CAJ | 학술논문

目的研究缺氧对人肝癌细胞SMMC-7721黏着斑激酶(FAK)表达的影响以及FAK表达对SMMC-7721细胞侵袭能力的影响.方法通过1%体积分数O2的低氧培养建立人肝癌细胞SMMC-7721物理缺氧模型,Western blot检测FAK的表达.构建针对FAK mRNA的干扰质粒pshRNA-FAK及阴性对照质粒pGensil-2,并将其转染至SMMC-7721细胞,G418筛选稳定转染细胞株.Western blot检测FAK蛋白表达的变化,细胞迁移和侵袭实验检测缺氧条件下细胞迁移和侵袭能力的改变.在正常条件下将FAK真核表达质粒pcDNA3-FAK转染至SMMC-7721细胞,观测其侵袭能力的改变.根据数所资料的不同分别采用t检验、单因素方差分析,LSD法及Dunnett法进行统计学处理. 结果低氧培养的SMMC-7721细胞FAK蛋白表达水平逐渐升高,24 h后较0 h时明显升高(P<0.01).SMMC-7721细胞稳定转染pshRNA-FAK后,FAK蛋白表达显著下降,抑制率达74.6%±5.1%,在正常及缺氧条件下都对FAK表达有显著抑制作用.细胞迁移实验结果显示,缺氧显著促进SMMC-7721细胞迁移能力(t=18.66,P<0.01),侵袭实验结果与迁移实验结果一致.转染pshRNA-FAK对促进SMMC-7721细胞在缺氧环境中的迁移能力有显著抑制作用,透膜细胞数(353±36)个较对照组(392±31)个明显降低(F=173.983,P<0.05);细胞侵袭实验显示,转染pshRNA-FAK对促进SMMC-7721细胞侵袭能力有显著抑制作用,透膜细胞数(160±12)个较对照组(194±13)个明显降低(F=59.674,P<0.05).同时转染真核表达质粒pcDNA3-FAK显著促进SMMC-7721细胞侵袭能力.结论缺氧促进SMMC-7721细胞侵袭可能与FAK表达水平升高相关,FAK表达的上调可能是缺氧促进肝癌细胞侵袭转移的机制之一.
목적 연구결양대인간암세포SMMC-7721점착반격매(FAK)표체적영향이급FAK표체대SMMC-7721세포침습능력적영향.방법 통과1%체적분수O2적저양배양건립인간암세포SMMC-7721물리결양모형,Western blot검측FAK적표체.구건침대FAK mRNA적간우질립pshRNA-FAK급음성대조질립pGensil-2,병장기전염지SMMC-7721세포,G418사선은정전염세포주.Western blot검측FAK단백표체적변화,세포천이화침습실험검측결양조건하세포천이화침습능력적개변.재정상조건하장FAK진핵표체질립pcDNA3-FAK전염지SMMC-7721세포,관측기침습능력적개변.근거수소자료적불동분별채용t검험、단인소방차분석,LSD법급Dunnett법진행통계학처리. 결과저양배양적SMMC-7721세포FAK단백표체수평축점승고,24 h후교0 h시명현승고(P<0.01).SMMC-7721세포은정전염pshRNA-FAK후,FAK단백표체현저하강,억제솔체74.6%±5.1%,재정상급결양조건하도대FAK표체유현저억제작용.세포천이실험결과현시,결양현저촉진SMMC-7721세포천이능력(t=18.66,P<0.01),침습실험결과여천이실험결과일치.전염pshRNA-FAK대촉진SMMC-7721세포재결양배경중적천이능력유현저억제작용,투막세포수(353±36)개교대조조(392±31)개명현강저(F=173.983,P<0.05);세포침습실험현시,전염pshRNA-FAK대촉진SMMC-7721세포침습능력유현저억제작용,투막세포수(160±12)개교대조조(194±13)개명현강저(F=59.674,P<0.05).동시전염진핵표체질립pcDNA3-FAK현저촉진SMMC-7721세포침습능력.결론 결양촉진SMMC-7721세포침습가능여FAK표체수평승고상관,FAK표체적상조가능시결양촉진간암세포침습전이적궤제지일.
Objective To study focal adhesion kinase (FAK) expression in hypoxia-stressed SMMC 7721 cells and the role of FAK expression in the hypoxia-induced invasion of SMMC-7721 cells. Methods SMMC-7721 cells were cultured in 21% O2 or 1% O2. FAK expression was determined by Western blot. The siRNA expression vector pshRNA-FAK targeting to FAK and the control vector pGensil-2 were transfected into SMMC-7721 cells. The hypoxia-induced migration and invasion ability of SMMC-7721 cells transfected with pshRNA-FAK were analyzed. In normoxia, invasion of SMMC-7721 cells transfected with pcDNA3FAK was analyzed. Results The expression of FAK was increased significantly in SMMC-7721 cells 24 h after hypoxia stress (P < 0.01). The level of FAK protein was decreased by 74.6% ±5.1% after the pshRNAFAK transfection in normoxia and hypoxia. The migration and invasion of SMMC-7721 cells was increased in 1% O2(P < 0.01). However, the migration and invasion of SMMC-7721 cells transfected with pshRNAFAK was decreased in 1% O2 (P < 0.05). Overexpression of FAK significantly stimulated the invasion of SMMC-7721 cells. Conclusion Up-regulation of FAK may play an important role in the invasion of SMMC-7721 cells induced by hypoxia.