CAJ | 학술논문

目的探讨创伤愈合过程中单核巨噬细胞集落刺激因子(GMCSF)调控新生血管化的分子机制.方法将成纤维细胞分为空白对照组、GMCSF组、GMCSF+ Go6976组以及Go6976组,空白组应用GMCSF溶解液PBS处理;GMCSF组应用50 ng/L的GMCSF处理;GMCSF+Go6976组先加入50 ng/L的GMCSF和10μmol/L的Go6976处理;Go6976组用10 μmol/L的Go6976处理.应用GMCSF作用成纤维细胞后,采用Western印迹法观察蛋白激酶磷酸化水平;采用酶联免疫吸附试验检测血管内皮生长因子(VEGF)表达,采用凝胶电泳迁移试验检测核因子-κB的活化.结果 GMCSF作用成纤维细胞45 min蛋白激酶C蛋白相对表达量0.434±0.056,与0 min 0.224±0.035比较,差异有统计学意义(P＜0.05);ELISA显示,GMCSF+ Go6976处理组VEGF表达量为39.26土7.63,与GMCSF组的86.75±10.78比较,差异有统计学意义(P＜0.05);凝胶电泳迁移实验结果显示,GMCSF组核因子-κB为82835±4565,GMCSF+Go6976组为71279±3658,差异有统计学意义(P＜0.05).结论 GMCSF可通过PKC活化核因子-κB,从而诱导人皮肤成纤维细胞VEGF的表达.
목적 탐토창상유합과정중단핵거서세포집락자격인자(GMCSF)조공신생혈관화적분자궤제.방법 장성섬유세포분위공백대조조、GMCSF조、GMCSF+ Go6976조이급Go6976조,공백조응용GMCSF용해액PBS처리;GMCSF조응용50 ng/L적GMCSF처리;GMCSF+Go6976조선가입50 ng/L적GMCSF화10μmol/L적Go6976처리;Go6976조용10 μmol/L적Go6976처리.응용GMCSF작용성섬유세포후,채용Western인적법관찰단백격매린산화수평;채용매련면역흡부시험검측혈관내피생장인자(VEGF)표체,채용응효전영천이시험검측핵인자-κB적활화.결과 GMCSF작용성섬유세포45 min단백격매C단백상대표체량0.434±0.056,여0 min 0.224±0.035비교,차이유통계학의의(P＜0.05);ELISA현시,GMCSF+ Go6976처리조VEGF표체량위39.26토7.63,여GMCSF조적86.75±10.78비교,차이유통계학의의(P＜0.05);응효전영천이실험결과현시,GMCSF조핵인자-κB위82835±4565,GMCSF+Go6976조위71279±3658,차이유통계학의의(P＜0.05).결론 GMCSF가통과PKC활화핵인자-κB,종이유도인피부성섬유세포VEGF적표체.
Objective To explore the molecular mechanisms for GMCSF regulating angiogenesis during wound healing.Methods Human fibroblasts were divided into control group,GMCSF group,GMCSF+Go6976 group and Go6976 group.The control group was treated with GMCSF solution PBS,the GMCSF group treated with 50 ng/L GMCSF,the GMCSF+ Go6976 group treated with 50 ng/L GMCSF and 10 μmol/L Go6976,and the Go6976 group treated with 10 μmol/L Go6976.The total VEGF proteins were extracted from the fibroblasts treated with GMCSF.Western blotting was employed to determine levels of PKC.The fibroblasts were treated by CMCSF.VEGF protein was analyzed by ELISA;NF-κB activation was analyzed by EMSA.Results The relative expression level of reached PKC 0.434±0.056 to the peak at 45 min in the fibroblasts affected by GMCSF,com-pared with that of PKC (0.224 ±0.035),with significant difference (P＜0.05).Compared with the GMCSF group (VEGF protein expression 86.75± 10.78),the GMCSF+Go6976 group (VEGF protein expression 39.26±7.63) showed that the expression level of VEGF significantly inhibited (P＜0.05) in ELISA.In EMSA,GMCSF group of nuclear factor kappa B was 82835 ±4565;and GMCSF+group (71279 ± 3658) Go6976 binding activity of nuclear factor kappa B and DNA was significantly lower than that in the GMCSF group (P＜0.05).Conclusions GMCSF up-regulates production of VEGF through activating PKC-NF-κB signal pathway in human fibroblasts.