丙酮酸激酶M1(PKM1)对眼黑色素瘤细胞迁移的影响  

Effects of PKM1 gene on the migration of ocular melanoma cells

作　　者：刘园园 朱晓雨 刘莹 徐小洁 李玲 叶棋浓 刘丹 

机构地区：[1]锦州医科大学附属第一医院,辽宁省锦州市121000

出　　处：《眼科新进展》2018年第1期27-30,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:81472589;81672602;81402345;81502264)~~

摘　　要：目的构建带有pXJ-40-myc标签的丙酮酸激酶M1(pyruvate kinase M1,PKM1)基因真核表达载体,并初步探讨PKM1对眼黑色素瘤的影响。方法将传代培养的眼B16黑色素瘤细胞随机分为实验组(转染pXJ-40-myc-PKM1重组质粒)和对照组(转染pXJ-40-myc空载质粒);以人肝cDNA文库为模板应用PCR扩增出PKM1基因的CDS编码区序列,应用菌液PCR、双酶切鉴定,分别将pXJ-40-myc-PKM1重组质粒和pXJ-40-myc空载质粒瞬时转染眼B16黑色素瘤细胞,Western blot检测其表达,应用划痕实验法检测PKM1对眼B16黑色素瘤细胞转移的影响。结果 PCR技术扩增出长1800 bp的PKM1基因,与预期大小一致;与对照组相比,菌液PCR结果为阳性,双酶切切出的条带分别为4000 bp和1800 bp;与对照组相比,Western blot检测结果为重组质粒pXJ-40-myc-PKM1在眼B16黑色素瘤细胞成功表达。与对照组相比,划痕实验结果为重组质粒pXJ-40-myc-PKM1抑制眼B16黑色素瘤细胞的迁移。结论成功构建了pXJ-40-mycPKM1真核表达载体,并证明该基因抑制眼B16黑色素瘤细胞的迁移。Objective T o construct the eukaryotic expression vector of pyruvate kinase M1( PKM1) gene labeled w ith pX J-40-myc and detect its biological activity in ocular B16 melanoma cells. Methods O cular B16 melanoma cells w ere randomly divided into experimental and control group,and the experimental group w as transfected w ith pX J-40-myc-PKM1 plasmid and the control group w as transfected w ith pX J-40-myc plasmid.T hen PKM1 gene w as amplified by PC R w ith human liver cDNA library as the template. T he recombinant plasmid pX J-40-myc-PKM1 w as identified by bacteria PC R and double enzyme digestion,follow ed by transfection of pX J-40-myc-PKM1 and pX J-40-myc plasmid into B16 melanoma cells,and finally,the expression of PKM1 protein w as verified by the W estern blot,w hile w ound healing assay w as used to detect the effects of PKM1 on the migration of ocular melanoma cells. Results T he length of PKM1 gene was 1800 bp,which was consistent with the expected size. Compared with the control group,the result of bacteria PCR was positive. T he length of double enzyme digestion was 4000 bp and 1800 bp respectively. Western blot results showed that recombinant plasmid pXJ-40-mycPKM1 was successfully expressed in ocular B16 melanoma cells. Compared with the control group,wound healing assay showed that recombinant plasmid could inhibit the migration of ocular B16 melanoma cells. Conclusion T he eukaryotic expression vector of pXJ-40-mycPKM1 is successfully constructed,which can suppress the migration of ocular B16 melanoma cells.

关 键 词：眼B16黑色素瘤细胞 丙酮酸激酶M1基因 克隆 真核表达 

分 类 号：R739.7[医药卫生—肿瘤]