骨桥蛋白反义基因在食管癌浸润与转移中的作用

目的:探讨骨桥蛋白(OPN)反义基因对食管癌细胞增殖和转移的影响，为食管癌基因治疗提供理论依据。方法： PCR扩增获得人OPN基因，连接pcDNA3.1(+)载体，酶切、测序。脂质体介导将pcDNA3.1 -ANOPN基因转入ECA 109，G418筛选获得稳定表达ANOPN基因的转染细胞ECA-ANOPN、表达空载体的ECA-vect细胞及空白对照ECA 。RT-PCR检测ANOPN mRNA、免疫组织化学检测ANOPN基因蛋白表达。体外观察各组细胞生长速度、倍增时间，Transwell小室法检测细胞黏附、侵袭迁移能力的差异。结果：载体构建正确，测序结果与GenBank中公布的序列同源性为100%。与ECA-vect、ECA组比较，ECA-ANOPN细胞OPN的表达率明显降低（P<0.05），生长速度明显减慢（P<0.05）,倍增时间增加（P<0.05），透膜细胞数明显降低（P<0.01）。结论：ANOPN基因的稳定表达明显抑制ECA 109细胞的恶性表型。

Effect of anti-sense osteopontin on metastasis and infiltration of esophagus cancer

Objective To investigate the effect of anti-sense osteopontin(ANOPN)on proliferation and metastasis of esophagus cancer cells.Methods An OPN gene recombinant expression vector plasmid was constructed by RT-PCR from human umbilical vein endothelial cell gene and cloned into a mammalian expression vector pcDNA3.1(+).PcDNA3.1-ANOPN was introduced by LipofectinTM.Positive cell clones(ECA-ANOPN),vector-transfected cells ECA-vect and blank cell ECA were used as three groups.RT-PCR and immunocytochemistry assay were used to investigate the expressions of OPN mRNA and protein.The metastasis characteristics of cells were studied by Transwell method.Results The vector was constructed successfully,the sequencing result was identical with that reported in GenBank.Compared with vector-transfected cells(ECA-vect cells) and ECA cells,the growth rate of ECA-ANOPN cells was significantly slowed(P<0.05),the expression rate was decreased(P<0.05),their doubling-time increased(P<0.05),and the number of ECA-ANOPN cells in mucosa was decreased(P<0.01).Conclusion The stable expression of ANOPN gene can significantly suppress the malignant phenotype of ECA109 cells.