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Transgelin基因真核表达载体的构建及其对胃腺癌AGS细胞增殖的影响 被引量：4

Construction of Transgelin Plamid Vector and Study of Its Effect on Proliferation of Ggastric Adenocarcinomas Cells
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摘要 目的 构建人肌动蛋白凝胶蛋白（Transgelin）基因编码区序列（cDNA）的真核表达载体,并观察其对胃腺癌细胞增殖的影响.方法 从健康人肠组织中提取总RNA,应用RT-PCR方法扩增TransgelincDNA的全长序列后克隆入pcDNA3.1/myc-His（-）A质粒中,构建成Transgelin基因的真核表达载体,然后转染入AGS细胞中,新霉素筛选稳定转染细胞克隆,通过MTT实验,研究转染Transgelin基因后细胞增殖的变化.结果 重组载体经酶切鉴定和测序证实目的 基因正确尤误,Western印迹检测结果显示Transgelin融合蛋白在AGS细胞中具有良好的表达,而转染空载体及未转染细胞对照则未见有此融合蛋白质条带;RT-PCR结果显示Transgelin基因在其稳定转染的AGS细胞克隆中表达上调,与空载体稳定转染及未转染细胞比较,差异具有统计学意义（P＜0.05）.Transgelin表达上调的稳定克隆组,AGS细胞的增殖活性明显降低,MTT结果显示其增殖活性显著低于空载体稳定转染组及未转染亲代细胞组,差异具有统计学意义（P＜0.05）,而后两者之间差异无统计学意义（P＞0.05）.结论 Transselin表达上调可导致胃腺癌细胞增殖降低. Objective To eonstrut eukaryotie expression vector peDNA3.1/mye-His （-） A- Transgelin, and to study the effect of Transgelin on proliferation of gastric adenoearcinoma （AGS） cells. Methods Total RNA was extracted from human colon. Transgelin eDNA was amplified by RT-PCR, and inserted into peDNA3.1/mye-His （-） A vector. The recombinant plasmid pcD- NA3. 1/myc-His （-） A-Transgelin was transfected into AGS cells by lipofeetamine. The stable transfeetants of AGS cells were selected with G418. Western blot and RT-PCR were used to detect expression of Transgelin protein and mRNA. MTT assay was used to evaluate proliferative effect of Transgelin on AGS cells. Results The recombinant expression vector peDNA3.1/mye-His （-） A- Transgelin was successfully constructed. The expression of transgelin-mye fusion protein was specifically detected in AGS cells stably transfected with Transgelin expression vector, but not in empty vector transfeeted or untransfected AGS cells. In addition, mRNA expression level of Transgelin was significantly higher in stable transfeetants with the Transgelin vector than that in control cells （P 〈 0. 05 ） . MTT assay showed that proliferation of Transgelin stably expressing AGS cells was considerably decreased, compared to empty vector transfected or in untransfeeted cells （P 〈 0. 05）.Conclusion Transgelin can inhibit gastric adenocarcinoma cell proliferation.
作者 钱凤英 黄俏佳
机构地区 福建医科大学福总临床医学院(南京军区福州总医院)分子医学研究中心
出处 《医学分子生物学杂志》 CAS CSCD 2010年第6期517-522,共6页 Journal of Medical Molecular Biology
基金 福建省自然科学基金（No.2009J01181）,南京军区医药卫生科研基金（No.08MA100）,南京军区福州总医院专项基金（No.2004037）
关键词 肌动蛋白凝胶蛋白 基因克隆 基因转染 四唑盐比色测定 Transgelin gene cloning gene transfection MTT assay
分类号 Q782 [生物学—分子生物学]
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参考文献14

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共引文献2

同被引文献44

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医学分子生物学杂志
2010年 第6期

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