北京大学学报(医学版)
北京大學學報(醫學版)
북경대학학보(의학판)
Journal of Peking University (Health Sciences)
2015年
5期
737-742
,共6页
贾双双%李伟阳%刘欣%李丽英
賈雙雙%李偉暘%劉訢%李麗英
가쌍쌍%리위양%류흔%리려영
转化生长因子β1%间充质细胞%细胞分化%活性氧%成纤维细胞
轉化生長因子β1%間充質細胞%細胞分化%活性氧%成纖維細胞
전화생장인자β1%간충질세포%세포분화%활성양%성섬유세포
Transforming growth factor beta 1%Mesenchymal stromal cells%Cell differentiation%Reac-tive oxygen species%Fibroblasts
目的:研究转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells ,BMSCs)向肌成纤维细胞分化的机制。方法:采用全骨髓贴壁培养法体外培养小鼠原代BMSCs ,将对数生长期的P3~P5代BMSCs作为实验细胞,使用不同浓度的TGF-β1诱导BMSCs向肌成纤维细胞分化,并在此基础上观察加入自由基清除剂 N-乙酰-半胱氨酸( N-acetylcysteine ,NAC)对其分化的影响。采用实时荧光定量PCR及Western blot技术检测BMSCs分化指标α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原[col-lagenα1(Ⅰ),Col α1(Ⅰ)]和Ⅲ型胶原[collagen α1(Ⅲ), Col α1(Ⅲ)]的表达情况。使用2’,7’-dichlorohydrofluorescein diacetate(DCFH-DA)预孵育BMSCs 15 min,然后加入TGF-β1处理不同时间,检测TGF-β1刺激下BMSCs中活性氧( reactive oxygen species ,ROS)的产生,并使用高内涵方法对BMSCs中产生的ROS进行统计分析。结果:TGF-β1可以剂量依赖地诱导BMSCs向肌成纤维细胞分化,上调α-SMA、Col α1(Ⅰ)和Col α1(Ⅲ)的表达。 TGF-β1诱导BMSCs分化的作用可以被NAC阻断。 TGF-β1在BMSCs中能够引起ROS的产生,且该过程迅速而短暂,当TGF-β1作用30 min时,其在BMSCs中诱发的ROS达到峰值。结论:TGF-β1通过产生ROS介导BMSCs向肌成纤维细胞分化。
目的:研究轉化生長因子-β1(transforming growth factor-β1,TGF-β1)誘導骨髓間充質榦細胞(bone marrow-derived mesenchymal stem cells ,BMSCs)嚮肌成纖維細胞分化的機製。方法:採用全骨髓貼壁培養法體外培養小鼠原代BMSCs ,將對數生長期的P3~P5代BMSCs作為實驗細胞,使用不同濃度的TGF-β1誘導BMSCs嚮肌成纖維細胞分化,併在此基礎上觀察加入自由基清除劑 N-乙酰-半胱氨痠( N-acetylcysteine ,NAC)對其分化的影響。採用實時熒光定量PCR及Western blot技術檢測BMSCs分化指標α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型膠原[col-lagenα1(Ⅰ),Col α1(Ⅰ)]和Ⅲ型膠原[collagen α1(Ⅲ), Col α1(Ⅲ)]的錶達情況。使用2’,7’-dichlorohydrofluorescein diacetate(DCFH-DA)預孵育BMSCs 15 min,然後加入TGF-β1處理不同時間,檢測TGF-β1刺激下BMSCs中活性氧( reactive oxygen species ,ROS)的產生,併使用高內涵方法對BMSCs中產生的ROS進行統計分析。結果:TGF-β1可以劑量依賴地誘導BMSCs嚮肌成纖維細胞分化,上調α-SMA、Col α1(Ⅰ)和Col α1(Ⅲ)的錶達。 TGF-β1誘導BMSCs分化的作用可以被NAC阻斷。 TGF-β1在BMSCs中能夠引起ROS的產生,且該過程迅速而短暫,噹TGF-β1作用30 min時,其在BMSCs中誘髮的ROS達到峰值。結論:TGF-β1通過產生ROS介導BMSCs嚮肌成纖維細胞分化。
목적:연구전화생장인자-β1(transforming growth factor-β1,TGF-β1)유도골수간충질간세포(bone marrow-derived mesenchymal stem cells ,BMSCs)향기성섬유세포분화적궤제。방법:채용전골수첩벽배양법체외배양소서원대BMSCs ,장대수생장기적P3~P5대BMSCs작위실험세포,사용불동농도적TGF-β1유도BMSCs향기성섬유세포분화,병재차기출상관찰가입자유기청제제 N-을선-반광안산( N-acetylcysteine ,NAC)대기분화적영향。채용실시형광정량PCR급Western blot기술검측BMSCs분화지표α-평활기기동단백(α-smooth muscle actin,α-SMA)、Ⅰ형효원[col-lagenα1(Ⅰ),Col α1(Ⅰ)]화Ⅲ형효원[collagen α1(Ⅲ), Col α1(Ⅲ)]적표체정황。사용2’,7’-dichlorohydrofluorescein diacetate(DCFH-DA)예부육BMSCs 15 min,연후가입TGF-β1처리불동시간,검측TGF-β1자격하BMSCs중활성양( reactive oxygen species ,ROS)적산생,병사용고내함방법대BMSCs중산생적ROS진행통계분석。결과:TGF-β1가이제량의뢰지유도BMSCs향기성섬유세포분화,상조α-SMA、Col α1(Ⅰ)화Col α1(Ⅲ)적표체。 TGF-β1유도BMSCs분화적작용가이피NAC조단。 TGF-β1재BMSCs중능구인기ROS적산생,차해과정신속이단잠,당TGF-β1작용30 min시,기재BMSCs중유발적ROS체도봉치。결론:TGF-β1통과산생ROS개도BMSCs향기성섬유세포분화。
Objective:The aim of this study was to investigate the mechanism underlying transforming growth factor-β1 ( TGF-β1 ) induced differentiation of bone marrow-derived mesenchymal stem cells (BMSCs)into myofibroblasts.Methods:Primary mouse BMSCs were isolated from bone marrow by flushing the tibias and femurs of mice , and passage 3 to passage 5 of BMSCs were used in the experiments . BMSCs differentiation into myofibroblast was induced by different doses of TGF-β1.In addition, reactive oxygen species (ROS) inhibitor (N-acetylcysteine, NAC) was added to test its effect on the action of TGF-β1.Expressions of BMSCs differentiation parameters , α-smooth muscle actin (α-SMA), collagenα1(Ⅰ) [Col α1(Ⅰ)] and collagen α1(Ⅲ) [Col α1(Ⅲ)] were measured by real-time quantitative PCR (RT-qPCR) and Western blot analysis.BMSCs were preloaded for 15 min with 2’, 7’-dichlorohydro-fluorescein diacetate ( DCFH-DA) , then stimulated with TGF-β1 for different times , and fluorescence of ROS was measured using high content analysis .Results:TGF-β1 stimulated differentiation of BMSCs into myofibroblasts and up-regulated expression of α-SMA, Col α1(Ⅰ) and Col α1(Ⅲ) in a dose-dependent manner , which blocked by ROS inhibitor NAC .In addition , TGF-β1 could induce a significant rapid and transient increase in ROS production in BMSCs , and the effect of TGF-β1 on ROS production was peaked at 30 min.Conclusion:TGF-β1 induced differentiation of BMSCs into myofibroblasts via production of ROS.