丁酸钠对Caco-2细胞增殖与凋亡的影响  

Effects of sodium butyrate on proliferation and apoptosis of Caco-2 cells

作　　者：黄晓忠[1] 夏立广[1] 侯龙龙[1] 胡书奇 朱利斌[1] 李仲荣[1] 林锦[2] 

机构地区：[1]温州医科大学附属第二医院育英儿童医院,325027 [2]纽约大学西奈山医学院

出　　处：《中华小儿外科杂志》2015年第2期121-126,共6页Chinese Journal of Pediatric Surgery

基　　金：浙江省自然科学基金（LY12H04005）、浙江省医药卫生支撑学科基金（11-ZC27）、浙江省高校重中之重学科临床医学儿科学开放基金

摘　　要：目的观察丁酸钠及p38MAPK特异性抑制剂SB203580对Caco-2细胞的作用情况，探索短链脂肪酸损害肠上皮屏障的可能机制，进而为防治新生儿坏死性小肠结肠炎寻找新靶点提供依据。方法使用不同浓度丁酸钠和SB203580作用于体外培养的Caco-2细胞及单层Caco-2细胞肠屏障模型，通过Cell Counting Kit（CCK-8）法检测细胞活性变化；通过Annexin V-FITC／PI双染流式细胞仪术与吖啶橙（Acridine Orange，AO）荧光染色检测与观察细胞凋亡情况；应用Western Blot法检测细胞内p38MAPK、p-p38MAPK的表达。结果①低浓度丁酸钠组（2mmol／L）作用Caco-2细胞72h，CCK-8的吸光度为0．78±0．049，较对照组的0．97±0．016吸光度要低（P〈0．01）；②AO凋亡核染发现5mmol／L丁酸钠作用Caco-2细胞72h，出现典型的凋亡细胞核形态学变化；③2mmol／L丁酸钠组作用Caco-2细胞72h，细胞凋亡率为（14±3．21）％，与对照组的（13．77±4．92）%相比差异无统计学意义（P〉0．05）；而中、高浓度丁酸钠（5mmol／L和8mmol／L）诱导的细胞凋亡率分别为（46．96±7．28）％和（71．97±6．65）％，与对照组相比差异有统计学意义（Pd0．01），SB203580可降低5mmol／L丁酸钠组的凋亡率。5mmol／L丁酸钠作用Caco-2细胞24h后，细胞凋亡率为（9．54±3．29）％，与对照组的（9．72±2．72）％凋亡率相比差异无统计学意义（P〉0．05）；作用48h和72h后的Caco-2细胞凋亡率分别为（26．75±3．89）％和（48．33±7．08）％，两者凋亡率均比对照组高（P〈0．01）；④丁酸钠以时间和浓度依赖方式激活p38MAPK，SB203580可抑制5mmol／L丁酸钠刺激后细胞内p-p38MAPK的表达，细胞内p38MAPK及β-Tubulin表达不受作用时间和干预药物浓度的影响。结论中、高浓度丁酸钠通过诱导大量Caco-2细胞凋亡来破坏单层Caco-2细胞肠屏障功能，在该过程中p38MAPK可能起重要作用。Objective To observe the effects of sodium butyrate and SB203580 （a specific inhibitor of p38MAPK） on Caco-2 cells and explore the mechanisms of short-chain fatty acids impairing intestinal epithelial barriers so as to reveal new targets for preventing and treating neonatal necrotizing enterocolitis （NEC）. Methods Cell viability was measured by cell counting kit-8 assay （CCK-8）. The induction of apoptosis was determined by flow cytometry with the stains of propidium iodide and annexin V-fluorescein isothiocyanate. The cells were stained with acridine orange （AO） for detecting cell apoptosis. The expressions of p38MAPK and p-p38MAPK were detected by Western blot. Results After a 72-hour treatment of sodium butyrate （2 mmol/L）, the absorbance of CCK-8 was 0. 78 ±0.049 versus 0. 97±0. 016 in control group （0 mmol/L sodium butyrate）. And the absorbance of CCK-8 significantly decreased in 2 mmol/L group （P〈0. 01）. A 72-hour treatment of 5 mmol/L sodium butyrate, typical morphological changes of nucleus appeared in apoptotic cell. After treating for 2h with 2, 5 and 8 retool/L, the ratios of apoptosis were （14 ± 3. 21）%, （46. 96 ± 7. 28）% and （71.97 ± 6. 65）% respectively. No obvious difference existed between the apoptotic rate （13.77 ± 4. 92）% of control group （0 mmol/L sodium butyrate） and that of 2 mmol/L （P〉0. 05）. However, the concentrations of 5 and 8 mmol/L had significantly different results （P〈0. 01）. And SB203580 could decrease the ratio of apoptosis in 5 mmol/L group. After treating with 5 mmol/L for 24, 48 and 72 h, the ratios of apoptosis were （9. 54 ± 3. 29）%, （26. 75 ± 3.89）% and （48. 33 ± 7. 08）% respectively. No obvious difference existed between the apoptotic rate （9. 72 ± 2. 72）% of control group （0 h） and that of 24 h （P〉0. 05）. However, there were significantly different results at 48 h and 72 h （P〈0. 01）. Sodium butyrate activated p38MAPK in a duration-dose dependent fashion with elongating tim

关 键 词：脂肪酸类 挥发性 肠黏膜 丝裂原激活蛋白激酶类 

分 类 号：R722.1[医药卫生—儿科]