粒细胞巨噬细胞刺激因子对大鼠创面哺乳动物西罗莫司靶蛋白信号通路的作用
립세포거서세포자격인자대대서창면포유동물서라막사파단백신호통로적작용
Effect of granulocyte-macrophage colony-stimulating factor on the mammalian target of sirolimus signaling pathway in the wound of rat
  • 万方数据
    中华烧伤杂志 중화소상잡지
    16
    2012年 1期 36-41 ,共6页

    崔文慧%黄宏%徐祥%郭敏%代卉%简宇%郭韡%邢伟%蒋建新%杨戎

    최문혜%황굉%서상%곽민%대훼%간우%곽위%형위%장건신%양융

    粒细胞巨噬细胞集落刺激因子%信号传导%哺乳动物西罗莫司(雷帕霉素)靶蛋白%创面愈合 粒細胞巨噬細胞集落刺激因子%信號傳導%哺乳動物西囉莫司(雷帕黴素)靶蛋白%創麵愈閤
    립세포거서세포집락자격인자%신호전도%포유동물서라막사(뢰파매소)파단백%창면유합
    Granulocyte-macrophage colony-stimulating factor%Singal transduction%Mammalian target of sirolimus(rapamycin)signaling pathway%Wound healing
    目的 了解应用粒细胞巨噬细胞刺激因子(GM-CSF)对大鼠创面愈合的影响及对哺乳动物西罗莫司(雷帕霉素)靶蛋白(mTOR)信号通路的作用. 方法 将50只SD大鼠按照随机数字表法分为治疗组和对照组,每组25只,于其背部制作约2 cm×2 cm全层皮肤缺损创面.治疗组大鼠创面外涂重组人粒细胞巨噬细胞刺激因子( rhGM-CSF)凝胶,10 μg/cm2,rhGM-CSF实际作用量为1×10-4 μg/cm2；对照组创面涂抹不含任何药物的凝胶基质,10 μg/cm2.2组每日给药1次直至创面愈合.于致伤后1、3、5、7、14 d,每组每时相点处死5只大鼠:(1)观察并计算创面愈合率.(2)于前4个时相点取创面组织标本,行HE染色观察组织病理学改变,ELISA法检测GM-CSF含量,蛋白质印迹法检测GM-CSF、CD31及mTOR信号通路相关分子P70S6K、磷酸化(p-)P70S6K、4E-BP1、p-4E-BP1、mTOR、p-mTOR表达量.对数据行t检验. 结果 (1)伤后1d,2组大鼠创面愈合率接近(t=0.307,P＞0.05)；伤后3～ 14 d,治疗组创面愈合率明显高于对照组(t值为2.704～ 4.030,P ＜0.05或P ＜0.01).(2)HE染色可见,与对照组同时相点相比,治疗组创面肉芽组织及微血管数量明显增多,创缘角化上皮细胞增殖明显.(3)ELISA法和蛋白质印迹检测结果显示,2组大鼠创面GM-CSF含量及蛋白表达量均在伤后3d达到峰值,对照组分别为(720.9±0.9)pg/mL、2.45±0.10,治疗组分别为(910.5±1.3)pg/mL、2 80±0.48.治疗组各时相点GM-CSF含量均明显高于对照组(t值为105.743～298.971,P值均为0.000),治疗组伤后1、5、7 d GM-CSF蛋白表达量明显高于对照组(t值为4.070～5.275,P值均小于0.01).(4)治疗组伤后1、3、7d创面组织中CD31表达量均明显高于对照组(t值为7.237～26.401,P值均小于0.01).(5)治疗组伤后各时相点创面组织中mTOR和p-mTOR表达量均高于对照组(t值为2.921 ～23.143,P ＜0.05或P＜0.01).治疗组P70S6K表达量伤后3、5、7d高于对照组(t值为2.950～5.275,P＜0 05或P＜0.01),p-P70S6K表达量于伤后1、3、7d高于对照组(t值为3.307 ～22.793,P＜0.05或P＜0.01).治疗组伤后1、3、5 d 4E-BP1表达量均明显低于对照组(t值为2.449～6.431,P＜0.05或P＜0.01),p-4E-BP1表达量于伤后1、3、7 d高于对照组(t值为5.522～11.613,P值均小于0.01). 结论 GM-CSF能促进大鼠创面组织mTOR蛋白及其下游信号分子P70S6K、4E-BP1的磷酸化,从而激活mTOR信号通路,促进创面愈合.
    목적 료해응용립세포거서세포자격인자(GM-CSF)대대서창면유합적영향급대포유동물서라막사(뢰파매소)파단백(mTOR)신호통로적작용. 방법 장50지SD대서안조수궤수자표법분위치료조화대조조,매조25지,우기배부제작약2 cm×2 cm전층피부결손창면.치료조대서창면외도중조인립세포거서세포자격인자( rhGM-CSF)응효,10 μg/cm2,rhGM-CSF실제작용량위1×10-4 μg/cm2；대조조창면도말불함임하약물적응효기질,10 μg/cm2.2조매일급약1차직지창면유합.우치상후1、3、5、7、14 d,매조매시상점처사5지대서:(1)관찰병계산창면유합솔.(2)우전4개시상점취창면조직표본,행HE염색관찰조직병이학개변,ELISA법검측GM-CSF함량,단백질인적법검측GM-CSF、CD31급mTOR신호통로상관분자P70S6K、린산화(p-)P70S6K、4E-BP1、p-4E-BP1、mTOR、p-mTOR표체량.대수거행t검험. 결과 (1)상후1d,2조대서창면유합솔접근(t=0.307,P＞0.05)；상후3～ 14 d,치료조창면유합솔명현고우대조조(t치위2.704～ 4.030,P ＜0.05혹P ＜0.01).(2)HE염색가견,여대조조동시상점상비,치료조창면육아조직급미혈관수량명현증다,창연각화상피세포증식명현.(3)ELISA법화단백질인적검측결과현시,2조대서창면GM-CSF함량급단백표체량균재상후3d체도봉치,대조조분별위(720.9±0.9)pg/mL、2.45±0.10,치료조분별위(910.5±1.3)pg/mL、2 80±0.48.치료조각시상점GM-CSF함량균명현고우대조조(t치위105.743～298.971,P치균위0.000),치료조상후1、5、7 d GM-CSF단백표체량명현고우대조조(t치위4.070～5.275,P치균소우0.01).(4)치료조상후1、3、7d창면조직중CD31표체량균명현고우대조조(t치위7.237～26.401,P치균소우0.01).(5)치료조상후각시상점창면조직중mTOR화p-mTOR표체량균고우대조조(t치위2.921 ～23.143,P ＜0.05혹P＜0.01).치료조P70S6K표체량상후3、5、7d고우대조조(t치위2.950～5.275,P＜0 05혹P＜0.01),p-P70S6K표체량우상후1、3、7d고우대조조(t치위3.307 ～22.793,P＜0.05혹P＜0.01).치료조상후1、3、5 d 4E-BP1표체량균명현저우대조조(t치위2.449～6.431,P＜0.05혹P＜0.01),p-4E-BP1표체량우상후1、3、7 d고우대조조(t치위5.522～11.613,P치균소우0.01). 결론 GM-CSF능촉진대서창면조직mTOR단백급기하유신호분자P70S6K、4E-BP1적린산화,종이격활mTOR신호통로,촉진창면유합.
    Objective To investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF)on wound healing and mammalian target of sirolimus(rapamycin)signaling pathway in rats.Methods Fifty SD rats were divided into control group( n =25 )and treatment group( n =25)according to the random number table.All rats were inflicted with 2 cm × 2 cm full-thickness skin wound on the back.Recombinant human GM-CSF gel(10 μg/cm2)was applied onto the wounds in treatment group,and the actual quantity was 1 × 10-4 μg/cm2.Gel vehicle( 10 μg/cm2 )without any medicine was applied onto the wounds in control group.The treatment was conducted once a day up to the day of wound healing.Five rats from two groups were sacrificed on post injury day(PID)1,3,5,7,14 respectively to observe and determine the wound healing rate.Wound tissue samples were collected at the former 4 time points to observe the histopathological changes with HE staining,and to detect the content of GM-CSF with enzyme-linked immunosorbent assay,and the expression levels of GM-CSF,CD31,and the mTOR signal pathway associated molecules P70S6K,phosphorylated(p-)P70S6K,4E-BP1,p-4E-BP1,mTOR,p-mTOR with Western blotting.Data were processed with t test. Results ( 1 )Wound healing rates in control group and treatment group were close on PID 1( t =0.307,P ＞ 0.05 ).Wound healing rate in treatment group was obviously higher than that in control group on PID 3,5,7,and 14( with t values from 2.704 to 4.030,P ＜0.05 or P＜ 0.01 ).(2)Compared with those in control group,more abundant granulation tissue was observed in treatment group,in which an increase in the number of microveseels and obvious proliferation of keratinized epithelial cells in wound margin were observed at each time point.(3)The content and the protein expression level of GM-CSF peaked on PID 3 in two groups,and they were(720.9 ± 0.9)pg/mL,2.45 ±0.10 in control group and(910.5 ± 1.3)pg/mL,2.80 ± 0.48 in treatment group.The content of GM-CSF in treatment group was significantly higher than that in control group at each time point( with t values from 105.743 to 298.971,P values all equal to 0.000).The protein expression level of GM-CSF in treatment group was significantly higher than that in control group on PID 1,5,and 7( with t values from 4.070 to 5.275,P values all below 0.01 ).(4)The expression level of CD31 in treatment group was obviously higher than that in control group on PID 1,3,and 7( with t values from 7.237 to 26.401,P values allbelow 0.01 ).(5)The expression levels of mTOR and p-mTOR in treatment group were significantly higher than those in control group at each time point(with t values from 2.921 to 23.143,P ＜ 0.05 or P ＜ 0.01 ).In treatment group,the expression level of P70S6K was obviously higher than that in control group on PID 3,5,and 7(with t values from 2.950 to 5.275,P ＜ 0.05 or P ＜ 0.01 ),and the expression level of pP70S6K was significantly higher than that in control group on PID 1,3,and 7( with t values from 3.307 to 22.793,P ＜ 0.05 or P ＜ 0.01 ).In treatment group,the expression level of4E-BP1 was significantly lower than that in control group on PID 1,3,and 5( with t values from 2.449 to 6.431,P ＜ 0.05 or P ＜ 0.01 ),but the expression level of p-4E-BP1 was significantly higher than that in control group on PID 1,3,and 7 (with t values from 5.522 to 11.613,P values all below 0.01 ). Conclusions GM-CSF can promote wound healing in rats by activating mTOR signaling pathway through phosphorylating mTOR proteins and its downstream signal molecules P70S6K and 4E-BP1.
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