摘要
目的探讨建立人真皮微血管内皮细胞的缺氧后处理模型的方法。方法采用酶消化加磁珠分选的方法，获得人真皮微血管内皮细胞，用5％ECM培养基培养，取第6～8代细胞备用，在进行缺氧处理后复氧前，将细胞进行3次一定时间的缺氧／复氧交替处理；将细胞接种于6孔板，1个6孔板为1组，共6组。1组：不作任何处理；2组：缺氧8h＋复氧24h；3组：缺氧8h＋2min×3次缺氧后处理；4组：缺氧8h＋5min×3次缺氧后处理；5组：缺氧8h＋10min×3次缺氧后处理；6组：缺氧8h＋20min×3次缺氧后处理。各组经过8h缺氧箱＋缺氧Buffer的缺氧处理和24h复氧后，检测乳酸脱氢酶（LDH）随缺氧时间含量的变化；Tunel法染色计算各组的细胞凋亡率；用Western Blot的方法检测Bcl-2、Bax和caspase-3以及活化easpase-3的蛋白含量表达。实验数据采用SPSS18．0进行统计分析，2组间比较采用t检验，多组间比较采用方差分析。结果在连续的缺氧过程中，LDH含量在8h（1563±83．35）IU／L与0h（582．85±58．25）IU／L比较，差异有统计学意义（P=0．0001）。Western Blot结果：2组Bax／Bcl-2表达含量为0．38±0．02，2组与3组0．23±0．01和4组0．22±0．02比较，差异有统计学意义（P=0．012，P=0．005），2组与5组0．33±0．02和6组0．34±0．01比较，差异无统计学意义（P〉0．05）。2组活化的caspase-3／easpase-3比例为6．30±1．50，与3组2．17±0．26和4组2．63±0。31比较，差异有统计学意义（P=0．008，P=0．019）；2组与5组4．36±0．29和6组4．97±0．51比较，差异无统计学意义（P〉0．05）。结论成功地建立了人真皮微血管内皮细胞缺氧后处理模型。
Objective To establish a model of hypoxic postconditioning of dermal microvascular endothelial cells. Methods During reoxygenation after hypoxia, cells received three times of hypoxic/ reoxygenation alternate treatment, for a certain time. The cells were seeded on 6-well plates, with one plate for one group. They were divided into 6 groups as group 1 （Control） , group 2 （ 8h hypoxia ＋ 24h reoxygenation）, group 3 （8h hypoxia ＋ 2 rain x 3 times post-hypoxia treatment） , group 4 （ 8h hypoxia ＋ 5 min × 3 times post-hypoxia treatment） , group 5 （8h hypoxia ＋ 10 min × 3 times post-bypoxia treatment） , 6 group （8h hypoxia ＋ 20 min × 3 times post-hypoxia treatment）. Each group underwent 8 b bypoxia ＋ 24 h hypoxia Buffer and reoxygenation. Lactate dehydrogenase （LDH） was detected during the process. Apoptosis rate was calculated by staining Tunel method. Bcl-2, Bax and activated caspase-3 protein were detected by Western Blot. Results In the continuous hypoxia process, the LDH was （1563 ±83.35）IU/L at 8h and （582.85 ±58.25）IU/L at Oh,showing a statistical difference （P = 0. 0001 ）. Western blotting results showed that the expression of Bax / Bcl-2 in group 2 was 0. 38 ±0. 02, showing a significant difference when compared with that in group 3 （0. 23 ±0. 01 ） and group 4 （0.22 ±0. 02） （P =0. 012, P=0.005）, while not when compared with that in group 5 （0.33 ±0.02） and 6 groups （0.34±0.01） （P 〉0. 05）. The ratio of Activated caspase-3/caspase-3 in group 2（6.30 ±1.50） was significantly higher than that in group3 （2. 17 ±0.26） and group 4 （2.63 ±0.31） （P =0.008,P=0.019）; while not in group5 （4.36±0.29） andgroup6 （4.97±0.51） （P 〉 0.05）. Conclusions The model of hypoxic postconditioning of human dermal microvascular endothelial cells is successfully established.
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2015年第5期369-374,共6页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金（81272130）
关键词
微血管内皮细胞
外科皮瓣
细胞缺氧
细胞凋亡
Microvascular endothelial cells
Surgical flaps
Cell bypoxia
Apoptosis
