丝裂霉素C预防硬膜外瘢痕粘连的分子生物学机制研究
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摘要
目的:本实验旨在通过体外培养人成纤维细胞,通过施加不同浓度的丝裂霉素C(MMC)作用细胞一定的时间后,观察丝裂霉素C对成纤维细胞抑制其正常增殖能力,诱导其凋亡的作用及探讨其中的分子生物学机制。
方法:1.体外用MEM培养液+10%胎牛血清培养人皮肤成纤维细胞并传致5-8代,分别用含有0、0.001、0.01、0.05、0.1、0.2、0.3 mg/ml MMC的MEM培养液处理种于96孔板中的成纤维细胞12h后,再于每孔加入CCK-8试剂100ul,放入细胞培养箱内1小时后用酶标仪检测不同浓度MMC处理组在450/630nm处的吸光度值,计算MMC对成纤维细胞的生长抑制效果;2.将正处于对数增长期的成纤维细胞均匀地种于消毒后置于6孔板中的圆形盖玻片上,以0、0.001、0.01、0.05、0.1、0.2 mg/ml MMC处理细胞爬片12小时后用预冷的PBS洗净培养液以Hoechest33342细胞核染色,在荧光显微镜下观察凋亡细胞核形态并用image pro-plus软件计数各组凋亡细胞百分率;3.按不加药培养细胞12h做对照组、0.2 mg/ml MMC培养细胞12h、PI3K/Akt抑制剂LY294002 100nmol/L与MEK/ERK抑制剂PD98059 100nmol/L分别处理细胞1h后换新鲜培养基培养12h分为4组,用预冷PBS洗净培养液,每瓶细胞加入100ul细胞裂解液,裂解20分钟后4oC 12000rpm离心提取胞浆蛋白,Western bolt免疫印迹法检测caspase-3,p-ERK1/2,p-Akt及Bad表达量,流式细胞法检测细胞凋亡率。
结果:不同浓度的MMC对人成纤维细胞的增殖抑制作用与药物浓度呈正相关性,在0.2mg/ml时MMC对细胞活性抑制率为50.21%;0.3mg/ml时达80.71%;Hoechest33342细胞核染色随MMC浓度升高出现凋亡细胞核特征的染色质凝聚,边集,呈亮蓝色核的比率逐渐增高,image pro-plus软件计数凋亡细胞显示0.05、0.1、0.2 mg/ml MMC处理组凋亡率显著高于其他低浓度组,差异有统计学意义(p<0.05)。Western blot检测细胞相关凋亡信号途径的蛋白表达结果显示:0.2mg/ml MMC组,PD98059组,LY294002组caspase-3表达均显著高于对照组,p-ERK1/2,p-Akt表达均较对照组降低,0.2mg/ml MMC组Bad表达水平显著高于其他三组。流失细胞法测细胞凋亡率结果显示0.2mg/ml MMC组凋亡率明显高于对照组和抑制剂预处理组。
结论:1.一定浓度的MMC可通过诱导成纤维细胞发生凋亡而抑制其正常增殖;
2.其效应途径可以解释为部分是通过降低ERK1/2, Akt的磷酸化水平使胞浆内Bad表达水平升高而导致凋亡执行蛋白caspase-3的表达水平增加,诱导成纤维细胞由正常增生状态转而发生凋亡减少胶原纤维生成,从而抑制瘢痕形成。
3.MMC抑制硬膜外瘢痕粘连的分子生物学机制其中之一是作为抗肿瘤药的丝裂霉素在术后使大量迁移至伤口炎症中心,并受到各种炎症介质和细胞生长因子刺激的成纤维细胞活性受到抑制,发生凋亡,减少了有效生成大量瘢痕组织的成纤维细胞数量,使后期为数不多的成纤维细胞不能产生过多的胶原纤维而造成瘢痕与硬膜的粘连,从而可以解决术后粘连带来的下腰椎手术失败综合症。
Objective: To elucidate the mechanism of mitomycin C prevention of peridural adhesion after laminectomy on inducing apoptosis of fibroblast
Methods The cultured fibroblasts were exposed for 12h with 0mg/ml,0.001 mg/ml,0.01mg/ml,0.05 mg/ml,0.1 mg/ml,0.2 mg/ml,0.3 mg/ml mitomycin C, respectively. The cell proliferation was detected by CCK-8 assay and Apoptotic cell death was analyzed by Hoechst 33342 stain. The expression of caspase-3 and the activation of ERK1/2, Akt ,Bad were analyzed by Western blot
Results Treatment of fibroblast with MMC induced a significant decreased of proliferation. fibroblasts exposed for 0.001 mg/ml to 0.2 mg/ml MMC, Staining with Hoechst 33342 MMC displayed nuclei with abnormal morphologies:“bean”-shaped nuclei with highly condensed chromatin or nuclei with irregular clumps of dense chromatin. 0.2mg/ml MMC,PD98059(ERK kinase inhibitor),LY294002(AKT kinase inhibitor) induced a significant increased of caspase-3 than control, accompanied by an increase in cell apoptosis.0.2mg/ml MMC PD98059,LY294002 caused decrease of p-ERK1/2 and p-Akt,at the same time, 0.2mg/ml MMC increased Bad than PD98059,LY294002 and control.
Conclusion 0.2 mg/ml MMC can induce fibroblast apoptosis by activation of caspase-3 in which requires inactivation of p-ERK1/2, p-Akt and increase the Bad. MMC can inhibit fibroblast proliferation and induce its apoptosis, which may be one of mechanism of that MMC prevent peridural adhesion after laminectomy
方法:1.体外用MEM培养液+10%胎牛血清培养人皮肤成纤维细胞并传致5-8代,分别用含有0、0.001、0.01、0.05、0.1、0.2、0.3 mg/ml MMC的MEM培养液处理种于96孔板中的成纤维细胞12h后,再于每孔加入CCK-8试剂100ul,放入细胞培养箱内1小时后用酶标仪检测不同浓度MMC处理组在450/630nm处的吸光度值,计算MMC对成纤维细胞的生长抑制效果;2.将正处于对数增长期的成纤维细胞均匀地种于消毒后置于6孔板中的圆形盖玻片上,以0、0.001、0.01、0.05、0.1、0.2 mg/ml MMC处理细胞爬片12小时后用预冷的PBS洗净培养液以Hoechest33342细胞核染色,在荧光显微镜下观察凋亡细胞核形态并用image pro-plus软件计数各组凋亡细胞百分率;3.按不加药培养细胞12h做对照组、0.2 mg/ml MMC培养细胞12h、PI3K/Akt抑制剂LY294002 100nmol/L与MEK/ERK抑制剂PD98059 100nmol/L分别处理细胞1h后换新鲜培养基培养12h分为4组,用预冷PBS洗净培养液,每瓶细胞加入100ul细胞裂解液,裂解20分钟后4oC 12000rpm离心提取胞浆蛋白,Western bolt免疫印迹法检测caspase-3,p-ERK1/2,p-Akt及Bad表达量,流式细胞法检测细胞凋亡率。
结果:不同浓度的MMC对人成纤维细胞的增殖抑制作用与药物浓度呈正相关性,在0.2mg/ml时MMC对细胞活性抑制率为50.21%;0.3mg/ml时达80.71%;Hoechest33342细胞核染色随MMC浓度升高出现凋亡细胞核特征的染色质凝聚,边集,呈亮蓝色核的比率逐渐增高,image pro-plus软件计数凋亡细胞显示0.05、0.1、0.2 mg/ml MMC处理组凋亡率显著高于其他低浓度组,差异有统计学意义(p<0.05)。Western blot检测细胞相关凋亡信号途径的蛋白表达结果显示:0.2mg/ml MMC组,PD98059组,LY294002组caspase-3表达均显著高于对照组,p-ERK1/2,p-Akt表达均较对照组降低,0.2mg/ml MMC组Bad表达水平显著高于其他三组。流失细胞法测细胞凋亡率结果显示0.2mg/ml MMC组凋亡率明显高于对照组和抑制剂预处理组。
结论:1.一定浓度的MMC可通过诱导成纤维细胞发生凋亡而抑制其正常增殖;
2.其效应途径可以解释为部分是通过降低ERK1/2, Akt的磷酸化水平使胞浆内Bad表达水平升高而导致凋亡执行蛋白caspase-3的表达水平增加,诱导成纤维细胞由正常增生状态转而发生凋亡减少胶原纤维生成,从而抑制瘢痕形成。
3.MMC抑制硬膜外瘢痕粘连的分子生物学机制其中之一是作为抗肿瘤药的丝裂霉素在术后使大量迁移至伤口炎症中心,并受到各种炎症介质和细胞生长因子刺激的成纤维细胞活性受到抑制,发生凋亡,减少了有效生成大量瘢痕组织的成纤维细胞数量,使后期为数不多的成纤维细胞不能产生过多的胶原纤维而造成瘢痕与硬膜的粘连,从而可以解决术后粘连带来的下腰椎手术失败综合症。
Objective: To elucidate the mechanism of mitomycin C prevention of peridural adhesion after laminectomy on inducing apoptosis of fibroblast
Methods The cultured fibroblasts were exposed for 12h with 0mg/ml,0.001 mg/ml,0.01mg/ml,0.05 mg/ml,0.1 mg/ml,0.2 mg/ml,0.3 mg/ml mitomycin C, respectively. The cell proliferation was detected by CCK-8 assay and Apoptotic cell death was analyzed by Hoechst 33342 stain. The expression of caspase-3 and the activation of ERK1/2, Akt ,Bad were analyzed by Western blot
Results Treatment of fibroblast with MMC induced a significant decreased of proliferation. fibroblasts exposed for 0.001 mg/ml to 0.2 mg/ml MMC, Staining with Hoechst 33342 MMC displayed nuclei with abnormal morphologies:“bean”-shaped nuclei with highly condensed chromatin or nuclei with irregular clumps of dense chromatin. 0.2mg/ml MMC,PD98059(ERK kinase inhibitor),LY294002(AKT kinase inhibitor) induced a significant increased of caspase-3 than control, accompanied by an increase in cell apoptosis.0.2mg/ml MMC PD98059,LY294002 caused decrease of p-ERK1/2 and p-Akt,at the same time, 0.2mg/ml MMC increased Bad than PD98059,LY294002 and control.
Conclusion 0.2 mg/ml MMC can induce fibroblast apoptosis by activation of caspase-3 in which requires inactivation of p-ERK1/2, p-Akt and increase the Bad. MMC can inhibit fibroblast proliferation and induce its apoptosis, which may be one of mechanism of that MMC prevent peridural adhesion after laminectomy
引文
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