5-氟尿嘧啶诱导脂多糖激活的巨噬细胞凋亡的研究  

Effect of 5-fluorouracfl on lipopolysaccharide-induced apoptosis of macrophages in vitro

作　　者：王维明[1] 周香[3] 孙洪伟[1] 陈必成[1] 周蒙滔[2] 

机构地区：[1]温州医学院附属第一医院肝胆胰外科,325000 [2]温州医学院附属文成院区普外科,325000 [3]温州医学院附属第二医院胃肠外科,325000

出　　处：《中华实验外科杂志》2013年第2期258-261,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81070372）;浙江省自然科学基金重点资助项目（Z2100853）;卫生部省部共建项目（WKJ2012-2-033）;浙江省重中之重学科（外科学）开放基金项目

摘　　要：目的探讨5．氟尿嘧啶（5-Fu）对脂多糖（LPS）激活的大鼠巨噬细胞凋亡的影响及其机制。方法自SD大鼠腹腔提取巨噬细胞，鉴定并判断其活力；LPS（1mg／L）刺激大鼠巨噬细胞建立体外炎症模型；设空白对照组、LPS组及不同浓度的LPS＋5一Fu组，细胞计数试剂盒（CCK一8）检测细胞活性，Hoechst染色观察细胞凋亡，并测定半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3活性，实时定量聚合酶链反应（Real．timePCR）法测定B淋巴细胞／白血病-2（bcl-2）、bax及p53基因表达的改变，Westernblot测定bcl-2、bax、P—Caspase-3及p53／p—p53蛋白表达的变化。结果大鼠腹腔成功提取巨噬细胞；CCK一8结果显示5-Fu能促进LPS激活的巨噬细胞凋亡（0．749±0．011、0．694±0．009、0．549±0．009、0．525±0．010、0．441±0．014、0．336±0．012比1．036±0．47，P〈0．05）；Hoechst染色也观察到LPS＋5．Fu组巨噬细胞凋亡显著增加；LPS＋5-Fu组Caspase-3活性强于LPS组（12．29±0．34、12．75±0．44、15．25±0．58、17．01±0．54比10．92±0．24，P〈0．05）；LPS＋5-Fu组bcl-2mRNA的表达显著低于LPS组，且呈剂量依赖性关系（1．99±0．11、1．14±0．11、0．76±0．04、0．37±0．05比4．14±0．27，P〈0．05），bax及p53mRNA的表达则显著高于LPS组，亦呈剂量依赖性关系（38．32±1．70、63．24±4．82、73．83±5．79、166．00±7．00比3．19±0．29，P〈0．05和3．60±0．18、5．47±0．25、6．30±0．13、8．49-t-O．38比3．16±0．06，P〈0．05）；Westernblot结果也显示：5-Fu作用LPS激活的巨噬细胞bcl-2蛋白表达显著下降，bax、P—Caspase-3及p53／p．p53蛋白的表达显著升高。结论5一Fu的确能诱导LPS激活的大鼠巨噬细胞凋亡，其机制可能与p53途径有关，通过影响Caspase-3、bcl-2及bax活化而促进凋亡。Objective To investigate the effects of 5-fluorouracil （5-Fu） on lipopolysaccharide （LPS）-induced apoptosis of abdominal macrophages in rats and the possible mechanism. Methods Ab- dominal macrophages extracted from SD rats were identified, their vitality was estimated. The. abdominal macrophages in rats were stimulated with LPS （1 mg/L） to establish inflammatory model in vitro. Macro- phages were randomized into blank control group, LPS group and LPS （ different concentrations） ＋ 5-Fu group. Cell counting kit-8 （CCK-8） was used to test cell viability. Hoechst dyeing was introduced to ob- serve cell apoptosis, and cysteinyl aspartate-specifie protease （Caspase）-3 kit was applied to measure Caspase-3 activity. The expression of B lymphocytes/leukemia-2 （ bcl-2）, bax and p53 genes was detected by using real-time polymerase chain reaction （real-time PCR）, and that of bcl-2, bax, p-Caspase-3 and p53/p-p53 proteins by using Western blotting. Results Abdominal macrophages were successfully extrac- ted from rats. CCK-8 revealed that 5-Fu could promote apoptosis of LPS-stimulated maerophages （0. 749 ± 0.011, 0.694 ±0.009, 0.549 ±0.009, 0.525 ±0.010, 0.441 ±0.014, 0.336 ±0.012 vs. 1.036± 0. 47 ,P 〈 O. 05 ）. Hoechst dyeing exhibited that 5-Fu could increase the apoptosis of cells activated with LPS. The expression of Caspase-3 mRNA and protein in LPS ＋ 5-Fu groups was higher than that in LPS group （12.29±0.34, 12.75±0.44, 15.25 ±0.58, 17.01 ±0.54 vs 10.92 ±0.24,P〈0.05）. The expressionof bax and p53 mRNA and protein was significantly higher in LPS ＋ 5-Fu groups than that in LPS group （38.32＋1.70, 63.24 ＋4.82, 73.83 ＋5.79, 166.00 ±7.00 vs 3.19 ±0.29,P〈0.05, and 3.60 ± 0. 18, 5.47 ± 0. 25, 6. 30 ± 0. 13, 8.49 ± 0. 38 vs 3. 16 ± 0. 06, P 〈 0. 05）, and the expression of bcl-2 mRNA and protein was the opposite （ 1.99 ± 0. 11, 1.14 ± 0. 11, 0. 76 ± 0. 04, 0. 37 ± 0. 05 vs 4. 14 ± 0. 27,P 〈 0. 05 ）. Conclusion 5-Fu could obviously acceler

关 键 词：5-氟尿嘧啶 脂多糖 巨噬细胞 脱噬作用 

分 类 号：R285.5[医药卫生—中药学]