CAJ | 학술논문

目的应用黏着斑激酶相关非激酶(FRNK)表达质粒瞬时转染纤维连接蛋白(FN)刺激的肝星状细胞(HSC),探讨膜型基质金属蛋白酶1(MT1-MMP)在FRNK诱导HSC凋亡中的作用.方法在体外,以FN刺激HSC增殖,采用脂质体介导的方法用FRNK表达质粒瞬时转染HSC,应用膜联蛋白/碘化丙啶双标记流式细胞术和透射电镜技术检测细胞的凋亡,蛋白免疫印迹及RT-PCR方法检测FRNK、FAK、p-FAK(Tyr397)、MT1-MMP蛋白及其mRNA表达.结果 FRNK表达质粒成功转染HSC,在翻译后水平抑制FAK磷酸化.与空质粒组比较,FRNK表达质粒转染HSC 48小时后,HSC凋亡率由(9.28±1.05)%增加至(25.37±1.92)0A(P<0.01).FRNK抑制FAK磷酸化后在翻译和转录水平上调MT1-MMP表达,2.26±0.14 vs 1.09±0.15(P<0.01);1.58±0.18 vs1.00±0.10(P<0.01).结论在脂质体介导下瞬时转粢FRNK表达质粒可诱导HSC发生凋亡,上调MT1-MMP可能是其机制之一.
목적 응용점착반격매상관비격매(FRNK)표체질립순시전염섬유련접단백(FN)자격적간성상세포(HSC),탐토막형기질금속단백매1(MT1-MMP)재FRNK유도HSC조망중적작용.방법 재체외,이FN자격HSC증식,채용지질체개도적방법용FRNK표체질립순시전염HSC,응용막련단백/전화병정쌍표기류식세포술화투사전경기술검측세포적조망,단백면역인적급RT-PCR방법검측FRNK、FAK、p-FAK(Tyr397)、MT1-MMP단백급기mRNA표체.결과 FRNK표체질립성공전염HSC,재번역후수평억제FAK린산화.여공질립조비교,FRNK표체질립전염HSC 48소시후,HSC조망솔유(9.28±1.05)%증가지(25.37±1.92)0A(P<0.01).FRNK억제FAK린산화후재번역화전록수평상조MT1-MMP표체,2.26±0.14 vs 1.09±0.15(P<0.01);1.58±0.18 vs1.00±0.10(P<0.01).결론 재지질체개도하순시전자FRNK표체질립가유도HSC발생조망,상조MT1-MMP가능시기궤제지일.
Objective To investigate the effect of breaking the phosphorylation of focal adhesion kinase(FAK) by FAK related non-kinase(FRNK) on the apoptosis and the expression of membrane-type matrix metalloproteinase-1 (MT1-MMP) in hepatic stellate cell(HSC) in vitro. Methods After fibronectin(FN) stimulated HSC,FRNK plasmid mediated by cationic liposome was transfected into HSC. The apoptosis of FRNK-induced HSC was examined by annexin-V/propidium iodide double-labeled flow cytometry (FCM), gel electrophoresis and transmission electron microscope. And the levels of FRNK,FAK,p-FAK(Tyr397) and MT1-MMP in HSC were assayed by Western blot on protein level, and by RT-PCR on mRNA level, respectively. Results The expression of FRNK was enhanced after FRNK had been transiently transfected into HSC in vitro. The apoptotic rate in HSC exposed to FRNK plasmid for 48 hours was higher than that in non-FRNK plasmid group, (25.37±1.92)%vs (9.28±1.05) % (P＜0.01),and accompanied by a significant increase of MT1-MMP activity both in the protein and in the mRNA level,2.26±0.14 vs 1.09±k0.15 (P <0.01)) 1.58±0. 18 vs 1.00±0. 10 (P <0.01). Conclusion FRNK can induce HSC apoptosis and MT1-MMP is perhaps involved in the process.