反义血管内皮生长因子165真核表达载体对肝癌细胞增殖的抑制作用

Inhibitory effect of antisense vascular endothelial growth factor 165 eukaryotic expression vector on proliferation of hepatocellular carcinoma cells.

作者信息

Gu Song, Liu Chang-Jian, Qiao Tong, Sun Xue-Mei, Chen Lei-Lei, Zhang Le

机构信息

Department of Vascular Surgery, Gulou Hospital, Affiliated Hospital of Medical College, Nanjing University, Nanjing 210008, Jiangsu Province, China.

出版信息

World J Gastroenterol. 2004 Feb 15;10(4):535-9. doi: 10.3748/wjg.v10.i4.535.

DOI:10.3748/wjg.v10.i4.535

PMID:14966912

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4716975/

Abstract

AIM

To construct antisense VEGF(165) eukaryotic expression vector PCDNA(3)-as-VEGF(165) and to study its expression and effect on the proliferation of hepatocarcinoma SMMC-7721 cells.

METHODS

VEGF(165) cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA(3) to construct PCDNA(3)-as-VEGF(165). Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cation lipofectamine 2000 mediated methods to evaluate the expression of VEGF protein and the inhibitory effect on the proliferation of hepatocarcinoma SMMC-7721 cells.

RESULTS

The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR. VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after PCDNA(3)-as-VEGF(165) transfection. The expression of VEGF protein was dramatically inhibited (142.01+/-7.95 vs 1 625.52+/-64.46 pg/ml(-1), P<0.01) 2 days after transfection, which correlated with the dose of PCDNA(3)-as-VEGF(165)5 gene. VEGF protein was most expressed in PCDNA(3) transferred SMMC-7721 cells but few in PCDNA(3)-as-VEGF(165) transferred cells by immunohistochemical staining. The apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98+/-0.86% vs 4.86+/-0.27%, P<0.01) and the survival rate was notably decreased (80.99+/-3.20% vs 93.52+/-3.93%, P<0.05) due to antisense VEGF(165) by flow cytometry (FCM). The transfection of antisense VEGF(165) gene resulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection.

CONCLUSION

It is confirmed that antisense VEGF(165) can inhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF(165) gene therapy may play an important role in the treatment of human hepatocarcinoma.

摘要

目的

构建反义血管内皮生长因子（VEGF）（165）真核表达载体PCDNA（3）-as-VEGF（165），并研究其表达及对肝癌SMMC-7721细胞增殖的影响。

方法

将VEGF（165）cDNA插入真核表达载体PCDNA（3）的多克隆位点构建PCDNA（3）-as-VEGF（165）。然后用阳离子脂质体2000介导的方法将该载体转入人肝癌细胞株SMMC-7721，以评估VEGF蛋白的表达及对肝癌SMMC-7721细胞增殖的抑制作用。

结果

PCR检测显示正常培养的SMMC-7721细胞中存在VEGF cDNA。PCDNA（3）-as-VEGF（165）转染后，通过RT-PCR检测发现SMMC-7721细胞中VEGF mRNA表达显著降低。转染后2天，VEGF蛋白表达受到显著抑制（142.01±7.95 vs 1625.52±64.46 pg/ml-1，P<0.01），且与PCDNA（3）-as-VEGF（165）基因剂量相关。免疫组化染色显示，VEGF蛋白在转染PCDNA（3）的SMMC-7721细胞中表达最多，而在转染PCDNA（3）-as-VEGF（165）的细胞中表达较少。通过流式细胞术（FCM）检测发现，反义VEGF（165）可显著促进肝癌SMMC-7721细胞的凋亡率（17.98±0.86% vs 4.86±0.27%，P<0.01），显著降低细胞存活率（80.99±3.20% vs 93.52±3.93%，P<0.05）。反义VEGF（165）基因转染通过噻唑蓝（MTT）法对肝癌细胞增殖产生抑制作用，转染后第6天所有肝癌细胞死亡。