【摘要】 目的构建KiSS-1基因重组真核质粒,将其导入人高转移肝癌MHCC97-H细胞中,获得瞬时表达的细胞株,为进一步研究KiSS-1基因对人肝癌细胞侵袭转移的影响奠定基础。方法Trizol试剂法提取新鲜胎盘组织总RNA,经RT-PCR扩增目的片段,PCR产物及真核表达载体分别双酶切后进行连接,并转化入大肠杆菌Ecoli.Dh5α扩增以获得重组载体,采用脂质体介导转染技术将高纯度的KiSS-1质粒转染到人高转移肝癌细胞株中,用RT-PCR和Westernblot技术加以鉴定。结果RT-PCR获得预期大小的特异性DNA片段,经PCR、双酶切鉴定及测序,证实已将KiSS-1基因cDNA片段正确插入真核表达载体中;RT-PCR、免疫细胞化学和Westernblot证实KiSS-1基因已转染入MHCC97-H细胞中。结论成功获得pcDNA3.1/HisC/KiSS-1重组质粒和瞬时表达KiSS-1基因的肝癌细胞,为后续建立稳定转染的KiSS-1高表达阳性细胞克隆奠定基础,为体内外实验研究KiSS-1对人肝癌细胞增殖、侵袭和转移能力的影响提供前提条件。更多还原

【Abstract】 Objective To construct a recombinant eukaryotic expression vector containing the functional region of human KiSS-1 gene and then to transfect this vector into human hepatocellular carcinoma cell line MHCC97-H with high metastasis potential.Transient-expression cell lines are to be obtained so as to establish a foundation for a further study on the impact of KiSS-1 on hepatoma carcinoma cell invasion and metastasis.Methods Total RNA was extracted by using Trizol one-step method from fresh placenta tissues.cDNA was amplified via reverse transcription polymerase chain reaction(RT-PCR).The product of PCR,amplified by being transformed into E.coli Dh5α,was inserted into the eukaryotic expression vector after digestion and ligation using restriction endonucleases and ligase.Liposome was used to transfect this KiSS-1 vector into MHCC97-H cell 1ines and the result was detected by RT-PCR,immunocytochemistry(ICC)and Western blot.Results The correct KiSS-1 cDNA clone was obtained.It was confirmed that KiSS-1 cDNA was inserted into the eukaryotic expression vector correctly by employing PCR,double digestion identification and sequencing.RT-PCR,ICC and Western blot showed that the KiSS-1 gene had been transfected into MHCC97-H cell.Conclusions If recombinant eukaryotic expression vector pcDNA3.1/HisC/KiSS-1 and transient-expression KiSS-1 cell lines are successfully obtained,a foundation will be laid for a further study on how to establish KiSS-1 cell lines of stable transfection and how to observe the impact of KiSS-1 on hepatoma carcinoma cell proliferation,invasion and metastasis through in vivo and in vitro experiment.更多还原