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目的:构建人磷脂磷酸水解酶3(hLPP3)基因的真核表达载体pLenExpress-hLPP3并转染卵巢癌细胞系SKOV3细胞,初步观察重组表达载体对卵巢癌细胞的转染效率、目的基因表达情况及对卵巢癌细胞生长的影响。方法:采用RT-PCR法从正常胎盘组织中提取hLPP3基因,先克隆入克隆载体pGEM-T easy质粒得到pGEM-T-hLPP3,再用BamHⅠ和XhoⅠ进行双酶切,切下的hLPP3基因亚克隆入真核表达载体pLenExpress,得到pLenEx-press-hLPP3重组质粒,采用脂质体转染法转染卵巢癌细胞系SKOV3细胞。实验分为3组:A组为实验组(转染表达hLPP3基因重组质粒)、B组为空质粒对照(转染空表达质粒)、C组为无转染对照。荧光显微镜观察细胞转染效率,实时荧光定量PCR法检测hLPP3 mRNA的表达,化学法测定转染前后细胞上清液中溶血磷脂酸(LPA)含量的变化,流式细胞仪检测细胞周期及凋亡情况。结果:重组表达质粒经限制性酶切鉴定得到与hLPP3基因长度一致(936bp)的酶切产物,测序分析证实,目的基因序列与GenBank上登录的hLPP3基因(BC009196)序列完全一致。荧光显微镜下见A、B2组SKOV3细胞内有大量绿色荧光信号,转染率分别为67%和69%,C组细胞中无荧光信号。实时荧光定量PCR测定显示A组细胞中hLPP3 mRNA的相对表达量较B、C组明显增加(A、B、C组分别为0.5109±0.0584、0.1499±0.0255、0.1433±0.0181,P<0.05)。A组卵巢癌细胞上清液中的LPA含量较B、C组明显下降[A、B、C组分别为(2.37±0.76)μmol/L、(6.71±2.03)μmol/L、(6.84±1.49)μmol/L,P<0.05];流式细胞仪检测显示A组卵巢癌细胞凋亡率明显增加[A、B、C组分别为(30.50±2.12)%、(1.26±0.43)%、(0.10±0.03)%,P<0.05],细胞周期无明显变化。结论:成功构建了表达目的基因hLPP3的重组真核表达载体pLenEx-press-hLPP3,并能高效转染卵巢癌细胞系SKOV3细胞,使细胞中hLPP3基因表达水平明显升高。增强hLPP3基因表达可以降低细胞外LPA水平,诱导细胞凋亡,抑制卵巢癌细胞的生长。
OBJECTIVE: To construct the eukaryotic expression vector pLenExpress-hLPP3 of human phospholipid phosphorylase 3 (hLPP3) gene and transfect it into ovarian cancer cell line SKOV3 to observe the transfection efficiency, target gene expression, On ovarian cancer cell growth. Methods: The hLPP3 gene was extracted from normal placenta tissue by RT-PCR and cloned into pGEM-T easy plasmid to obtain pGEM-T-hLPP3. The recombinant plasmid was double digested with BamHⅠand XhoⅠ. The hLPP3 gene subclone Into the eukaryotic expression vector pLenExpress, pLenEx-press-hLPP3 recombinant plasmids were obtained and transfected into ovarian cancer cell line SKOV3 by lipofection. The experiment was divided into three groups: group A was experimental group (transfected with hLPP3 gene recombinant plasmid), group B was empty plasmid control (transfected with empty expression plasmid), group C was without transfection control. The transfection efficiency of cells was observed by fluorescence microscopy, the expression of hLPP3 mRNA was detected by real-time fluorescence quantitative PCR, the content of lysophosphatidic acid (LPA) in the supernatants of the cells before and after transfection was measured by chemical method, the cell cycle and apoptosis were detected by flow cytometry Happening. Results: Restriction endonuclease digestion analysis showed that the recombinant plasmid was identical to the length of hLPP3 gene (936bp). Sequencing analysis confirmed that the sequence of the target gene was identical with the sequence of hLPP3 gene (BC009196) registered in GenBank. Under the fluorescence microscope, a large number of green fluorescent signals were observed in SKOV3 cells in group A and B2, the transfection rates were 67% and 69%, respectively. There was no fluorescence signal in C cells. Real-time quantitative PCR showed that the relative expression of hLPP3 mRNA in group A was significantly higher than that in group B and C (0.5109 ± 0.0584,0.1499 ± 0.0255,0.1433 ± 0.0181, P <0.05, respectively). The level of LPA in supernatant of ovarian cancer cell in group A was significantly lower than that in group B and C (6.73 ± 2.03 and 0.457 μmol / L, ) μmol / L, P <0.05]. The apoptosis rate of ovarian cancer cells in group A was significantly increased by flow cytometry [(30.50 ± 2.12)%, (1.26 ± 0.43)%, 0.10 ± 0.03)%, P <0.05], no significant changes in cell cycle. CONCLUSION: The recombinant eukaryotic expression vector pLenEx-press-hLPP3 expressing the target gene hLPP3 was successfully constructed and transfected into ovarian cancer cell line SKOV3 with high efficiency. The hLPP3 gene expression level was significantly increased. Enhancement of hLPP3 gene expression can reduce the level of extracellular LPA, induce apoptosis and inhibit the growth of ovarian cancer cells.