中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年
20期
3715-3722
,共8页
周代星%李智慧%占成业%张莉伟
週代星%李智慧%佔成業%張莉偉
주대성%리지혜%점성업%장리위
组织构建%组织构建与中医药%丹参酮ⅡA%心肌成纤维细胞%心肌纤维化%转化生长因子β1%Smad3%Smad7%结缔组织生长因子%Ⅰ型胶原%信号通路%细胞培养%省级基金
組織構建%組織構建與中醫藥%丹參酮ⅡA%心肌成纖維細胞%心肌纖維化%轉化生長因子β1%Smad3%Smad7%結締組織生長因子%Ⅰ型膠原%信號通路%細胞培養%省級基金
조직구건%조직구건여중의약%단삼동ⅡA%심기성섬유세포%심기섬유화%전화생장인자β1%Smad3%Smad7%결체조직생장인자%Ⅰ형효원%신호통로%세포배양%성급기금
背景:转化生长因子β-Smads信号通路是心肌纤维化的关键通路;丹参酮ⅡA具有抑制心肌纤维化作用.目的:验证丹参酮ⅡA对转化生长因子β1致心肌纤维化的作用并探讨其机制.方法:取出生24 h内的SD大鼠心脏,利用酶消化法结合差速贴壁体外培养心肌成纤维细胞.取上述第3代心肌成纤维细胞,用5μg/L转化生长因子β1培养15,30,60,120 min或6,12,24 h后收集细胞,用不同浓度(10-5 mol/L、10-4结果与结论:转化生长因子β1在一定范围内以时间依赖方式诱导结缔组织生长因子、Ⅰ型胶原、磷酸化Smad3及Smad7的表达,刺激终末结缔组织生长因子、Ⅰ型胶原mRNA的表达量明显上升(均P <0.01);磷酸化Smad3及Smad7蛋白表达量在刺激后1 h达到峰值,表达量显著增加(均P <0.01).高浓度丹参酮ⅡA预处理可显著下调磷酸化Smad3、结缔组织生长因子及Ⅰ型胶原表达(均P <0.01).两种浓度的丹参酮ⅡA预处理均可上调Smad7蛋白表达(P <0.05,P <0.01).提示丹参酮ⅡA对心肌纤维化有抑制作用,可能与其上调Smad7蛋白表达,抑制转化生长因子β1诱导的Smad3磷酸化,部分阻断转化生长因子β1-Smads信号通路有关. mol/L)丹参酮ⅡA预处理2 h后再加入5μg/L转化生长因子β1培养120 min或24 h后收集细胞,并设空白对照组.免疫细胞化学染色法进行细胞鉴定,反转录聚合酶链反应检测结缔组织生长因子及Ⅰ型胶原蛋白mRNA表达,Western Blot检测Smad7及磷酸化Smad3蛋白表达.免疫组织化学染色及免疫荧光法检测磷酸化Smad3、结缔组织生长因子及Ⅰ型胶原蛋白表达.
揹景:轉化生長因子β-Smads信號通路是心肌纖維化的關鍵通路;丹參酮ⅡA具有抑製心肌纖維化作用.目的:驗證丹參酮ⅡA對轉化生長因子β1緻心肌纖維化的作用併探討其機製.方法:取齣生24 h內的SD大鼠心髒,利用酶消化法結閤差速貼壁體外培養心肌成纖維細胞.取上述第3代心肌成纖維細胞,用5μg/L轉化生長因子β1培養15,30,60,120 min或6,12,24 h後收集細胞,用不同濃度(10-5 mol/L、10-4結果與結論:轉化生長因子β1在一定範圍內以時間依賴方式誘導結締組織生長因子、Ⅰ型膠原、燐痠化Smad3及Smad7的錶達,刺激終末結締組織生長因子、Ⅰ型膠原mRNA的錶達量明顯上升(均P <0.01);燐痠化Smad3及Smad7蛋白錶達量在刺激後1 h達到峰值,錶達量顯著增加(均P <0.01).高濃度丹參酮ⅡA預處理可顯著下調燐痠化Smad3、結締組織生長因子及Ⅰ型膠原錶達(均P <0.01).兩種濃度的丹參酮ⅡA預處理均可上調Smad7蛋白錶達(P <0.05,P <0.01).提示丹參酮ⅡA對心肌纖維化有抑製作用,可能與其上調Smad7蛋白錶達,抑製轉化生長因子β1誘導的Smad3燐痠化,部分阻斷轉化生長因子β1-Smads信號通路有關. mol/L)丹參酮ⅡA預處理2 h後再加入5μg/L轉化生長因子β1培養120 min或24 h後收集細胞,併設空白對照組.免疫細胞化學染色法進行細胞鑒定,反轉錄聚閤酶鏈反應檢測結締組織生長因子及Ⅰ型膠原蛋白mRNA錶達,Western Blot檢測Smad7及燐痠化Smad3蛋白錶達.免疫組織化學染色及免疫熒光法檢測燐痠化Smad3、結締組織生長因子及Ⅰ型膠原蛋白錶達.
배경:전화생장인자β-Smads신호통로시심기섬유화적관건통로;단삼동ⅡA구유억제심기섬유화작용.목적:험증단삼동ⅡA대전화생장인자β1치심기섬유화적작용병탐토기궤제.방법:취출생24 h내적SD대서심장,이용매소화법결합차속첩벽체외배양심기성섬유세포.취상술제3대심기성섬유세포,용5μg/L전화생장인자β1배양15,30,60,120 min혹6,12,24 h후수집세포,용불동농도(10-5 mol/L、10-4결과여결론:전화생장인자β1재일정범위내이시간의뢰방식유도결체조직생장인자、Ⅰ형효원、린산화Smad3급Smad7적표체,자격종말결체조직생장인자、Ⅰ형효원mRNA적표체량명현상승(균P <0.01);린산화Smad3급Smad7단백표체량재자격후1 h체도봉치,표체량현저증가(균P <0.01).고농도단삼동ⅡA예처리가현저하조린산화Smad3、결체조직생장인자급Ⅰ형효원표체(균P <0.01).량충농도적단삼동ⅡA예처리균가상조Smad7단백표체(P <0.05,P <0.01).제시단삼동ⅡA대심기섬유화유억제작용,가능여기상조Smad7단백표체,억제전화생장인자β1유도적Smad3린산화,부분조단전화생장인자β1-Smads신호통로유관. mol/L)단삼동ⅡA예처리2 h후재가입5μg/L전화생장인자β1배양120 min혹24 h후수집세포,병설공백대조조.면역세포화학염색법진행세포감정,반전록취합매련반응검측결체조직생장인자급Ⅰ형효원단백mRNA표체,Western Blot검측Smad7급린산화Smad3단백표체.면역조직화학염색급면역형광법검측린산화Smad3、결체조직생장인자급Ⅰ형효원단백표체.
@@@@BACKGROUND: Transforming growth factor β-Smads is the critical path of cardiac fibrosis, and tanshinone ⅡA can limit cardiac fibrosis. OBJECTIVE: To explore the effect of tanshinone ⅡA on the cardiac fibrosis induced by transforming growth factor-β1 and the possible mechanisms. METHODS: Cardiac fibroblasts were isolated from cardiac tissues of neonatal Sprague-Dawley rats by the trypsin digestion and differential adhesion method. Passage 3 cel s were treated with 5 μg/L transforming growth factor β1 alone or pretreated with tanshinone ⅡA at different concentrations (10-5 mol/L and 10-4 RESULTS AND CONCLUSION: Transforming growth factor-β1 induced the expression of connective tissue growth factor, type Ⅰ col agen, phosphorylated Smads and Smad7 in a time-dependant manner. The mRNA expression of connective tissue growth factor and type Ⅰ col agen was significantly increased 24 hours after transforming growth factor-β1 stimulation (P < 0.01 for al ). The protein expression of phosphorylated Smad3 and Smad7 reached a peak 1 hour after transforming growth factor-β1 stimulation, much higher than the baseline level (P < 0.01 for al ). Pretreatment with high concentration of tanshinone ⅡA resulted in a decrease in the expression of phosphorylated Smad3, connective tissue growth factor and type Ⅰ col agen (P < 0.01). The protein expression of Smad7 was substantial y upregulated after pretreatment with two concentrations of tanshinone ⅡA as compared with that in the cel s at 2 hours post transforming growth factor-β1 stimulation (P < 0.05, P < 0.01). Tanshinone ⅡA may exert an inhibitory effect on cardiac fibrosis by upregulating the expression of Smad7, suppressing the transforming growth factor-β1-induced phosphorylated Smad3 and partial y blocking the transforming growth factor-β1-Smads signaling pathway. mol/L). Immunocytochemistry was used for cel identification, reverse transcription-PCR for detection of the mRNA expression of connective tissue growth factor and type Ⅰ col agen, western blotting for detection of the protein expression of Smad7 and phosphorylated Smad3, and immunohistochemistry and immunofluorescence for detection of the protein expression of phosphorylated Smad3, connective tissue growth factor and type Ⅰ col agen.
