CAJ | 학술논문

目的：探讨瘦素（leptin）对大鼠肝纤维化星状细胞（HSC）的作用及相关信号转导机制。方法采用改良原位灌注、Optiprep密度梯度离心法分离纯化大鼠HSC。通过台盼蓝拒染试验评估细胞存活率，α-平滑肌肌动蛋白（α- SMA）免疫组化鉴定，光镜观察形态学变化。将40只SD大鼠分为4个处理组：对照组、血管紧张素Ⅱ（AngⅡ）组（加入10-7mol/L AngⅡ）、Leptin组（加入100ng/ml Leptin）、Leptin+AngⅡ组（加入100ng/ml Leptin+10-7mol/L AngⅡ）。分别采用3H- TdR和3H- Pro掺入法进行HSC增殖和胶原合成的测定。Western blot检测各处理组p- ERK1/ERK1、p- ERK2/ERK2、AngⅡ蛋白表达情况。结果大鼠HSC存活率＞90%，传代1次后HSC纯度＞95%。与对照组相比，AngⅡ组、Leptin组、Leptin+AngⅡ组的HSC增殖和胶原合成均明显增加（均P＜0.05）；与AngⅡ组、Leptin组相比，Leptin+AngⅡ组HSC增殖和胶原合成明显增加（均P＜0.05）。Western blot显示，AngⅡ组、Leptin组、Leptin+AngⅡ组的p- ERK1/ERK1、p- ERK2/ERK2、AngⅡ蛋白水平均较对照组明显升高（均P＜0.05）；与AngⅡ组、Lep-tin组相比，Leptin+AngⅡ组p- ERK1/ERK1、p- ERK2/ERK2、AngⅡ蛋白水平明显升高（均P＜0.05）。结论瘦素可通过上调AngⅡ水平，激活ERK信号转导通路，刺激HSC增殖和胶原合成，导致肝纤维化。
목적：탐토수소（leptin）대대서간섬유화성상세포（HSC）적작용급상관신호전도궤제。방법채용개량원위관주、Optiprep밀도제도리심법분리순화대서HSC。통과태반람거염시험평고세포존활솔，α-평활기기동단백（α- SMA）면역조화감정，광경관찰형태학변화。장40지SD대서분위4개처리조：대조조、혈관긴장소Ⅱ（AngⅡ）조（가입10-7mol/L AngⅡ）、Leptin조（가입100ng/ml Leptin）、Leptin+AngⅡ조（가입100ng/ml Leptin+10-7mol/L AngⅡ）。분별채용3H- TdR화3H- Pro참입법진행HSC증식화효원합성적측정。Western blot검측각처리조p- ERK1/ERK1、p- ERK2/ERK2、AngⅡ단백표체정황。결과대서HSC존활솔＞90%，전대1차후HSC순도＞95%。여대조조상비，AngⅡ조、Leptin조、Leptin+AngⅡ조적HSC증식화효원합성균명현증가（균P＜0.05）；여AngⅡ조、Leptin조상비，Leptin+AngⅡ조HSC증식화효원합성명현증가（균P＜0.05）。Western blot현시，AngⅡ조、Leptin조、Leptin+AngⅡ조적p- ERK1/ERK1、p- ERK2/ERK2、AngⅡ단백수평균교대조조명현승고（균P＜0.05）；여AngⅡ조、Lep-tin조상비，Leptin+AngⅡ조p- ERK1/ERK1、p- ERK2/ERK2、AngⅡ단백수평명현승고（균P＜0.05）。결론수소가통과상조AngⅡ수평，격활ERK신호전도통로，자격HSC증식화효원합성，도치간섬유화。
Objective To investigate the effect of leptin on hepatic stel ate cells(HSC) in liver fibrosis rats and its relation to ERK signal transduction pathway. Methods The purified HSCs were obtained by the modified in situ perfusion and Optiprep density gradient centrifugation. The survival rate of HSCs was evaluated by trypan blue exclusion test, the identification of HSCs was tested by α- SMA immunocytochemical staining, and the morphological changes were observed by light microscope. The cultured HSCs were divided into four groups: the control group, the angiotensin II group (Ang II, 10-7mol/L), the Leptin group (Leptin, 100ng/ml), and the Leptin+Ang II group [Leptin (100ng/ml)+AngⅡ(10-7mol/L)]. The HSCs proliferation and col agen syn-thesis were measured by 3H- TdR and 3H- Pro incorporation methods. The protein levels of p- ERK1/ERK1, p- ERK2/ERK2 and Ang II were detected by Western blot. Results The survival rate of rat HSCs was>90%and the purity of HSCs was>95%after passage 1. The findings of HSCs proliferation and col agen synthesis showed that compared with control group, the proliferation and col agen synthesis of HSCs were significantly increased in the Ang II, Leptin and Leptin+Ang II groups (P<0.05);compared with the Ang II, Leptin groups, the HSCs proliferation and col agen synthesis were significantly increased in leptin+Ang II group (P<0.05). Western blot analysis showed that compared with the control group, the protein levels of p- ERK1/ERK1, p- ERK2/ERK2 and Ang II were significantly increased in Ang II, Leptin and Leptin+Ang II groups(P<0.05);compared with Ang II, Leptin groups, the protein levels of p- ERK1/ERK1, p- ERK2/ERK2 and Ang II were significantly increased in Leptin+Ang II group (P<0.05). Conclusion Leptin may up- regulate the Ang II levels, activate the ERK signal transduction pathway, stimulate the HSCs prolifera-tion and col agen synthesis, and then result in the liver fibrosis.