中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
1期
42-46
,共5页
刘萍%王密%陆小丹%张淑娟%唐望先
劉萍%王密%陸小丹%張淑娟%唐望先
류평%왕밀%륙소단%장숙연%당망선
肝硬化%血吸虫病%细胞增殖%内源性大麻酚类%肝星状细胞%血吸虫肝纤维化%P-Erk
肝硬化%血吸蟲病%細胞增殖%內源性大痳酚類%肝星狀細胞%血吸蟲肝纖維化%P-Erk
간경화%혈흡충병%세포증식%내원성대마분류%간성상세포%혈흡충간섬유화%P-Erk
Liver cirrhosis%Schistosomasis%Cell proliferation%Endocannabinoids%Hepatic stellate cells%Schistosome-induced liver fibrosis%Phosphorylation-Erk
目的 观察内源性大麻素(AEA)对血吸虫肝纤维化小鼠原代肝星状细胞(HSC)增殖及磷酸化-Erk(P-Erk)的影响,为大麻素在肝纤维化领域的研究提供理论依据. 方法 (1)腹部皮肤贴敷尾蚴建立血吸虫肝纤维化模型；(2) HE和Masson染色证实肝纤维化形成；(3)非连续密度梯度离心法提取原代HSC；(4)α平滑肌肌动蛋白(α-SMA)和结蛋白免疫荧光双重染色鉴定原代HSC；(5)二甲基偶氮唑盐法检测AEA对HSC增殖的影响；(6)Western blot检测AEA对HSC磷酸化-Erk表达的影响.多个样本的均数比较采用单因素方差分析,若方差齐则采用LSD检验,若方差不齐采用Dunnett T3检验.结果 (1)成功建立血吸虫肝纤维化模型:HE染色显示嗜酸性肉芽肿形成,Masson染色显示出现围绕血管的胶原纤维沉积；(2)成功提取高纯度的原代HSC:纯度约为95％；(3)AEA能呈浓度依赖性地抑制HSC的增殖:5、10、20、40、60、80、100、120 μmol/L组AEA对HSC的抑制率分别为5.14％±1.61％、10.34％±3.22％、14.12％±3.98％、18.64％±3.67％、23.86％±3.06％、62.81％±7.75％、70.39％±7.11％、72.31％±6.71％；(4)AEA能呈浓度和时间依赖性地增加P-Erk的表达:20、60、120 μ mol/L组的平均灰度值分别为39.90±4.61、43.45±0.91、52.91±1.97,与阴性对照组(19.21±1.60)相比,P值均＜0.05；在15、30 min,1、3、6、12、24、48h的平均灰度值分别为85.05±15.80、103.41±11.89,118.02±12.24、109.17±15.69、100.86±10.55、71.70±12.87、34.62±14.85、22.84±11.73,与阴性对照组(19.21±1.60)相比,在15、30min,1、3、6、12h,6个时间点比较,P值均＜0.05.结论 内源性大麻素AEA能通过激活Erk信号通路,抑制血吸虫肝纤维化小鼠原代HSC的增殖.
目的 觀察內源性大痳素(AEA)對血吸蟲肝纖維化小鼠原代肝星狀細胞(HSC)增殖及燐痠化-Erk(P-Erk)的影響,為大痳素在肝纖維化領域的研究提供理論依據. 方法 (1)腹部皮膚貼敷尾蚴建立血吸蟲肝纖維化模型；(2) HE和Masson染色證實肝纖維化形成；(3)非連續密度梯度離心法提取原代HSC；(4)α平滑肌肌動蛋白(α-SMA)和結蛋白免疫熒光雙重染色鑒定原代HSC；(5)二甲基偶氮唑鹽法檢測AEA對HSC增殖的影響；(6)Western blot檢測AEA對HSC燐痠化-Erk錶達的影響.多箇樣本的均數比較採用單因素方差分析,若方差齊則採用LSD檢驗,若方差不齊採用Dunnett T3檢驗.結果 (1)成功建立血吸蟲肝纖維化模型:HE染色顯示嗜痠性肉芽腫形成,Masson染色顯示齣現圍繞血管的膠原纖維沉積；(2)成功提取高純度的原代HSC:純度約為95％；(3)AEA能呈濃度依賴性地抑製HSC的增殖:5、10、20、40、60、80、100、120 μmol/L組AEA對HSC的抑製率分彆為5.14％±1.61％、10.34％±3.22％、14.12％±3.98％、18.64％±3.67％、23.86％±3.06％、62.81％±7.75％、70.39％±7.11％、72.31％±6.71％；(4)AEA能呈濃度和時間依賴性地增加P-Erk的錶達:20、60、120 μ mol/L組的平均灰度值分彆為39.90±4.61、43.45±0.91、52.91±1.97,與陰性對照組(19.21±1.60)相比,P值均＜0.05；在15、30 min,1、3、6、12、24、48h的平均灰度值分彆為85.05±15.80、103.41±11.89,118.02±12.24、109.17±15.69、100.86±10.55、71.70±12.87、34.62±14.85、22.84±11.73,與陰性對照組(19.21±1.60)相比,在15、30min,1、3、6、12h,6箇時間點比較,P值均＜0.05.結論 內源性大痳素AEA能通過激活Erk信號通路,抑製血吸蟲肝纖維化小鼠原代HSC的增殖.
목적 관찰내원성대마소(AEA)대혈흡충간섬유화소서원대간성상세포(HSC)증식급린산화-Erk(P-Erk)적영향,위대마소재간섬유화영역적연구제공이론의거. 방법 (1)복부피부첩부미유건립혈흡충간섬유화모형；(2) HE화Masson염색증실간섬유화형성；(3)비련속밀도제도리심법제취원대HSC；(4)α평활기기동단백(α-SMA)화결단백면역형광쌍중염색감정원대HSC；(5)이갑기우담서염법검측AEA대HSC증식적영향；(6)Western blot검측AEA대HSC린산화-Erk표체적영향.다개양본적균수비교채용단인소방차분석,약방차제칙채용LSD검험,약방차불제채용Dunnett T3검험.결과 (1)성공건립혈흡충간섬유화모형:HE염색현시기산성육아종형성,Masson염색현시출현위요혈관적효원섬유침적；(2)성공제취고순도적원대HSC:순도약위95％；(3)AEA능정농도의뢰성지억제HSC적증식:5、10、20、40、60、80、100、120 μmol/L조AEA대HSC적억제솔분별위5.14％±1.61％、10.34％±3.22％、14.12％±3.98％、18.64％±3.67％、23.86％±3.06％、62.81％±7.75％、70.39％±7.11％、72.31％±6.71％；(4)AEA능정농도화시간의뢰성지증가P-Erk적표체:20、60、120 μ mol/L조적평균회도치분별위39.90±4.61、43.45±0.91、52.91±1.97,여음성대조조(19.21±1.60)상비,P치균＜0.05；재15、30 min,1、3、6、12、24、48h적평균회도치분별위85.05±15.80、103.41±11.89,118.02±12.24、109.17±15.69、100.86±10.55、71.70±12.87、34.62±14.85、22.84±11.73,여음성대조조(19.21±1.60)상비,재15、30min,1、3、6、12h,6개시간점비교,P치균＜0.05.결론 내원성대마소AEA능통과격활Erk신호통로,억제혈흡충간섬유화소서원대HSC적증식.
Objective To investigate the potential therapeutic properties of the endogenous cannabinoid N-arachidonic acid aminoethanols (anandamide,AEA) in liver fibrosis by observing its affects on proliferation of and expression of phosphorylated-Erk (pErk) in primary hepatic stellate cells (HSCs) from a mouse model of schistosome-induced liver fibrosis.Methods The schistosome-induced liver fibrosis model was established by attaching cercaria to the skin on the ventral side of the mouse and allowing infection to occur via direct penetration.Six weeks later,the model was confirmed by pathological analysis of liver,with Masson trichrome staining showing collagen fiber deposition around the blood vessels and hematoxylin-eosin staining showing eosinophilic granuloma formation.Primary HSCs were isolated by discontinuous density gradient centrifugation,confirmed by immunofluorescence detection of double-staining for n-smooth muscle actin and desmin (95％ purity),and cultured in the presence of absence of various concentrations of AEA.Proliferative ability was evaluated by MTT assay and the expression of pErk was observed by Westem blotting.Results AEA treatment inhibited the proliferation of the primary HSCs in a concentration-dependent manner (AEA:5μmol/L,inhibition:7.68％; 10 μmol/L,11.65％; 20μmol/L,14.70％; 40μmol/L,15.07％; 60 μmol/L,18.18％;80 μmol/L,20.26％; 100 μmol/L,20.17％; 120 μmol/L,29.24％).AEA treatment increased pERK expression in both a concentration-dependent manner (AEA:20 μmol/L,average gray value:39.90 ± 4.61; 60 μmol/L,43.45 ± 0.91; 120 μrnol/L,52.91 ± 1.97; vs.negative control,allP＜ 0.05) and a time-dependent manner (time:15 min,average gray value:85.05 ± 15.80; 30 min,103.41 ± 11.89; 1 h,118.02 ± 12.24; 3 h,109.17 ± 15.69;6h,100.86 ± 10.55; 12h,71.70 ± 12.87; 24h,34.62 ± 14.85; 48 h,22.84 ± 11.73; vs.negative control,all except 48 h had P ＜ 0.05).Conclusion AEA can suppress the proliferative capacity of primary HSCs from schistosome-induced fibrotic livers through activation of the Erk signaling pathway.
