摘要
目的研究Smad4基因对人类胃癌细胞株血管生成相关因子的影响。方法构建Smad4真核表达载体pcDNA3．1（-）-Smad4质粒，并用脂质体介导转染方法将pcDNA3．1（-）-Smad4质粒和空载质粒pcDNA3．1（-）导入培养的Smad4基因表达缺失的胃癌MKN28细胞株中，经G418筛选，获得稳定表达Smad4基因的Smad4^＋-MKN28细胞和作为对照的Smad4^--MKN28细胞。分别应用逆转录（RT）-PCR法和Western印迹法检测体外培养亲本细胞、Smad4^＋-MKN28细胞和Smad4^--MKN28细胞的血管内皮生长因子（VEGF）和凝血酶敏感蛋白1（TSP1）基因的mRNA和蛋白的表达。结果RT—PCR结果显示，VEGF在稳定转染pcDNA3．1（-）-Smad4重组质粒的Smad4^＋-MKN28细胞（0．41±0．14）较亲本细胞（0．71±0．45）和转染空载质粒的Smad4^--MKN28细胞（0．76±0．28）表达明显下降（P〈0．05），而TSP1的表达明显增高（分别为0．71±0．45、0．41±0．14和0．76±0．28，P值均〈0．05），Western印迹结果示其编码蛋白也出现相应的变化。结论恢复Smad4表达后此细胞株促进血管生成的基因表达下降，抑制血管生成的基因表达上调，提示Smad4基因可能通过抑制肿瘤血管生成而使肿瘤的生长受到抑制。
Objective To study the effect of Smad4 gene on angiogenesis related factors in hmnan gastric cancer cell line. Methods Recombinant eukaryotic expressing plasmid pcDNA3.1 （-）- Smad4 containing Smad4 gene and empty vector pcDNA3. 1 （-） were introduced into human gastric cancer cell line MKN28 using lipofectam and selected by G418, respectively. Two cell lines were obtained as follows： Smad4^＋-MKN28 cell line which was MKN28 transfected with a stable hybrid containing Smad4 gene and Smad4^--MKN28 cell line with empty plasmid as control. The transcription and expression of VEGF and TSP1 were investigated by RT-PCR and Western blot. Results The mRNA expression of TSP1 in Smad4^＋-MKN28 cells was higher （P〈0.05） than that in control ceils, while VEGF was lower （P〈0. 05）. Western blot showed the consistent results as measurement by RT-PCR. Conclusion Smad4 restoration in gastric cancer cells reduced angiogenesis rates through down-regulation of angiogenesis activitor and up-regulation of angiogenesis inhibitor.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2009年第6期385-388,共4页
Chinese Journal of Digestion
