中华整形外科杂志
中華整形外科雜誌
중화정형외과잡지
Chinese Journal of Plastic Surgery
2015年
5期
369-374
,共6页
鲁昆%褚小玲%王绿娅%林煌
魯昆%褚小玲%王綠婭%林煌
로곤%저소령%왕록아%림황
微血管内皮细胞%外科皮瓣%细胞缺氧%细胞凋亡
微血管內皮細胞%外科皮瓣%細胞缺氧%細胞凋亡
미혈관내피세포%외과피판%세포결양%세포조망
Microvascular endothelial cells%Surgical flaps%Cell hypoxia%Apoptosis
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24 h;3组:缺氧8 h+2 min×3次缺氧后处理;4组:缺氧8 h+5 min×3次缺氧后处理;5组:缺氧8 h+10 min×3次缺氧后处理;6组:缺氧8 h+20 min×3次缺氧后处理.各组经过8h缺氧箱+缺氧Buffer的缺氧处理和24 h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caspase-3的蛋白含量表达.实验数据采用SPSS 18.0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8 h(1563±83.35)IU/L与0 h(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/caspase-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.
目的 探討建立人真皮微血管內皮細胞的缺氧後處理模型的方法.方法 採用酶消化加磁珠分選的方法,穫得人真皮微血管內皮細胞,用5％ ECM培養基培養,取第6～8代細胞備用,在進行缺氧處理後複氧前,將細胞進行3次一定時間的缺氧/複氧交替處理;將細胞接種于6孔闆,1箇6孔闆為1組,共6組.1組:不作任何處理;2組:缺氧8h+複氧24 h;3組:缺氧8 h+2 min×3次缺氧後處理;4組:缺氧8 h+5 min×3次缺氧後處理;5組:缺氧8 h+10 min×3次缺氧後處理;6組:缺氧8 h+20 min×3次缺氧後處理.各組經過8h缺氧箱+缺氧Buffer的缺氧處理和24 h複氧後,檢測乳痠脫氫酶(LDH)隨缺氧時間含量的變化;Tunel法染色計算各組的細胞凋亡率;用Western Blot的方法檢測Bcl-2、Bax和caspase-3以及活化caspase-3的蛋白含量錶達.實驗數據採用SPSS 18.0進行統計分析,2組間比較採用t檢驗,多組間比較採用方差分析.結果在連續的缺氧過程中,LDH含量在8 h(1563±83.35)IU/L與0 h(582.85±58.25) IU/L比較,差異有統計學意義(P =0.0001).Western Blot結果:2組Bax/Bcl-2錶達含量為0.38±0.02,2組與3組0.23±0.01和4組0.22±0.02比較,差異有統計學意義(P =0.012,P=0.005),2組與5組0.33±0.02和6組0.34±0.01比較,差異無統計學意義(P＞0.05).2組活化的caspase-3/caspase-3比例為6.30±1.50,與3組2.17 ±0.26和4組2.63 ±0.31比較,差異有統計學意義(P =0.008,P=0.019);2組與5組4.36±0.29和6組4.97±0.51比較,差異無統計學意義(p＞0.05).結論 成功地建立瞭人真皮微血管內皮細胞缺氧後處理模型.
목적 탐토건립인진피미혈관내피세포적결양후처리모형적방법.방법 채용매소화가자주분선적방법,획득인진피미혈관내피세포,용5％ ECM배양기배양,취제6～8대세포비용,재진행결양처리후복양전,장세포진행3차일정시간적결양/복양교체처리;장세포접충우6공판,1개6공판위1조,공6조.1조:불작임하처리;2조:결양8h+복양24 h;3조:결양8 h+2 min×3차결양후처리;4조:결양8 h+5 min×3차결양후처리;5조:결양8 h+10 min×3차결양후처리;6조:결양8 h+20 min×3차결양후처리.각조경과8h결양상+결양Buffer적결양처리화24 h복양후,검측유산탈경매(LDH)수결양시간함량적변화;Tunel법염색계산각조적세포조망솔;용Western Blot적방법검측Bcl-2、Bax화caspase-3이급활화caspase-3적단백함량표체.실험수거채용SPSS 18.0진행통계분석,2조간비교채용t검험,다조간비교채용방차분석.결과재련속적결양과정중,LDH함량재8 h(1563±83.35)IU/L여0 h(582.85±58.25) IU/L비교,차이유통계학의의(P =0.0001).Western Blot결과:2조Bax/Bcl-2표체함량위0.38±0.02,2조여3조0.23±0.01화4조0.22±0.02비교,차이유통계학의의(P =0.012,P=0.005),2조여5조0.33±0.02화6조0.34±0.01비교,차이무통계학의의(P＞0.05).2조활화적caspase-3/caspase-3비례위6.30±1.50,여3조2.17 ±0.26화4조2.63 ±0.31비교,차이유통계학의의(P =0.008,P=0.019);2조여5조4.36±0.29화6조4.97±0.51비교,차이무통계학의의(p＞0.05).결론 성공지건립료인진피미혈관내피세포결양후처리모형.
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 min × 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-hypoxia treatment),6 group (8h hypoxia +20 min ×3 times post-hypoxia treatment).Each group underwent 8 h hypoxia + 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 0h,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 group3 (0.23±0.01) and group4 (0.22 + 0.02) (P=0.012,P=0.005),while not when compared with that in group5 (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 group4 (2.63 ±0.31) (P=0.008,P=0.019);while not in group5 (4.36±0.29) and group6 (4.97±0.51) (P ＞ 0.05).Conclusions The model ofhypoxic postconditioning of human dermal microvascular endothelial cells is successfully established.
