照组、ATRA组瘤体积均显著增加(P<0.01)，5-Fu组瘤体积轻度增加(P>0.05)，而AF组治疗后瘤体积小于治疗前，尽管没有统计学的显著性，但可能提示ATRA与5-Fu联用对肿瘤生长有更强的抑制作用.
1996年，Xu et al[31]用ATRA诱导白血病HL-60细胞分化时，发现HL-60细胞端粒酶活性显著下降. 1997年杨骅et al[32]报道用ATRA诱导分化大肠癌时，大肠癌细胞端粒酶活性下降. 但这些都是在体外实验取得的结果. 目前尚未见ATRA对胃癌细胞端粒酶活性的影响，特别是在体内实验条件下ATRA对裸鼠胃癌移植瘤端粒酶活性影响的报道. 本研究发现ATRA在抑制胃癌移植瘤生长的同时伴有端粒酶活性的降低. 1997年Ku et al[33]报道5-Fu在抑制鼻咽癌细胞生长时未发现其端粒酶活性下降. 1998年Asai et al[34]在比较食管癌细胞对5-Fu化疗敏感性、食管癌细胞的增殖、分化与端粒长度及端粒酶活性的关系时发现，5-Fu不影响端粒酶活性. 本研究在体内实验条件下亦证实上述实验结果. 5-Fu能明显抑制胃癌细胞生长，但对其端粒酶活性无影响，ATRA与5-Fu二者合用与ATRA单独应用对端粒酶活性的影响相比较，二者无差别，说明二者合用既无协同作用，也无拮抗作用.
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国内外多位作者报道ATRA对多种肿瘤细胞均有诱导分化、抑制生长的作用[35-43]. 近10+a来人们一直在探讨ATRA的作用机制. 关于ATRA的作用机制，近几年研究最为活跃的是受体与基因调节[44-47]. 关于ATRA的作用机制，近几年研究最为活跃的是受体与基因调节. 现已发现二大类维甲酸特异性受体即维甲酸受体(RARs)和视黄醇受体RXRs，RARs又包括RARα，RARβ，RARγ. Naka et al[48]发现ATRA能在转录水平调节对其敏感的胃癌细胞RARs及RXR-α mRNA的生成. Liu et al[49]发现ATRA能在转录水平调节对其敏感的细胞产生RAR-β2 mRNA. 也有作者发现，ATRA可直接调控三磷酸肌醇(IP3)受体基因转录，使IP3受体数目增加，功能表达增强，细胞内钙动员增加，提示ATRA可通过下调节蛋白激酶C(PKC)信息途径发挥作用[50]. 本研究发现ATRA在抑制胃癌移植瘤生长的同时，移植瘤胃癌细胞端粒酶活性下降. 端粒酶作为肿瘤细胞特异性标志，而ATRA能作用于该靶点，为阐明ATRA的作用机制提供了一条新的思路. 根据以往的研究成果和本实验的研究结果，我们推测ATRA的作用机制可能是：ATRA在转录水平调节维甲酸受体mRNA的生成，之后翻译成维甲酸受体，ATRA与维甲酸受体结合通过IP3途径下调节PKC，PKC又进一步调节端粒酶，使端粒酶活性下降，抑制肿瘤细胞生长. 我们发现5-Fu能明显抑制胃癌细胞生长，但对胃癌细胞端粒酶活性无影响. 该实验结果与Ku[33]、Asai[34]的报道一致，虽然所采用的肿瘤细胞株不同，但同样能说明问题. 5-Fu作为消化道肿瘤的首选化疗药物，能特异地作用于细胞周期中的S期，通过抑制胸腺嘧啶合成酶阻止DNA复制，阻止细胞分裂，抑制细胞生长，导致细胞死亡，这是5-Fu的经典作用机制. 本研究发现5-Fu对移植瘤胃癌细胞端粒酶活性无影响，但能显著抑制胃癌移植瘤的生长，说明5-Fu抑制胃癌细胞生长不是通过抑制端粒酶途径来实现的. 我们目前仍认为5-Fu是通过经典的作用机制来实现其抗肿瘤作用. 我们先前在体外实验中发现ATRA与5-Fu合用具有协同抗肿瘤效应，但在体内实验中却未发现二者的协同抗肿瘤作用. 分析可能的原因是：①在体内实验条件下，药物经过体内代谢过程并受到体内各种药物外因素的影响，其作用方式、效应都可能与体外实验不同，因而也有可能ATRA与5-Fu联用在体内实验条件下确实没有协同抗肿瘤效应. ②用药剂量的因素：用药剂量较大，本实验所采用的ATRA的剂量是参考Jiang et al[2]报道的剂量，5-Fu的剂量是参考Kubota et al[3]报道的剂量. 而该二位作者研究的都是某一种药物对裸鼠移植瘤生长的抑制作用，采用的是能产生比较明显作用的较大剂量，我们知道在某种药物已达其最大效应时是观察不出它和其他药物合用的协同作用的. ③用药时间的因素：用药时间过长、过短均不利于观察药物的协同作用,用药时间过短，可能因最大疗效未出现而观察不到协同作用，而当用药时间过长时又可因无论单用还是合用药物的作用已达极限而掩盖了曾经存在的协同作用. 本实验因条件的限制，只选用了一种剂量和一个观察时间终点，如果实施一项采用分级剂量和不同用药时间的研究，将有可能解决这一问题.
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