中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2009年
9期
678-682
,共5页
细胞增殖%纤连蛋白类%西罗莫司%成肌纤维细胞%结缔组织生长因子%ERK1/2信号通路
細胞增殖%纖連蛋白類%西囉莫司%成肌纖維細胞%結締組織生長因子%ERK1/2信號通路
세포증식%섬련단백류%서라막사%성기섬유세포%결체조직생장인자%ERK1/2신호통로
Cell proliferation%Fibronectins%Sirolimus%Myofibroblasts%Connective tissue growth factor%ERK1/2 signaling pathway
目的 探讨雷帕霉素(RAPA)对于结缔组织生长因子(CTGF)生物学作用的影响及可能机制.方法 分别用RAPA 20 μg/L和40 μg/L预处理成肌纤维细胞(MyoF)30min后再加入CTGF 100 μg/L,与单独加入CTGF(CTGF组)和未加任何刺激的MyoF(对照组)进行比较.采用BrdU掺入法检测细胞的增殖反应;用Western印迹法检测绌胞上清中纤连蛋白(FN)的水平及细胞ERK1/2信号的磷酸化.结果 与对照组相比,CTGF(100 μg/L)显著促进MyoF增殖(P<0.01),增加上清中FN的蛋白水平(P<0.05).与CTGF组相比,RAPA20 μg/L及40 μg/L预处理可使细胞增殖率显著下降,分别下降了62%和70%(均P<0.05),但两个剂量之间差异无统计学意义;使细胞上清中FN的蛋白水平分别下降了15%和44%,后者与CTGF组的差异有统计学意义(P<0.05).CTGF(100 μg/L)刺激10 min可敛MyoF的ERK1/2发生磷酸化,RAPA 40 μg/L预处理细胞30 min可显著降低CTGF诱导的ERK1/2磷酸化.以ERK1/2活化特异抑制剂PD98059(50 μmol/L)预处理30 min后,可以抑制CTGF诱导的细胞增殖效应(7%±5%比85%±7%,P<0.01)和FN的分泌效应(1.0±0.1比1.6±0.3,P<0.05).结论 RAPA具有部分抑制CTGF的促增殖和促细胞外基质分泌的作用,其作用可能通过抑制ERK1/2信号通路实现.
目的 探討雷帕黴素(RAPA)對于結締組織生長因子(CTGF)生物學作用的影響及可能機製.方法 分彆用RAPA 20 μg/L和40 μg/L預處理成肌纖維細胞(MyoF)30min後再加入CTGF 100 μg/L,與單獨加入CTGF(CTGF組)和未加任何刺激的MyoF(對照組)進行比較.採用BrdU摻入法檢測細胞的增殖反應;用Western印跡法檢測絀胞上清中纖連蛋白(FN)的水平及細胞ERK1/2信號的燐痠化.結果 與對照組相比,CTGF(100 μg/L)顯著促進MyoF增殖(P<0.01),增加上清中FN的蛋白水平(P<0.05).與CTGF組相比,RAPA20 μg/L及40 μg/L預處理可使細胞增殖率顯著下降,分彆下降瞭62%和70%(均P<0.05),但兩箇劑量之間差異無統計學意義;使細胞上清中FN的蛋白水平分彆下降瞭15%和44%,後者與CTGF組的差異有統計學意義(P<0.05).CTGF(100 μg/L)刺激10 min可斂MyoF的ERK1/2髮生燐痠化,RAPA 40 μg/L預處理細胞30 min可顯著降低CTGF誘導的ERK1/2燐痠化.以ERK1/2活化特異抑製劑PD98059(50 μmol/L)預處理30 min後,可以抑製CTGF誘導的細胞增殖效應(7%±5%比85%±7%,P<0.01)和FN的分泌效應(1.0±0.1比1.6±0.3,P<0.05).結論 RAPA具有部分抑製CTGF的促增殖和促細胞外基質分泌的作用,其作用可能通過抑製ERK1/2信號通路實現.
목적 탐토뢰파매소(RAPA)대우결체조직생장인자(CTGF)생물학작용적영향급가능궤제.방법 분별용RAPA 20 μg/L화40 μg/L예처리성기섬유세포(MyoF)30min후재가입CTGF 100 μg/L,여단독가입CTGF(CTGF조)화미가임하자격적MyoF(대조조)진행비교.채용BrdU참입법검측세포적증식반응;용Western인적법검측출포상청중섬련단백(FN)적수평급세포ERK1/2신호적린산화.결과 여대조조상비,CTGF(100 μg/L)현저촉진MyoF증식(P<0.01),증가상청중FN적단백수평(P<0.05).여CTGF조상비,RAPA20 μg/L급40 μg/L예처리가사세포증식솔현저하강,분별하강료62%화70%(균P<0.05),단량개제량지간차이무통계학의의;사세포상청중FN적단백수평분별하강료15%화44%,후자여CTGF조적차이유통계학의의(P<0.05).CTGF(100 μg/L)자격10 min가렴MyoF적ERK1/2발생린산화,RAPA 40 μg/L예처리세포30 min가현저강저CTGF유도적ERK1/2린산화.이ERK1/2활화특이억제제PD98059(50 μmol/L)예처리30 min후,가이억제CTGF유도적세포증식효응(7%±5%비85%±7%,P<0.01)화FN적분비효응(1.0±0.1비1.6±0.3,P<0.05).결론 RAPA구유부분억제CTGF적촉증식화촉세포외기질분비적작용,기작용가능통과억제ERK1/2신호통로실현.
Objective To investigate the inhibitory effect and associated mechanism of rapamycin on proliferation and extracellular matrix (ECM) secretion in myofibroblasts stimulated by connective tissue growth factor (CTGF). Methods Primary cultivated myofibroblasts were divided into 6 groups: control, CTGF (100 μg/L), rapamycin 20 μg/L+CTGF 100 μg/L, rapamycin 40 μg/L +CTGF 100 μg/L, rapamycin 20 μg/L, and rapamycin 40 μg/L alone. 5'-bromodeoxyuridine (BrdU) incorporation assay was used to detect the myofibroblast proliferation.Western blot was used to analysis the secretory FN protein in the supernatant medium of cultured myofibroblasts and the ERK1/2 phosphorylation in myofibroblasts. Results CTGF (100 μg/L)incubation significantly increased the number of Brdu positive myofibroblasts(P<0.01) and the level of FN protein secretory (P<0.05) in cell supernatant medium compared with control group,respectively. The number of Brdu positive myofibroblasts markedly decreased by 62% and 70% (P <0.05) in rapamycin 20 μg/L+CTGF 100 μg/L and rapamycin 40 μg/L+CTGF 100 μg/L groups, respectively. The FN protein levels in supernatant were decreased by 15% and 44% compared with CTGF 100 μg/L group, respectively; but the difference of FN protein levels was significant only in rapamycin 40 μg/L group (P<0.05). CTGF could activate ERK1/2 at 10 minutes; but as myofibroblasts were pretreated with rapamycin 40 μg/L for 30 min, it abolished CTGF-induced ERK1/2 phosphoralation. PD98059, the specific inhibitor of ERK1/2, could block the effect of CTGF-induced proliferation (7%±5% vs 85%±7%, P<0.01) and FN secretion (1.0±0.1 vs 1.6±0.3, P<0.05). Conclusions Rapamycin partially suppresses the proliferation and ECM secretion of myofibroblasts induced by CTGF. Its effect may be through inhibiting CTGF-induced activation of ERKI/2 signaling pathway.
