| 摘要: |
| 目的:观察外源基因hugl-1转染对结直肠癌LS174细胞生物学行为的影响，探讨hugl-1基因与结直肠肿瘤的相关性。方法:构建重组质粒pcDNA3.0-hugl-1，转染至结直肠癌细胞株LS174细胞中，RT-PCR、Western印迹检测转染后hugl-1 mRNA及蛋白的表达;通过软琼脂集落形成试验、划痕修复试验、细胞黏附试验及Transwell细胞侵袭试验等进一步对转染后LS174细胞增殖、黏附、运动和侵袭能力进行分析，并与空质粒载体及未转染LS174细胞作对照。结果:成功构建重组质粒pcDNA3.0-hugl-1;RT-PCR、Western印迹检测结果显示，重组体pcDNA3.0-hugl-1转染细胞株中hugl-1 mRNA及蛋白表达明显高于空载体转染及未转染组细胞(P＜0.05)。重组体转染组细胞克隆形成率(32.23%)与未转染及空载体转染组细胞相比(35.76%、33.91%)无明显改变;重组体转染组LS174细胞克隆的迁移细胞数(82.14±7.62)明显低于空载体组(135.61±3.74)及未转染组细胞(142.37±6.12，P<0.05);转染后120 min，重组体转染组LS174细胞克隆黏附力明显高于空载体组及未转染组(P<0.05);重组体转染组穿膜细胞数(63.7±8.0)明显少于空载体组及未转染组(158.3±16.5、156.3±13.0，P<0.05)。结论:人hugl-1基因表达上调能降低结直肠癌细胞迁移运动和侵袭能力，增加细胞黏附能力,但对肿瘤细胞增殖能力无明显影响；其表达降低可使肿瘤细胞播散。 |
| 关键词: 结直肠肿瘤 hugl-1基因 软琼脂集落形成 划痕修复 细胞黏附试验 Transwell细胞侵袭试验 |
| DOI：10.3724/SP.J.1008.2008.01475 |
| 投稿时间：2008-03-03修订日期：2008-10-26 |
| 基金项目:上海市科委基金（No.2006074） |
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| Biological effects of recombinant pcDNA3.0-hugl-1 plasmid on colorectal cancer cell line LS174 |
| HUANG Shi-yong,HAO Li-qiang﹡,MENG Rong-gui |
| (Department of Anorectal Surgery,Changhai Hospital,Second Military Medical University,Shanghai 200433,China) |
| Abstract: |
| Objective:To observe the biological behaviors of colorectal cancer LS174 cells before and after pcDNA3.0-hugl-1 transfection,so as to investigate the association of hugl-1 with colorectal cancer.Methods: The eukaryotic expression vector pcDNA3.0-hugl-1 was constructed and transfected into LS174 cells.RT-PCR and Western blotting methods were used to analyze the expression of hugl-1 mRNA and protein in LS174 cells before and after transfection.Soft agar colony formation assay,wound-healing experiment,adhesion assay and Matrigel invasion assays were used to study the effects of hugl-1 expression on the proliferation,adhesion，movement and invasion in LS174 cells.Results: The recombinant plasmid pcDNA3.0-hugl-1 was successfully constructed.RT-PCR and Western blotting showed that the hugl-1 expression was higher in cells transfected with pcDNA3.0-hugl-1 than in those un-transfected or empty vector-transfected cells (P＜0.05).The colony forming rates showed no significant difference between the 3 groups (35.76%,33.91% and 32.23%). Wound healing assays showed a significant reduction in the migrated cells in pcDNA3.0-hugl-1 transfected cell clones(82.14±7.62) compared with un-transfected cell lines(142.37±6.12)and empty vector transfected cell line (135.61±3.74,P＜0.05). Attachment assays revealed enhanced adhesion of the pcDNA3.0-hugl-1-transfected cell clones compared with the other 2 groups.Matrigel invasion assay showed a significant reduction in the invasive ability of the pcDNA3.0-hugl-1-transfected cell (63.7±8.0) in comparison to the un-transfected cells(156.3±13.0)and pcDNA(-) cells(158.3±16.5,P＜0.05).Conclusion: Overexpression of hugl-1 eukaryotic expression vector can decrease migratory and invasive ability of LS174 cells,but has no influence on cell proliferation. |
| Key words: colorectal neoplasms hugl-1 gene soft agar colony formation assay wound-healing adhesion assay Transwell invasion |
