Genetherapy for gastrointestinal cancer with caspase-3 gene transfection

设计了一套适合于聚合酶链式反应(PCR)扩增caspase-3基因的引物。以健康人的DNA为模板，通过聚合酶链式反应(PCR)获得caspase-3基因的全长序列。Caspase-3的腺病毒载体使用Adeno-X^&lt；TM&gt；表达系统(Clontech实验室公司)制备。Western blotting显示，除一株(Kato-III)外，所有受检的6株胃癌细胞株均表达大量caspase-3蛋白。在Kato-3细胞中检测到微弱的caspase-3蛋白。因此，使用Kato-III细胞进行caspase-3基因转染。用0.1、1和10微克/毫升的5-FU和10微克/毫升的顺铂处理亲本Kato-III细胞和转导caspase-3基因的细胞(Kato-III/Cas3)48h。用四甲基偶氮唑盐比色法测定细胞抑制率。与Kato-III细胞相比，5-FU和顺铂对Kato-III/Cas3细胞有明显的抑制作用。提示caspase-3基因导入与化疗联合应用可能是一种有效的抗癌治疗方法。然而，腺病毒载体在HEK293细胞培养过程中逐渐减少并消失。Caspase-3基因的新型腺病毒载体曾多次尝试，但均无济于事。成功构建了caspase-3表达载体。以日本包膜血凝病毒(HVJ-E)为第二优策略，将caspase-3基因导入胃癌细胞。然而，与亲本细胞相比，用这种方法转导caspase-3的细胞并没有表现出化疗对细胞抑制率的显著提高。

The primer set was designed to make an adequate caspase-3 cDNA by polymerase chain reaction (PCR). The caspase-3 cDNA was made by PCR using DNA derived from a healthy individual. Adenovirus vector for caspase-3 was made using Adeno-X^<TM> Expression System (Clontech Laboratories, Inc.). Western blotting showed that all of six gastric cancer cell lines examined but one (KATO-III) expressed much caspase-3 protein. In KATO-3 cells, a faint of caspase-3 protein was detected. Consequently, KATO-III cells were used for caspase-3 gene transfection. The parent KATO-III cells and the caspase-3 gene-transfected cells (KATO-III/cas3) were treated with 5-FU and cisplatin at the concentrations of 0.1, 1, and 10 microgram/ml for 48 hrs. The inhibition rates were measured by the MTT assay. KATO-III/cas3 cells were significantly inhibited, compared to KATO-III cells at any concentrations of 5-FU and cisplatin. These results indicated the caspase-3 gene transfection might be a useful anti-cancer treatment as the combination with chemotherapy. However, the adenovirus vector decreased gradually and disappeared during the culture in HEK293 cells. New adenovirus vectors for caspase-3 gene were tried to make again and again, but in vain. Caspase-3 plasmid could be made successfully. As the second best policy, the hemagglutinating virus of Japan-envelope (HVJ-E) was used to transfect caspase-3 plasmid into gastric cancer cells. However, the cells transfected caspase-3 by this method did not show the significant increment of inhibition rate by chemotherapy, compared to parent cells.