5’-DFUR在小鼠结直肠癌模型内转化分析
|
摘要
研究背景:
5'-脱氧氟尿苷(5'-deoxy-5-fluorouridine, 5'-DFUR)是临床治疗消化道恶性肿瘤的口服抗癌药物,为5-氟尿嘧啶(5-FU)的前体药物。其本身没有细胞毒作用,需要在细胞内经过胸苷磷酸化酶(thymidine phosphorylase,TP)转化为5-FU才能发挥抗肿瘤作用。已有文献报道乳腺癌和胃癌细胞可以表达TP活性,而大肠癌细胞是否表达TP则持论不同。我们在前期研究中发现大肠癌组织中TP活性主要由间质细胞中的巨噬细胞表达,而测定6株结肠癌细胞系也几乎没有TP蛋白表达。在癌细胞不表达TP的情况下5'-DFUR在结直肠癌组织中如何转化尚属疑问。我们前期体内实验对结肠癌小鼠动物模型应用化疗药物5'-DFUR进行治疗,结果发现与5-FU相比平均荷瘤生存期更长,平均瘤重轻,同期平均体重下降缓慢,提示5'-DFUR在小鼠结肠癌组织比正常组织中转化率高,抗癌选择性高。其原因可能是TP酶在癌组织中分布较正常组织多。前期体外实验把5'-DFUR加入培养基中同人血单核细胞一起培养24h,5'-DFUR对4种癌细胞的IC50明显下降,提示血液中单核细胞也可表达TP。由于尚未发现实验比较在癌组织和血液中TP含量,故两者TP的含量高低尚需要实验进一步证实。本实验应用高效液相色谱法(high performance liquid chromatography,HPLC)测定应用5'-DFUR后癌组织和血液中5-FU的转化情况,间接推断TP酶在癌组织和血液中分布差异,为进一步研究5'-DFUR在结直肠癌组织中转化及TP酶调控机制提供资料。
实验材料:
1、实验动物SPF级近交系BALB/c小鼠28只,6-8周龄,雄性,体重20.00±2.34g,购自广东省医学实验动物中心。
2、肿瘤细胞株BALB/c小鼠结肠腺癌细胞株(CT26),购自美国菌种保藏中心(American Type Culture Collection, ATCC)。
3、实验药物5'-DFUR由Roche公司日本研究中心提供; 5-FU注射液,江苏南通精华制药有限公司生产(批号: 080607);5-FU标准品购自Sigma有限公司提供(批号: 097K1352)。
4、实验仪器岛津高效液相系统;色谱柱:Diamonsil C18柱(250mm×4.6mm,5μm)
实验方法:
1、小鼠结肠癌CT-26细胞株的培养10%胎牛血清1640培养基,含青霉素100×103 U/L和链霉素100 mg/L,37℃,5%CO2水浴恒温培养箱中培养,隔日换液,2-3天酶消化法传代。
2、细胞悬液制备
制备模型当天取指数生长期的细胞,用0.25%胰蛋白酶消化,机械吹打成细胞悬液,2 000r/min离心5 min,弃上清液,加适量生理盐水调整细胞浓度至1×107个/ml,以台盼蓝测定细胞活力在95%以上。
3、结肠癌模型制作方法
将体外培养的CT26细胞悬液0.2ml注入小鼠(BALB/c)背部皮下,约2周后基本可以形成肉眼可见的肿瘤隆起。
4、动物分组及给药
荷瘤小鼠28只随机分为4组:①5'-DFUR给药15分钟组;②5'-DFUR给药30分钟组;③5-FU给药15分钟组;④5-FU给药30分钟组。根据动物体重,5-FU用量0.020mg/g ,配制浓度为1.0 mg/ml。5'-DFUR用量0.038mg/g;配置浓度为2.0mg/ml。各组分别腹腔注射给药15分钟、30分钟后处死小鼠立即取血和瘤组织。
5、标本处理
小鼠眼眶动静脉取血0.5 ml后放置入37℃水浴30分钟,3200rpm离心5min,取上清液4℃保存。肿瘤组织用滤纸吸干血迹后称重,然后按0.5g组织与4 ml生理盐水(1:8)加入匀浆器匀浆5min, 3200rpm离心5min,取上清液4℃保存。
6、制作血液和肿瘤组织的5-FU药物标准曲线
取未给药小鼠血清7份,每份90μL,分别加入由5-FU对照品和蒸馏水配制的系列标准液适量并混匀配成100μL,使血清中药物浓度分别为6.25,12.5,25.0,50.0,100.0,200.0,400.0μg·mL~(-1),制作血清标准曲线;取未给药小鼠肿瘤组织匀浆液7份,每份90μL,分别加入由5-FU对照品和蒸馏水配制的系列标准液适量并混匀配成100μL,使肿瘤匀浆液中药物浓度分别为1.0,2.0,4.0,8.0,16.0,32.0,64.0μg·mL~(-1),制作肿瘤标准曲线。
7、测量各标本浓度
取血清100μL,置于5mL玻璃试管中,加入乙酸乙酯2mL,漩涡振荡2min后,3200rpm离心5min,取上层析液置于另一玻璃试管中。再次加入乙酸乙酯2mL进行第二次提取,漩涡振荡2min后,3200rpm离心5min,取上层析液,然后合并两次提取的上层析液,离心浓缩挥干。加入100μL流动相定容,混匀取出,置于EP管中,10000rpm离心7min,取上层析液20μL进样。记录药物峰面积,代入相应标准曲线计算药物浓度;取肿瘤匀浆液100μL,以同样方法处理标本测量浓度。
8、观测指标
给药15分钟、30分钟处死组5'-DFUR组和5-FU组小鼠血液与癌组织5-FU浓度。
9、统计学方法
应用统计软件SPSS13.0数据包对5'-DFUR组和5-FU组小鼠血液与癌组织5-FU浓度采用配对样本t检验进行比较。当P<0.05时,认为差异有统计学意义。
结果:
1、注射药物5'-DFUR 15、30分钟后,癌组织转化的5-FU浓度分别54.64μg/g±12.80μg/g和45.58μg/g±18.82μg/g,血清中中5-FU浓度分别为8.83μg/ml±1.68μg/ml和9.82μg/ml±2.93μg/ml,15分钟、30分钟组癌组织5-FU浓度分别为血清的6.36、4.47倍(P<0.05);
2、注射药物5-FU 15、30分钟后,癌组织转化的5-FU浓度分别86.13μg/g±15.42μg/g和94.68μg/g±39.89μg/g,血清中5-FU浓度分别为133.35μg/ml±20.69μg/ml和112.70μg/ml±26.27μg/ml,15分钟、30分钟组血清5-FU浓度分别为癌组织的1.59、1.62倍(P<0.05)。
结论:
小鼠结肠癌模型体内,癌组织内5'-DFUR转化率高于血液,考虑分布在癌组织中的PyNPase酶比血液高。
Research background:
5'-deoxy-5-fluorouridine (5'-DFUR) is an oral anti-cancer drug, widely used in clinical treatment of gastrointestinal cancer. It is a prodrug of 5-fluorouracil (5-FU), must be activated by thymidine phosphorylase (TP) in cancer tissues and converted into 5-FU. It had been reported that breast cancer and gastric cancer cells express TP , but in colon cancer it is still controversial. We have found in previous studies that in colorectal cancer tissues, TP mainly expressed in stromal cells around cancer nests, especially in macrophages. Previous study has found TP was mainly produced by macrophages in stroma and six colon cancer lines hardly expressed TP. When cancer cells do not express TP , transformation of 5'-DFUR in tissue of colorectal cancer is not clear. Previous study in vivo found that average period of nature survival was longer, average tumor weight was lighter, the average body weight decreased slowly in mouse models of colon cancer given 5'-DFUR ,compared to 5-FU,suggesting that 5'-DFUR as a highly selective anti-cancer drug was transferd to 5-FU in cancer tissue higher than normal tissue . The reason may be the TP enzyme in cancer tissue is more than normal tissues. The early study in vitro 5'-DFUR cultured 24h with blood mononuclear cells, mean value of IC50 of four cancer cells lines decreased significantly,suggesting that blood monocytes can express TP, however ,whether specific content of TP in cancer tissue is higher than in blood still need further experiment to confirm. In this study, 5-FU transfered by 5'-DFUR in cancer tissue and blood was measured by HPLC(high performance liquid chromatography) in order to deduce indirectly distribution difference of TP in tumor tissue and blood , for further study of 5'-DFUR in the transformation of colorectal carcinoma and to provide information on regulation of TP.
Materials:
1、Animal
28 SPF-inbred BALB / c mice, 6-8 weeks, male, weight (20.00±2.34) g, purchased from Experimental Animal Center of Guangdong Province.
2、The tumor cell line
BALB/c mouse colon cancer cell line (CT26), purchased from the American Type Culture Collection(ATCC).
3、The experiment medicine
5’-deoxy-5-fluorouridine(5’-DFUR), from the Japanese company Roche Research Center; 5-fluorouracil (5-FU) injection solution, the Jiangsu Nantong Drugs Manufacture Limited Company (the batch number: 080607); control Standard of 5-fluorouracil (5-FU) purchased from Sigma Limited Company (the batch number: 097K1352).
4、Experimental apparatus
Shimadzu high performance liquid system ;Diamonsil C18 column (250mm×4.6mm, 5μm).
Methods:
1、Culture of mouse colon cancer cell line CT-26
1640 10% fetal bovine serum, containing penicillin 100×103 U / L and streptomycin 100 mg / L, cultured in 5% CO2 incubator at 37℃,bathed every other day and passaged by enzyme digestion every two or three days.
2、Preparation of cell suspension
Took exponentially growing cells on the day of making model . Digested with 0.25% trypsin and passage into cell suspension and centrifuged at 2 000r/min for 5 minutes . Discarded supernatant and added appropriate amount of saline to adjust the cell concentration to 1×107 pcs / ml.Cell viability measured by trypan blue was above 95%.
3、Colon cancer model making
Injected 0.2ml CT26 suspension into the mouse (BALB/c) the back hypodermic. About 2 weeks later,the tumor could be visible to the naked eye on the back of mouse.
4、Animal groups and drug administration
28 mice were randomly divided into 4 groups:①5'-DFUR group in 15 minutes ;②5'-DFUR group in 30 minutes ;③5-FU group in 15 minutes ;④5-FU group in 30 minutes . Based on animal weight ,5-FU dosage was 0.020mg / g and solution of concentration was 1.0mg / ml while 5'-DFUR dosage was 0.038mg / g and solution of concentration is 2.0mg/ml. Each group were killed respectively to get blood and tumor tissue on scheduled time after mice were injected intraperitoneally.
5、Taking samples
0.5 ml blood from Orbital venous of mice was placed into 37℃water bath for 30 minutes and was Centrifuged for 5min at 3200rpm.Supernatant was stored at 4℃. 0.5g tumor tissue was weight after the filter paper cleared blood.Every 0.5g tissue and 4ml normal saline (1:8) were added to homogenizer to fix for 5 minutes, centrifuged at 3200rpm for 5 minutes. Supernatant was stored at 4℃.
6、Production of blood and tumor tissue of standard curve of 5-FU
Different distilled water containing 5-FU standard were added to seven species of 90μL serum from untreated mice into 100μL.The drug concentrationsin serum were set into 6.25,12.5,25.0, 50.0,100.0,200.0,400.0μg mL-1 ,which was used to produce serum standard curve ;Different distilled water containing 5-FU standard were added to seven species of 90μL tumor homogenate from untreated mice into 100μL.The drug concentrations of tumor homogenate were set into 1.0,2.0,4.0,16.0,32.0,64.0,μg ? mL-1,which was used to produce tumor homogenate standard curve.
7、Measurement of concentration of samples 100μL serum was placed 5ml test tube and added 2mL of ethyl acetate . Mixture was centrifuged at 3200rpm for 5 min after vortex oscillation for 2 min.Supernatant was placed in another test tube . 2mL of ethyl acetate was added into the first test tube again for the second extraction, vortex oscillation,centrifugation in the same way. Merged the two supernatant and made it dry by centrifugation.Added 100μL mobile phase solution into dry test tube and mixed it. Mixed solution was placed in EP tube before 10000rpm centrifugation for 7min. 20μL of liquid supernatant was obtained to prepare for measure. Recorded the peak area of drugand calculated concentration by standard curve .the 5-FU of concentration of tumor homogenateare was measured in the same way.
8、Oservation index
5-FU concentration of tumor tissues and serum in 5'-DFUR group and 5-FU group.
9、Statistical analysis
5-FU concentration in tumor tissue and serum of 5'-DFUR group and 5-FU group made statistics analysis separately by using statistical software. SPSS13.0.Used the Paired-samples T test.When P<0.05, that had a significant difference.
Results:
1、At 15,30 minutes after injecting drug 5'-DFUR, 5-FU concentrations in tumor tissue are respectively 54.64μg/g±12.80μg/g and 45.58μg/g±18.82μg/g; 5-FU concentrations in serum are respectively 8.83μg/ml±1.68μg/ml and 9.82μg/ml±2.93μg/ml;5-FU concentration in tumor tissue are respectively 6.37,4.47 times as much as that in serum in 5'-DFUR-15min group and 5'-DFUR-30min group(P<0.05).
2、At 15,30 minutes after injecting drug 5-FU, 5-FU concentrations in tumor tissue are respectively 86.13μg/g±15.42μg/g and 94.68μg/g±39.89μg/g; 5-FU concentrations in serum are respectively 133.35μg/ml±20.69μg/ml and 112.70μg/ml±26.27μg/ml; 5-FU concentration in serum are respectively 1.59,1.62 times as much as that in tumor tissue in 5-FU-15min group and 5-FU-30min group(P <0.05).
Conclusion:
In vivo mouse model of colorectal cancer,conversion ratio of 5'-DFUR in tumor tissue is higher than that in the blood .As a result, we considere the distribution of PyNPase in tumor tissues higher than in blood .
5'-脱氧氟尿苷(5'-deoxy-5-fluorouridine, 5'-DFUR)是临床治疗消化道恶性肿瘤的口服抗癌药物,为5-氟尿嘧啶(5-FU)的前体药物。其本身没有细胞毒作用,需要在细胞内经过胸苷磷酸化酶(thymidine phosphorylase,TP)转化为5-FU才能发挥抗肿瘤作用。已有文献报道乳腺癌和胃癌细胞可以表达TP活性,而大肠癌细胞是否表达TP则持论不同。我们在前期研究中发现大肠癌组织中TP活性主要由间质细胞中的巨噬细胞表达,而测定6株结肠癌细胞系也几乎没有TP蛋白表达。在癌细胞不表达TP的情况下5'-DFUR在结直肠癌组织中如何转化尚属疑问。我们前期体内实验对结肠癌小鼠动物模型应用化疗药物5'-DFUR进行治疗,结果发现与5-FU相比平均荷瘤生存期更长,平均瘤重轻,同期平均体重下降缓慢,提示5'-DFUR在小鼠结肠癌组织比正常组织中转化率高,抗癌选择性高。其原因可能是TP酶在癌组织中分布较正常组织多。前期体外实验把5'-DFUR加入培养基中同人血单核细胞一起培养24h,5'-DFUR对4种癌细胞的IC50明显下降,提示血液中单核细胞也可表达TP。由于尚未发现实验比较在癌组织和血液中TP含量,故两者TP的含量高低尚需要实验进一步证实。本实验应用高效液相色谱法(high performance liquid chromatography,HPLC)测定应用5'-DFUR后癌组织和血液中5-FU的转化情况,间接推断TP酶在癌组织和血液中分布差异,为进一步研究5'-DFUR在结直肠癌组织中转化及TP酶调控机制提供资料。
实验材料:
1、实验动物SPF级近交系BALB/c小鼠28只,6-8周龄,雄性,体重20.00±2.34g,购自广东省医学实验动物中心。
2、肿瘤细胞株BALB/c小鼠结肠腺癌细胞株(CT26),购自美国菌种保藏中心(American Type Culture Collection, ATCC)。
3、实验药物5'-DFUR由Roche公司日本研究中心提供; 5-FU注射液,江苏南通精华制药有限公司生产(批号: 080607);5-FU标准品购自Sigma有限公司提供(批号: 097K1352)。
4、实验仪器岛津高效液相系统;色谱柱:Diamonsil C18柱(250mm×4.6mm,5μm)
实验方法:
1、小鼠结肠癌CT-26细胞株的培养10%胎牛血清1640培养基,含青霉素100×103 U/L和链霉素100 mg/L,37℃,5%CO2水浴恒温培养箱中培养,隔日换液,2-3天酶消化法传代。
2、细胞悬液制备
制备模型当天取指数生长期的细胞,用0.25%胰蛋白酶消化,机械吹打成细胞悬液,2 000r/min离心5 min,弃上清液,加适量生理盐水调整细胞浓度至1×107个/ml,以台盼蓝测定细胞活力在95%以上。
3、结肠癌模型制作方法
将体外培养的CT26细胞悬液0.2ml注入小鼠(BALB/c)背部皮下,约2周后基本可以形成肉眼可见的肿瘤隆起。
4、动物分组及给药
荷瘤小鼠28只随机分为4组:①5'-DFUR给药15分钟组;②5'-DFUR给药30分钟组;③5-FU给药15分钟组;④5-FU给药30分钟组。根据动物体重,5-FU用量0.020mg/g ,配制浓度为1.0 mg/ml。5'-DFUR用量0.038mg/g;配置浓度为2.0mg/ml。各组分别腹腔注射给药15分钟、30分钟后处死小鼠立即取血和瘤组织。
5、标本处理
小鼠眼眶动静脉取血0.5 ml后放置入37℃水浴30分钟,3200rpm离心5min,取上清液4℃保存。肿瘤组织用滤纸吸干血迹后称重,然后按0.5g组织与4 ml生理盐水(1:8)加入匀浆器匀浆5min, 3200rpm离心5min,取上清液4℃保存。
6、制作血液和肿瘤组织的5-FU药物标准曲线
取未给药小鼠血清7份,每份90μL,分别加入由5-FU对照品和蒸馏水配制的系列标准液适量并混匀配成100μL,使血清中药物浓度分别为6.25,12.5,25.0,50.0,100.0,200.0,400.0μg·mL~(-1),制作血清标准曲线;取未给药小鼠肿瘤组织匀浆液7份,每份90μL,分别加入由5-FU对照品和蒸馏水配制的系列标准液适量并混匀配成100μL,使肿瘤匀浆液中药物浓度分别为1.0,2.0,4.0,8.0,16.0,32.0,64.0μg·mL~(-1),制作肿瘤标准曲线。
7、测量各标本浓度
取血清100μL,置于5mL玻璃试管中,加入乙酸乙酯2mL,漩涡振荡2min后,3200rpm离心5min,取上层析液置于另一玻璃试管中。再次加入乙酸乙酯2mL进行第二次提取,漩涡振荡2min后,3200rpm离心5min,取上层析液,然后合并两次提取的上层析液,离心浓缩挥干。加入100μL流动相定容,混匀取出,置于EP管中,10000rpm离心7min,取上层析液20μL进样。记录药物峰面积,代入相应标准曲线计算药物浓度;取肿瘤匀浆液100μL,以同样方法处理标本测量浓度。
8、观测指标
给药15分钟、30分钟处死组5'-DFUR组和5-FU组小鼠血液与癌组织5-FU浓度。
9、统计学方法
应用统计软件SPSS13.0数据包对5'-DFUR组和5-FU组小鼠血液与癌组织5-FU浓度采用配对样本t检验进行比较。当P<0.05时,认为差异有统计学意义。
结果:
1、注射药物5'-DFUR 15、30分钟后,癌组织转化的5-FU浓度分别54.64μg/g±12.80μg/g和45.58μg/g±18.82μg/g,血清中中5-FU浓度分别为8.83μg/ml±1.68μg/ml和9.82μg/ml±2.93μg/ml,15分钟、30分钟组癌组织5-FU浓度分别为血清的6.36、4.47倍(P<0.05);
2、注射药物5-FU 15、30分钟后,癌组织转化的5-FU浓度分别86.13μg/g±15.42μg/g和94.68μg/g±39.89μg/g,血清中5-FU浓度分别为133.35μg/ml±20.69μg/ml和112.70μg/ml±26.27μg/ml,15分钟、30分钟组血清5-FU浓度分别为癌组织的1.59、1.62倍(P<0.05)。
结论:
小鼠结肠癌模型体内,癌组织内5'-DFUR转化率高于血液,考虑分布在癌组织中的PyNPase酶比血液高。
Research background:
5'-deoxy-5-fluorouridine (5'-DFUR) is an oral anti-cancer drug, widely used in clinical treatment of gastrointestinal cancer. It is a prodrug of 5-fluorouracil (5-FU), must be activated by thymidine phosphorylase (TP) in cancer tissues and converted into 5-FU. It had been reported that breast cancer and gastric cancer cells express TP , but in colon cancer it is still controversial. We have found in previous studies that in colorectal cancer tissues, TP mainly expressed in stromal cells around cancer nests, especially in macrophages. Previous study has found TP was mainly produced by macrophages in stroma and six colon cancer lines hardly expressed TP. When cancer cells do not express TP , transformation of 5'-DFUR in tissue of colorectal cancer is not clear. Previous study in vivo found that average period of nature survival was longer, average tumor weight was lighter, the average body weight decreased slowly in mouse models of colon cancer given 5'-DFUR ,compared to 5-FU,suggesting that 5'-DFUR as a highly selective anti-cancer drug was transferd to 5-FU in cancer tissue higher than normal tissue . The reason may be the TP enzyme in cancer tissue is more than normal tissues. The early study in vitro 5'-DFUR cultured 24h with blood mononuclear cells, mean value of IC50 of four cancer cells lines decreased significantly,suggesting that blood monocytes can express TP, however ,whether specific content of TP in cancer tissue is higher than in blood still need further experiment to confirm. In this study, 5-FU transfered by 5'-DFUR in cancer tissue and blood was measured by HPLC(high performance liquid chromatography) in order to deduce indirectly distribution difference of TP in tumor tissue and blood , for further study of 5'-DFUR in the transformation of colorectal carcinoma and to provide information on regulation of TP.
Materials:
1、Animal
28 SPF-inbred BALB / c mice, 6-8 weeks, male, weight (20.00±2.34) g, purchased from Experimental Animal Center of Guangdong Province.
2、The tumor cell line
BALB/c mouse colon cancer cell line (CT26), purchased from the American Type Culture Collection(ATCC).
3、The experiment medicine
5’-deoxy-5-fluorouridine(5’-DFUR), from the Japanese company Roche Research Center; 5-fluorouracil (5-FU) injection solution, the Jiangsu Nantong Drugs Manufacture Limited Company (the batch number: 080607); control Standard of 5-fluorouracil (5-FU) purchased from Sigma Limited Company (the batch number: 097K1352).
4、Experimental apparatus
Shimadzu high performance liquid system ;Diamonsil C18 column (250mm×4.6mm, 5μm).
Methods:
1、Culture of mouse colon cancer cell line CT-26
1640 10% fetal bovine serum, containing penicillin 100×103 U / L and streptomycin 100 mg / L, cultured in 5% CO2 incubator at 37℃,bathed every other day and passaged by enzyme digestion every two or three days.
2、Preparation of cell suspension
Took exponentially growing cells on the day of making model . Digested with 0.25% trypsin and passage into cell suspension and centrifuged at 2 000r/min for 5 minutes . Discarded supernatant and added appropriate amount of saline to adjust the cell concentration to 1×107 pcs / ml.Cell viability measured by trypan blue was above 95%.
3、Colon cancer model making
Injected 0.2ml CT26 suspension into the mouse (BALB/c) the back hypodermic. About 2 weeks later,the tumor could be visible to the naked eye on the back of mouse.
4、Animal groups and drug administration
28 mice were randomly divided into 4 groups:①5'-DFUR group in 15 minutes ;②5'-DFUR group in 30 minutes ;③5-FU group in 15 minutes ;④5-FU group in 30 minutes . Based on animal weight ,5-FU dosage was 0.020mg / g and solution of concentration was 1.0mg / ml while 5'-DFUR dosage was 0.038mg / g and solution of concentration is 2.0mg/ml. Each group were killed respectively to get blood and tumor tissue on scheduled time after mice were injected intraperitoneally.
5、Taking samples
0.5 ml blood from Orbital venous of mice was placed into 37℃water bath for 30 minutes and was Centrifuged for 5min at 3200rpm.Supernatant was stored at 4℃. 0.5g tumor tissue was weight after the filter paper cleared blood.Every 0.5g tissue and 4ml normal saline (1:8) were added to homogenizer to fix for 5 minutes, centrifuged at 3200rpm for 5 minutes. Supernatant was stored at 4℃.
6、Production of blood and tumor tissue of standard curve of 5-FU
Different distilled water containing 5-FU standard were added to seven species of 90μL serum from untreated mice into 100μL.The drug concentrationsin serum were set into 6.25,12.5,25.0, 50.0,100.0,200.0,400.0μg mL-1 ,which was used to produce serum standard curve ;Different distilled water containing 5-FU standard were added to seven species of 90μL tumor homogenate from untreated mice into 100μL.The drug concentrations of tumor homogenate were set into 1.0,2.0,4.0,16.0,32.0,64.0,μg ? mL-1,which was used to produce tumor homogenate standard curve.
7、Measurement of concentration of samples 100μL serum was placed 5ml test tube and added 2mL of ethyl acetate . Mixture was centrifuged at 3200rpm for 5 min after vortex oscillation for 2 min.Supernatant was placed in another test tube . 2mL of ethyl acetate was added into the first test tube again for the second extraction, vortex oscillation,centrifugation in the same way. Merged the two supernatant and made it dry by centrifugation.Added 100μL mobile phase solution into dry test tube and mixed it. Mixed solution was placed in EP tube before 10000rpm centrifugation for 7min. 20μL of liquid supernatant was obtained to prepare for measure. Recorded the peak area of drugand calculated concentration by standard curve .the 5-FU of concentration of tumor homogenateare was measured in the same way.
8、Oservation index
5-FU concentration of tumor tissues and serum in 5'-DFUR group and 5-FU group.
9、Statistical analysis
5-FU concentration in tumor tissue and serum of 5'-DFUR group and 5-FU group made statistics analysis separately by using statistical software. SPSS13.0.Used the Paired-samples T test.When P<0.05, that had a significant difference.
Results:
1、At 15,30 minutes after injecting drug 5'-DFUR, 5-FU concentrations in tumor tissue are respectively 54.64μg/g±12.80μg/g and 45.58μg/g±18.82μg/g; 5-FU concentrations in serum are respectively 8.83μg/ml±1.68μg/ml and 9.82μg/ml±2.93μg/ml;5-FU concentration in tumor tissue are respectively 6.37,4.47 times as much as that in serum in 5'-DFUR-15min group and 5'-DFUR-30min group(P<0.05).
2、At 15,30 minutes after injecting drug 5-FU, 5-FU concentrations in tumor tissue are respectively 86.13μg/g±15.42μg/g and 94.68μg/g±39.89μg/g; 5-FU concentrations in serum are respectively 133.35μg/ml±20.69μg/ml and 112.70μg/ml±26.27μg/ml; 5-FU concentration in serum are respectively 1.59,1.62 times as much as that in tumor tissue in 5-FU-15min group and 5-FU-30min group(P <0.05).
Conclusion:
In vivo mouse model of colorectal cancer,conversion ratio of 5'-DFUR in tumor tissue is higher than that in the blood .As a result, we considere the distribution of PyNPase in tumor tissues higher than in blood .
引文
1、De-Sen Wan.Epidemiologic trend of and strategies for colorectal cancer[J].Chinese Journal of Cancer, 2009, 28(9) :897-902.
2、Buys M,Zeleniuch J, Chalmers TC. Adjuvant therapy of colorectal cancer: Why we still don’t know [J] .JAMA, 1988, 259:3571.
3、Furugawa T, Yoshimura A, Sumizawa T, et al. Antigenic factor[J]. Nature, 1992, 356(637): 668.
4、Sumizawa T, Furuwa T, Haraguchi M, et al. Thymicine phosphorylase activity associated with platelet-derived endothelial cell growth factor [J]. J Biochem, 1993, 114(1): 9-14.
5、Griffiths I,Dachs GU,Bicknell R,et a1.The influence of oxygen tension and pH on the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human breast tumor cells grown in vitro and in vivo[J].Cancer Res, 1997, 57(4) : 570-572.
6、Yang Q, Sakurai T, Shan L, et a1.Thymicine phosphorylase expression correlates with tumor differentiation and bcl-2 in invasive breast cancer[J].Breast Cancer, 2000, 7 (3) : 210-214.
7、Eda H, Fujimoto K, Watanabe S, et al. Cytokine induces thymidine phosphorylase expression in tumor cell and make them more susceptible to 5’-deoxy-5-fluorouridine[J].Cancer Chemotherapy Pharmacology, 1993, 32: 333-338.
8、Konno S, Takebayashi Y, Aibac M, et a1.Clinicopathological and prognostic significance of thymidine phosphorylase and proliferating cell nuclear antigen in gastric carcinoma[J].Cancer Lett,2001,166 (1) : 103-111.
9、Takebayashi Y, Miyadera S, et al. Expression of thymidine phosphorylase in human gastric carcinoma[J].Jpn J Cancer Res, 1996, 87(3): 288-295.
10、Fujiwaki R, Hata K, Iida K, et al. Co-expression of vascular endothelial growth factor and thymidine phosphorylase in endometrial cancer[J].Acta Obstet Gynecol Scand, 1999, 78(8): 728-734.
11、Sivridis E, Giatrommanolaki A, Anastasiadis P, et al. Angiogenic co-operation of VEGR and stromal cell TP in endometrial carcinomas[J].J Pathol, 2002, 196(4): 416-422.
12、Yong X, Toshihiko K, Kazufumi S, et al. Macrophage infiltration-associated thymidine phosphorylase expression correlates with increased microvessel density and poor prognosisin astrocytic tumors[J].Clinical Cancer Research, 2001.12(7): 4021-4026.
13、Haraguchi M, Komuta K, Ueda T, et a1.Prognostic significance of the serum thymidine phosphorylase levels in venous blood drainage specimens in patients with colorectal cancer[J]. Hepatogastroenterology, 2008, 5(5):418-421.
15、王晓毓张鸿彬郑玲等.卡培他滨与5-氟尿嘧啶治疗晚期结肠癌的疗效观察.西部医学杂志,2008,20(3) : 558-559.
16、张继民,刘明姬,溝井賢幸等.单核-巨噬细胞表达的胸苷磷酸化酶增强去氧氟尿苷抗结肠癌细胞活性.中华医学杂志,2004,84 (12): 718-724.
17、Zhang JM, Mizoi T, Shiiba K, et al. Expression of thymidine phosphorylase by macrophages in colorectal cancer tissues [J]. World J Gastroenterology, 2004, 10(4): 545-549.
18、张继民,洪楚原,邓一文等.5’-脱氧氟尿苷和5-氟尿嘧啶对6株大肠癌细胞的抑制作用。广州医学院学报,2003,31(4):27-30.
19、Zhang JM, Mizoi T, Harade N, et al. Thymidine phosphorylase expressed in macrophage enhances antitumor effect of 5'-Deoxy-5-fluorouridine in human colorectal cancer cells [J]. Anticancer Research, 2003, 23(1A): 323-329.
20、张继民,刘明姬,溝井賢幸等.类巨噬细胞增强5’-脱氧氟尿苷抗结直肠癌细胞活性.中华胃肠外科杂志,2004,7(3): 218-221.
21、Bartsch R, Steger GG, Forstner B et al. Expression of thymidine phosphorylase in peripheral blood cells of breast cancer patients is not increased by paclitaxel [J].BMC Clin Pharmacol,2007,7(18):7
22、Bading JR, Shahinian AH, Paff MT et al. Validation of fluorouracil metabolite analysis in excised tumor. Lability of anabolites [J].Brioche Pharmacol, 2000, 60(7):963-967
23、Ito K, Wong L, Ando H et al. Pharmacodynamics induced by direct electric current for the treatment of 5-fluorouracil resistant tumor: an animal experiment [J].IntJ Colorectal Dis, 2001, 16(5):326-330
24、Pohlen U, Rieger H, Binnenhei M etal. Improvement in 5-fluorouracil (5-FU) and 5-fluoro-2'- deoxyuridine (FdUrd) concentration by 5-fluorouracil-polyethylene-glycol- liposomes in abdominal stop-flow: treatment of VX2 liver-tumor-bearing rabbits [J].J Chemother, 2005, 17(4): 428-434
25、Pisano R, Breda M, Grassy S et al. Hydrophilic interaction liquid chromatography- APCI-mass spectrometry determination of 5-fluorouracil in plasma and tissues [J].J Pharm Biomed Anal, 2005, 38(4):738-745
26、He W, Du Q, Cao DY et al. Study on colon-specific pectin/ ethyl cellulose film-coated 5-fluorouracil pellets in rats [J].Int J Pharm, 2008, 348(1-2):35-45.
27、金黑鹰,刘秀芳等.一种结直肠癌造口原位移植模型建立方法:中国2007100119273.0 [P]. 2007-8-15.
28、甘伙烨,何兴祥,邹湘才等.盲肠造疝原位接种瘤块法建立小鼠大肠癌肝转移模型.广东医学,2007,6(28):855-857.
29、Yo shimura A , Kuwazuru Y, Furukawa T, et al. Purification and tissue distribution of human thymidine phosphorylase: high expression in lymphocytes, reticulocytes and tumors [J]. Biochem Biophys Acta, 1990, 1034 (1): 107-113.
30、T0i M,Atiqur-Rabman M,BandoH,et a1.Thymidine phosphorylase (platelet-derived endothelial -cell growth factor)in cancer biology and treatment[J].Lancet Oncol, 2005, 6(3):158 -166.
31、Takebayashi Y,Natsugone S,Baba M,et a1.Thymidine phosphorylase in human esophageal squamous cell carcinoma[J].Cancer, 1999, 85(2): 282-289.
32、Hotta T,Taniguchi K,Kobayashi Y,et a1.Increased expression of thymidine phosphorylase in tumor tissue in proportion to DTHD-PASE-expression in primary normal tissue[J].Oncol Rep,2004, 12(3): 539-541.
33、Kuwabara K,Sasaki T,Kuwada Y,et a1.Expression of angiogenic factors in pancreatic ductal carcinoma: a correlative study with dial-co pathologic parameters and patient survival[J].Pancreas,2003, 26(4): 344-349.
34、Zhang JM,Mizoi T,Shiiba KI,et a1.Expression of thymidine phosphorylase by macrophages in colorectal cancer tissues[J].World J Gastroenterology,2004, 10(4): 545-549.
35、Kobayashi M,Okamoto K,Akimori T,et a1.Localization of thymidine phosphorylase in advanced gastric and colorectal cancer[J].J Molecular Histology,2004, 35(1): 69-74.
2、Buys M,Zeleniuch J, Chalmers TC. Adjuvant therapy of colorectal cancer: Why we still don’t know [J] .JAMA, 1988, 259:3571.
3、Furugawa T, Yoshimura A, Sumizawa T, et al. Antigenic factor[J]. Nature, 1992, 356(637): 668.
4、Sumizawa T, Furuwa T, Haraguchi M, et al. Thymicine phosphorylase activity associated with platelet-derived endothelial cell growth factor [J]. J Biochem, 1993, 114(1): 9-14.
5、Griffiths I,Dachs GU,Bicknell R,et a1.The influence of oxygen tension and pH on the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human breast tumor cells grown in vitro and in vivo[J].Cancer Res, 1997, 57(4) : 570-572.
6、Yang Q, Sakurai T, Shan L, et a1.Thymicine phosphorylase expression correlates with tumor differentiation and bcl-2 in invasive breast cancer[J].Breast Cancer, 2000, 7 (3) : 210-214.
7、Eda H, Fujimoto K, Watanabe S, et al. Cytokine induces thymidine phosphorylase expression in tumor cell and make them more susceptible to 5’-deoxy-5-fluorouridine[J].Cancer Chemotherapy Pharmacology, 1993, 32: 333-338.
8、Konno S, Takebayashi Y, Aibac M, et a1.Clinicopathological and prognostic significance of thymidine phosphorylase and proliferating cell nuclear antigen in gastric carcinoma[J].Cancer Lett,2001,166 (1) : 103-111.
9、Takebayashi Y, Miyadera S, et al. Expression of thymidine phosphorylase in human gastric carcinoma[J].Jpn J Cancer Res, 1996, 87(3): 288-295.
10、Fujiwaki R, Hata K, Iida K, et al. Co-expression of vascular endothelial growth factor and thymidine phosphorylase in endometrial cancer[J].Acta Obstet Gynecol Scand, 1999, 78(8): 728-734.
11、Sivridis E, Giatrommanolaki A, Anastasiadis P, et al. Angiogenic co-operation of VEGR and stromal cell TP in endometrial carcinomas[J].J Pathol, 2002, 196(4): 416-422.
12、Yong X, Toshihiko K, Kazufumi S, et al. Macrophage infiltration-associated thymidine phosphorylase expression correlates with increased microvessel density and poor prognosisin astrocytic tumors[J].Clinical Cancer Research, 2001.12(7): 4021-4026.
13、Haraguchi M, Komuta K, Ueda T, et a1.Prognostic significance of the serum thymidine phosphorylase levels in venous blood drainage specimens in patients with colorectal cancer[J]. Hepatogastroenterology, 2008, 5(5):418-421.
15、王晓毓张鸿彬郑玲等.卡培他滨与5-氟尿嘧啶治疗晚期结肠癌的疗效观察.西部医学杂志,2008,20(3) : 558-559.
16、张继民,刘明姬,溝井賢幸等.单核-巨噬细胞表达的胸苷磷酸化酶增强去氧氟尿苷抗结肠癌细胞活性.中华医学杂志,2004,84 (12): 718-724.
17、Zhang JM, Mizoi T, Shiiba K, et al. Expression of thymidine phosphorylase by macrophages in colorectal cancer tissues [J]. World J Gastroenterology, 2004, 10(4): 545-549.
18、张继民,洪楚原,邓一文等.5’-脱氧氟尿苷和5-氟尿嘧啶对6株大肠癌细胞的抑制作用。广州医学院学报,2003,31(4):27-30.
19、Zhang JM, Mizoi T, Harade N, et al. Thymidine phosphorylase expressed in macrophage enhances antitumor effect of 5'-Deoxy-5-fluorouridine in human colorectal cancer cells [J]. Anticancer Research, 2003, 23(1A): 323-329.
20、张继民,刘明姬,溝井賢幸等.类巨噬细胞增强5’-脱氧氟尿苷抗结直肠癌细胞活性.中华胃肠外科杂志,2004,7(3): 218-221.
21、Bartsch R, Steger GG, Forstner B et al. Expression of thymidine phosphorylase in peripheral blood cells of breast cancer patients is not increased by paclitaxel [J].BMC Clin Pharmacol,2007,7(18):7
22、Bading JR, Shahinian AH, Paff MT et al. Validation of fluorouracil metabolite analysis in excised tumor. Lability of anabolites [J].Brioche Pharmacol, 2000, 60(7):963-967
23、Ito K, Wong L, Ando H et al. Pharmacodynamics induced by direct electric current for the treatment of 5-fluorouracil resistant tumor: an animal experiment [J].IntJ Colorectal Dis, 2001, 16(5):326-330
24、Pohlen U, Rieger H, Binnenhei M etal. Improvement in 5-fluorouracil (5-FU) and 5-fluoro-2'- deoxyuridine (FdUrd) concentration by 5-fluorouracil-polyethylene-glycol- liposomes in abdominal stop-flow: treatment of VX2 liver-tumor-bearing rabbits [J].J Chemother, 2005, 17(4): 428-434
25、Pisano R, Breda M, Grassy S et al. Hydrophilic interaction liquid chromatography- APCI-mass spectrometry determination of 5-fluorouracil in plasma and tissues [J].J Pharm Biomed Anal, 2005, 38(4):738-745
26、He W, Du Q, Cao DY et al. Study on colon-specific pectin/ ethyl cellulose film-coated 5-fluorouracil pellets in rats [J].Int J Pharm, 2008, 348(1-2):35-45.
27、金黑鹰,刘秀芳等.一种结直肠癌造口原位移植模型建立方法:中国2007100119273.0 [P]. 2007-8-15.
28、甘伙烨,何兴祥,邹湘才等.盲肠造疝原位接种瘤块法建立小鼠大肠癌肝转移模型.广东医学,2007,6(28):855-857.
29、Yo shimura A , Kuwazuru Y, Furukawa T, et al. Purification and tissue distribution of human thymidine phosphorylase: high expression in lymphocytes, reticulocytes and tumors [J]. Biochem Biophys Acta, 1990, 1034 (1): 107-113.
30、T0i M,Atiqur-Rabman M,BandoH,et a1.Thymidine phosphorylase (platelet-derived endothelial -cell growth factor)in cancer biology and treatment[J].Lancet Oncol, 2005, 6(3):158 -166.
31、Takebayashi Y,Natsugone S,Baba M,et a1.Thymidine phosphorylase in human esophageal squamous cell carcinoma[J].Cancer, 1999, 85(2): 282-289.
32、Hotta T,Taniguchi K,Kobayashi Y,et a1.Increased expression of thymidine phosphorylase in tumor tissue in proportion to DTHD-PASE-expression in primary normal tissue[J].Oncol Rep,2004, 12(3): 539-541.
33、Kuwabara K,Sasaki T,Kuwada Y,et a1.Expression of angiogenic factors in pancreatic ductal carcinoma: a correlative study with dial-co pathologic parameters and patient survival[J].Pancreas,2003, 26(4): 344-349.
34、Zhang JM,Mizoi T,Shiiba KI,et a1.Expression of thymidine phosphorylase by macrophages in colorectal cancer tissues[J].World J Gastroenterology,2004, 10(4): 545-549.
35、Kobayashi M,Okamoto K,Akimori T,et a1.Localization of thymidine phosphorylase in advanced gastric and colorectal cancer[J].J Molecular Histology,2004, 35(1): 69-74.