乳酸脱氢酶A促进人脉络膜黑色素瘤细胞的生长

Lactate dehydrogenase A promotes the growth of human melanoma cell line MUM-2B

目的成功构建带有Flag标签的人乳酸脱氢酶A(lactate dehydrogenase A,LDHA)的真核表达载体,初步检测LDHA基因对人脉络膜黑色素瘤(choroidal melanoma,CM)MUM-2B细胞生长功能的影响。方法将军事科学院传代培养的人CM系随机分为实验组和对照组,实验组MUM-2B细胞瞬时转染构建成功带有Flag标签的人LDHA重组质粒(Flag-LDHA),对照组MUM-2B细胞瞬时转染Flag空载质粒;以带有GST标签的Flag-LDHA为模板,采用聚合酶链反应(PCR)技术扩增LDHA基因,并将其插入到Flag载体,应用菌液PCR、双酶切和测序方法鉴定重组质粒,鉴定成功后瞬时转染人CM,通过Western blot鉴定其表达;CCK8法检测并绘制生长曲线。结果PCR技术扩增得到长约1000 bp的LDHA基因的编码区序列,并成功克隆至Flag载体上;与对照组相比,实验组菌液PCR结果为阳性,双酶切鉴定结果为切开两条长约4000 bp的Flag载体和1000 bp的LDHA基因条带,测序鉴定结果为插入的序列与LDHA基因的编码序列完全一致。Western blot检测结果显示,与对照组相比,实验组MUM-2B细胞成功表达LDHA蛋白;CCK8法检测结果显示,实验组MUM-2B细胞较对照组MUM-2B细胞生长快。结论成功构建了Flag-LDHA真核表达载体,并发现LDHA基因能够促进人CM MUM-2B细胞的生长,为研究LDHA在人CM的发生发展中的功能奠定基础。

Objective To construct the eukaryotic expression vector of LDHA with Flag label and detect its effects on the grow th of human choroidal melanoma（ CM） MU M-2 B cells.Methods CM cells line MU M-2 B subcultured by the Military Academy of Sciences were divided into two groups： experimental group and control group. The experimental group was transiently transfected with Flag-LD HA plasmid,and the control group was transiently transfected with Flag plasmid. U sing the Flag-LD HA with GST label as a template,the LD HA gene was amplified by polymerase chain reaction（ PCR）,which then was inserted into eukaryotic expression vector of Flag,and the recombinant plasmid Flag-LD HA was identified by bacterial liquid PCR,double enzyme digestion and sequencing,both which were transiently transfected into human CM MU M-2 B cells after successful identification,and finally,its expression was determined by W estern blot. The biology behaviors of melanoma cell line MU M-2 B transfected with Flag-LD HA and Flag plasmid were analyzed by counting Kit-8（ CC K8） assays. Results The coding region sequence of LD HA gene of approximately 1000 bp was harvested from PCR amplification,which was successfully cloned into the Flag vector. Compared with the control group,the PCR result of the bacterial liquid in the experimental group was positive. The double enzyme digestion results show ed that eukaryotic expression vector of Flag with a length of about 4000 bp Flag vector and a 1000 bp LD HA gene band. And the sequencing results indicated that the inserted sequence was completely in consonance with the coding sequence of the LD HA gene. W estern blot results show ed the successful expression of recombinant plasmid Flag-LD HA in MU M-2 B melanoma cells. CC K8 assays demonstrated that Flag-LD HA recombinant plasmid could promote the grow th of melanoma cell line MU M-2 B. Conclusion The eukaryotic expression vector of Flag-LD HA was successfully constructed,which can promote the grow th of melanoma cell line MU M-2 B.T his will lay the foundation for studying the function of LD HA in the initiation and progression of human CM.