他克莫司影响成纤维细胞溶酶体功能与胶原生成的研究  

Effects of tacrolimus on lysosomal function and collagen formation in fibroblasts

作　　者：周凌 刘泽明 周伟[1] 陈丹洋 郭亮 ZHOU Ling;LIU Ze-ming;ZHOU Wei;CHEN Dan-yang;GUO Liang(Department of Plastic and Cosmetic,Zhongnan Hospital,Wuhan University,Wuhan 430071,Hubei Province,China)

机构地区：[1]武汉大学中南医院整形美容科

出　　处：《中国临床药理学杂志》2019年第24期3197-3200,共4页The Chinese Journal of Clinical Pharmacology

基　　金：湖北省自然科学基金面上基金资助项目(2018CKB904)

摘　　要：目的 探讨他克莫司对转化生长因子-β1(TGF-β1)诱导后成纤维细胞胶原生成的影响及其机制。方法 将人皮肤成纤维细胞分为4组:空白组、模型组、空白他克莫司组和模型他克莫司组。空白组细胞不做任何处理,模型组细胞用10 ng·m L-1TGF-β1处理24 h,空白他克莫司组用200 nmol·L-1他克莫司处理24 h,模型他克莫司组同时加入10 ng·m L-1TGF-β1和200 nmol·L-1他克莫司处理24 h。用比色法测定8种不同浓度(0为空白组,25,50,100,200,400,800,1600 nmol·L-1)他克莫司作用下成纤维细胞乳酸脱氢酶(LDH)释放量。以免疫印迹实验检测自噬相关蛋白、内质网凋亡蛋白、Ⅰ型胶原蛋白(COLⅠ)和转录因子EB(TFEB)的表达。结果 8种不同浓度他克莫司作用后,成纤维细胞LDH释放量分别为(99. 33±5. 52)%,(99. 32±4. 40)%,(99. 32±4. 95)%,(98. 66±4. 45)%,(98. 80±6. 31)%,(98. 51±4. 35)%,(106. 29±6. 03)%和(121. 36±8. 24)%;800 nmol·L-1以下浓度的他克莫司作用后,成纤维细胞LDH释放率与空白组比较,差异无统计学意义(P> 0. 05)。空白组、模型组和他克莫司模型组的LC3Ⅱ表达比值分别为1. 00±0. 23,3. 41±0. 25和4. 45±0. 44;这3组的溶酶体相关膜蛋白1(LAMP1)表达比值分别为1. 00±0. 25,3. 90±0. 43和2. 51±0. 22;这3组的内质网凋亡蛋白糖调节蛋白78(GRP78)表达比值为1. 00±0. 21,1. 94±0. 28和3. 10±0. 29;这3组的Caspase 3蛋白比值分别为1. 00±0. 22,1. 27±0. 38和3. 16±0. 24;这3组的COLⅠ蛋白表达比值分别为1. 00±0. 29,3. 26±0. 34和2. 09±0. 31;这3组的TFEB蛋白表达比值为1. 00±0. 26,2. 94±0. 44和1. 54±0. 19。模型组与空白组相比,诱导成纤维细胞自噬水平上调和TFEB蛋白表达增加,促进TFEB核易位和胶原沉积,上述蛋白表达差异均有统计学意义(均P <0. 01)。他克莫司降低溶酶体功能,增加内质网应激,造成细胞凋亡,减少TFEB蛋白表达,降低胞质及胞核内TFEB荧光强度,抑制胶原沉积,与模型组相Objective To investigate the inhibitory effect of tacrolimus on collagen production of fibroblasts induced by transforming growth factor-β1(TGF-β1)and its possible mechanism.Methods Human skin fibroblasts cells were divided into 4 groups:blank group,model group,tacrolimus-blank group and tacrolimus-model group.Cells in the blank group were not treated with any treatment.Cells in the model group were treated with 10 ng·m L-1 TGF-β1 for 24 h,cells in the tacrolimus-blank group were treated with 200 nmol·L-1 tacrolimus for 24 h,and cells in the tacrolimus-model group were treated with 10 ng·mL-1 TGF-β1 and 200 nmol·L-1 tacrolimus for 24 h.Lactic dehydrogenase(LDH)release of fibroblast under different concentrations(blank group with 0,and 25,50,100,200,400,800,1600 nmol·L-1)of tacrolimus were determined by colorimetry.The expression of autophagy related proteins,endoplasmic reticulum apoptosis protein,collagenⅠ(COLⅠ)and transcription factor EB(TFEB)were studied by Western blotting.Results LDH release of fibroblast treated with 8 kinds of concentrations of tacrolimus were(99.33±5.52)%(blank),(99.32±4.40)%,(99.32±4.95)%,(98.66±4.45)%,(98.80±6.31)%,(98.51±4.35)%,(106.29±6.03)%and(121.36±8.24)%,respectively.The LDH release rate of fibroblasts treated with tacrolimus at a concentration of less than 800 nmol·L-1 was not statistically significant different from that of the blank group(P>0.05).The relative expression of LC3Ⅱin fibroblasts in blank group,model group and tacrolimus-model group were 1.00±0.23,3.41±0.25 and 4.45±0.44.The relative expression of lysosome-associated membrane protein1(LAMP1)in the 3 groups were 1.00±0.25,3.90±0.43 and 2.51±0.22.The relative expression of glucose-regulated protein 78(GRP78)in the 3 groups were 1.00±0.21,1.94±0.28 and 3.10±0.29.The relative expression of Caspase 3 in the3 groups were 1.00±0.22,1.27±0.38 and 3.16±0.24.The relative expression of COLⅠin the 3 groups were1.00±0.29,3.26±0.34 and 2.09±0.31.The relative expression of TFEB in the 3 gro

关 键 词：他克莫司 成纤维细胞 转化生长因子Β1 转录因子EB 

分 类 号：R97[医药卫生—药品]