[1]李留霞),乔玉环)#,王淼),等.人磷脂磷酸水解酶3基因真核表达载体的构建及其对卵巢癌细胞生长的抑制作用*[J].郑州大学学报(医学版),2009,(01):69-73.
 LI Liuxia),QIAO Yuhuan),WANG Miao),et al.Construction of hLPP3 eukaryotic expression vector and its inhibition effect on ovarian cancer cell line[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2009,(01):69-73.
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人磷脂磷酸水解酶3基因真核表达载体的构建及其对卵巢癌细胞生长的抑制作用*()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2009年01期
页码:
69-73
栏目:
论著
出版日期:
2009-01-01

文章信息/Info

Title:
Construction of hLPP3 eukaryotic expression vector and its inhibition effect on ovarian cancer cell line
作者:
李留霞1)乔玉环1)#王淼2)郭瑞霞1)赵国强3)张孝艳1)周蔚1)张建好1)赵先兰1)
1)郑州大学第一附属医院妇产科郑州4500522)厦门市妇幼保健院厦门3600003)郑州大学微生物学与免疫学教研室郑州450001
Author(s):
LI Liuxia1)QIAO Yuhuan1)WANG Miao2)GUO Ruixia1)ZHAO Guoqiang3) ZHANG Xiaoyan1) ZHOU Wei1) ZHANG Jianhao1) ZHAO Xianlan1)
1)Department of Gynecology and Obstetrics, the First Affiliated Hospital,Zhengzhou University, Zhengzhou 4500522) Women and Children Hospital of Xiamen, Xiamen 3600003) Department of Microbiology and Immunology, Zhengzhou University, Zhengzhou 450001
关键词:
人磷脂磷酸水解酶3卵巢癌细胞系真核表达载体转染
Keywords:
human lipid phosphate phosphatase 3 gene ovarian cancer cell line eukaryotic expression vector transfection
分类号:
R737.31
摘要:
构建人磷脂磷酸水解酶3(hLPP3)基因的真核表达载体pLenExpresshLPP3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RTPCR法从正常胎盘组织中提取hLPP3基因,先克隆入克隆载体pGEMTeasy质粒得到pGEMThLPP3,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pLenExpresshLPP3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hLPP3mRNA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hLPP3mRNA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pLenExpresshLPP3,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。
Abstract:
To construct an eukaryotic expression vector of human lipid phosphate phosphatase 3 gene(hLPP3), pLenExpresshLPP3, and transfect it into ovarian cancer cell line SKOV3. The transfection efficacy of the recombinant vector, the expression of hLPP3 gene in the transfected SKOV3 cells and the inhibition effect on the growth and apoptosis of ovarian cancer cells were observed.Methods:The hLPP3 gene(936 bp) was obtained from human placenta tissue by RTPCR. The PCR product was cloned into pGEMT easy vector, digested by BamHⅠand XhoⅠrestrictive enzymes, subsequently subcloned into the pLenExpress vector to form a recombinant plasmid pLenExpress hLPP3, which was identified by PCR, restrictive enzyme cutting and sequencing. The SKOV3 cells were transfected by recombinant vector with the helping of lipid reagent lipofectamineTM2000.There were three groups: group A (the cells were transfected by pLenExpresshLPP3), group B (the cells were transfected by blank pLenExpress) and group C (nontransfection cells). The transfection efficacy was detected with fluorescent microscope.The expression of hLPP3 mRNA in the transfected cells was detected by Real time quantitative PCR, the LPA level in supernatants of the cells was determined using a biochemical method, and the cell cycle and apoptosis of the cells were measured by flow cytometry.Results:The recombinant expression vector pLenExpresshLPP3 was constructed successfully and identified to be coincided with the sequence of the hLPP3 gene in GenBank(BC009196)by endonuclease digestion and sequencing. A lot of green fluorescent signals were observed under the fluorescence microscope in group A and group B , and their positive rates were 67% and 69% respectively. While no signal in group C. The expression of hLPP3 mRNA in group A was significantly higher than those in group B and C( group A,B,C were 0.510 9± 0.058 4,0.149 9±0.025 5,0.143 3±0.018 1,respectively,P<0.05).The LPA levels in supernatants of group A was lower significantly than those in group B and C [group A,B,C were (2.37±0.76) μmol/L,(6.71±2.03)μmol/L and (6.84±1.49)μmol/L,P<0.05]. The apoptosis rate in group A was dramatic higher than those in group B and C [group A,B,C were (30.50±2.12)%,(1.26±0.43)%,(0.10±0.03)%,respectively,P<0.05].Conclusion: The recombinant eukaryotic expression vector pLenExpresshLPP3 was constructed successfully, which could be transfected into the ovarian cancer cell line SKOV3 with high efficacy. The elevated expression of hLPP3 in the SKOV3 cells transfected by recombinant vector could promote LPA hydrolization, induce cell apoptosis and inhibit ovarian cancer cells growth.

参考文献/References:

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备注/Memo

备注/Memo:
#通讯作者,女,1946年生,教授,研究方向:妇科肿瘤 *国家自然科学基金资助项目30571948
更新日期/Last Update: 2010-05-18