中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2010年
3期
204-208
,共5页
吴文杰%阳乔%曹芹芳%张耀文%夏雨佳%胡晓文%唐望先
吳文傑%暘喬%曹芹芳%張耀文%夏雨佳%鬍曉文%唐望先
오문걸%양교%조근방%장요문%하우가%호효문%당망선
癌,肝细胞%HepG2细胞%内源性大麻素
癌,肝細胞%HepG2細胞%內源性大痳素
암,간세포%HepG2세포%내원성대마소
Carcinoma,hepatocellular%HepG2 cell%Anandamide
目的 研究内源性大麻素(AEA)以脂质为基础的信号途径对肝癌细胞株HepG2的作用机制,探讨AEA在肝癌发生和发展中的作用.方法 免疫荧光检测脂肪酸水解酶、大麻素受体(CB)1和CB2在胎肝细胞株L02和肝癌细胞株HepG2中的定位.以不同浓度的AEA及膜胆固醇耗竭剂甲基-β-环糊精(MCD)处理,分为AEA 10 μmol/L组、AEA 20 μmol/L组、AEA40 μmol/L组,MCD 10 mmol/L+AEA 10 μmol/L组、MCD 10 mmol/L+AEA 20 μmol/L组和MCD 10 mmol/L+AEA 40 μmol/L组,分别孵育L02细胞和HepG2细胞,流式细胞术碘化丙啶单染法检测细胞的坏死率.Western Blot检测L02和HepG2细胞中脂肪酸水解酶、CB1和CB2的蛋白表达及其下游信号通路磷酸化P38促分裂原活化蛋白激酶(p-P38MAPK)和磷酸化c-Jun氨基端激酶(P-JNK)的表达变化.组间均数的比较采用独立样本t检验或单因素方差分析.结果 AEA可有效地导致肝癌细胞坏死,以浓度为AEA 40 μmol/L组达到最大效应,F=108.594,P<0.05,差异有统计学意义.MCD 10mmol/L预孵育后的HepG2细胞坏死率在AEA 10 μmol/L组、AEA 20 μmol/L组和AEA 40 μmol/L组分别为7.83%±2.13%,16.30%±0.94%,43.09%±5.10%,MCD处理前AEA 10 μmol/L组、AEA 20 μmol/L组、AEA 40 μmol/L组分别为13.64%±1.69%、20.28%±0.91%,52.71%±4.29%,处理前后比较,t值分别为3.702,5.274和3.503,P值均<0.05,差异均有统计学意义.同时,AEA能激活HepG2细胞中P38 MAPK和JNK,以AEA 40 μmol/L组作用明显,F值分别为11.908和26.054,P值均<0.05,差异均有统计学意义,MCD作用前p-P38 MAPK和p-JNK/β-肌动蛋白灰度值分别为1.63±0.06,1.60±0.31,MCD作用后p-P38 MAPK/β-肌动蛋白、p-JNK灰度值分别为1.14±0.01、1.17±0.29,作用前后相比,t值分别为2.801和12.829,P值均<0.05,差异均有统计学意义.结论 AEA可以有效地导致HepG2细胞坏死而对L02细胞无影响,AEA激活了HepG2细胞p-P38 MAPK和P-JNK相关信号传导途径,且此过程与脂筏有关.
目的 研究內源性大痳素(AEA)以脂質為基礎的信號途徑對肝癌細胞株HepG2的作用機製,探討AEA在肝癌髮生和髮展中的作用.方法 免疫熒光檢測脂肪痠水解酶、大痳素受體(CB)1和CB2在胎肝細胞株L02和肝癌細胞株HepG2中的定位.以不同濃度的AEA及膜膽固醇耗竭劑甲基-β-環糊精(MCD)處理,分為AEA 10 μmol/L組、AEA 20 μmol/L組、AEA40 μmol/L組,MCD 10 mmol/L+AEA 10 μmol/L組、MCD 10 mmol/L+AEA 20 μmol/L組和MCD 10 mmol/L+AEA 40 μmol/L組,分彆孵育L02細胞和HepG2細胞,流式細胞術碘化丙啶單染法檢測細胞的壞死率.Western Blot檢測L02和HepG2細胞中脂肪痠水解酶、CB1和CB2的蛋白錶達及其下遊信號通路燐痠化P38促分裂原活化蛋白激酶(p-P38MAPK)和燐痠化c-Jun氨基耑激酶(P-JNK)的錶達變化.組間均數的比較採用獨立樣本t檢驗或單因素方差分析.結果 AEA可有效地導緻肝癌細胞壞死,以濃度為AEA 40 μmol/L組達到最大效應,F=108.594,P<0.05,差異有統計學意義.MCD 10mmol/L預孵育後的HepG2細胞壞死率在AEA 10 μmol/L組、AEA 20 μmol/L組和AEA 40 μmol/L組分彆為7.83%±2.13%,16.30%±0.94%,43.09%±5.10%,MCD處理前AEA 10 μmol/L組、AEA 20 μmol/L組、AEA 40 μmol/L組分彆為13.64%±1.69%、20.28%±0.91%,52.71%±4.29%,處理前後比較,t值分彆為3.702,5.274和3.503,P值均<0.05,差異均有統計學意義.同時,AEA能激活HepG2細胞中P38 MAPK和JNK,以AEA 40 μmol/L組作用明顯,F值分彆為11.908和26.054,P值均<0.05,差異均有統計學意義,MCD作用前p-P38 MAPK和p-JNK/β-肌動蛋白灰度值分彆為1.63±0.06,1.60±0.31,MCD作用後p-P38 MAPK/β-肌動蛋白、p-JNK灰度值分彆為1.14±0.01、1.17±0.29,作用前後相比,t值分彆為2.801和12.829,P值均<0.05,差異均有統計學意義.結論 AEA可以有效地導緻HepG2細胞壞死而對L02細胞無影響,AEA激活瞭HepG2細胞p-P38 MAPK和P-JNK相關信號傳導途徑,且此過程與脂筏有關.
목적 연구내원성대마소(AEA)이지질위기출적신호도경대간암세포주HepG2적작용궤제,탐토AEA재간암발생화발전중적작용.방법 면역형광검측지방산수해매、대마소수체(CB)1화CB2재태간세포주L02화간암세포주HepG2중적정위.이불동농도적AEA급막담고순모갈제갑기-β-배호정(MCD)처리,분위AEA 10 μmol/L조、AEA 20 μmol/L조、AEA40 μmol/L조,MCD 10 mmol/L+AEA 10 μmol/L조、MCD 10 mmol/L+AEA 20 μmol/L조화MCD 10 mmol/L+AEA 40 μmol/L조,분별부육L02세포화HepG2세포,류식세포술전화병정단염법검측세포적배사솔.Western Blot검측L02화HepG2세포중지방산수해매、CB1화CB2적단백표체급기하유신호통로린산화P38촉분렬원활화단백격매(p-P38MAPK)화린산화c-Jun안기단격매(P-JNK)적표체변화.조간균수적비교채용독립양본t검험혹단인소방차분석.결과 AEA가유효지도치간암세포배사,이농도위AEA 40 μmol/L조체도최대효응,F=108.594,P<0.05,차이유통계학의의.MCD 10mmol/L예부육후적HepG2세포배사솔재AEA 10 μmol/L조、AEA 20 μmol/L조화AEA 40 μmol/L조분별위7.83%±2.13%,16.30%±0.94%,43.09%±5.10%,MCD처리전AEA 10 μmol/L조、AEA 20 μmol/L조、AEA 40 μmol/L조분별위13.64%±1.69%、20.28%±0.91%,52.71%±4.29%,처리전후비교,t치분별위3.702,5.274화3.503,P치균<0.05,차이균유통계학의의.동시,AEA능격활HepG2세포중P38 MAPK화JNK,이AEA 40 μmol/L조작용명현,F치분별위11.908화26.054,P치균<0.05,차이균유통계학의의,MCD작용전p-P38 MAPK화p-JNK/β-기동단백회도치분별위1.63±0.06,1.60±0.31,MCD작용후p-P38 MAPK/β-기동단백、p-JNK회도치분별위1.14±0.01、1.17±0.29,작용전후상비,t치분별위2.801화12.829,P치균<0.05,차이균유통계학의의.결론 AEA가이유효지도치HepG2세포배사이대L02세포무영향,AEA격활료HepG2세포p-P38 MAPK화P-JNK상관신호전도도경,차차과정여지벌유관.
Objective To study the effect of anandamide(AEA)on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer.Methods Localization of the fatty acid hydrolytic enzyme(FAAH),cannabinoid receptors1(CB1)and cannabinoid receptors2(CB2)proteins was detected in L02 and HepG2 cells using immunofluorescenee.L02 and HepG2 cells were treated with different concentrations of AEA and methyl-β-cyclodextrin,and the rates of cells necrosis were examined by PI stain.Meanwhile,the expression levels of FAAH,CB1 and CB2 receptor proteins,as well as P38 mitogen-activated protein kinase(p-P38 MAPK)and c-Jun-NH2-terminal kinase(p-JNK)proteins,were analyzed by Western blot.Results The FAAH,CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells.The expression level of FAAH protein was higher in HepG2 than in L02 cells.The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells.The expression level of CB2 receptor protein was high in both L02 and HepG2 cells.AEA treatment induced necrosis in HepG2 cells but not in L02 cells.Methyl-β-cyciodextrin treatment prevented necrosis in HepG2 cells(t=3.702;5.274;3.503,P < 0.05).The expression patterns of FAAH,CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot,which were consistent with the immunofluorescence results.AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependant manner in HepG2 cells (F=11.908;26.054,P < 0.05)and the increase can be partially by prevented by MCD(t=2.801;t=12.829,P < 0.05).Conclusion AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.