重组Caspase-3基因的构建及其诱导胰腺癌细胞凋亡的观察

Construction of Recombinant Caspases-3 Gene and the Test of Its Apoptotic Activity in Pancreatic Carcinoma Cell Strain

通过对 Caspase- 3(半胱氨酸蛋白酶 3)大亚基 (L S) ,小亚基 (SS)的重组 ,构建重组型 Caspase- 3基因 (r-caspase- 3) ,并探讨 r- Caspase- 3基因诱导胰腺癌细胞凋亡的可能性 ,以寻求肿瘤基因治疗的新途径。采用分子生物学方法克隆 Caspase- 3的大、小亚基 ,体外进行重新排列组合 ,使大、小亚基原来的排序颠倒 ,构建出 pc DNA3.1(+) / r- Caspase- 3真核表达质粒 ;利用脂质体瞬时转染人胰腺癌细胞 PC- ,RT- PCR检测 r- Caspase- 3m RNA的表达 ;流式细胞技术 (FCM)检测转染胰腺癌细胞凋亡状况。结果显示 :Caspase- 3的大、小亚基被完整克隆 ,r- Cas-pase- 3基因测序结果证实小亚基位于大亚基之前 ;RT- PCR扩增出 894 bp大小片段 ,流式细胞检测可见明显的凋亡峰出现。以上结果表明 ,重组 r- Caspase- 3基因其 m RNA可在胰腺癌细胞中表达并自催化诱导细胞凋亡 。

To explore the new gene therapic method for pancreatic carcinoma, the recombinant Caspases 3 gene (r Caspases 3) was constructed by molecular biologic method. The eukaryotic expression plasmid pcDNA3.1(+)/r Caspase 3 was constructed by rearrangement of the large subunit and small subunit of caspases 3, and then it was transfected into pancreatic carcinoma cells strain (PC II). After being transfected , the expression of r Caspase 3 mRNA in pancreatic carcinoma cells was detected by RT PCR and its apoptotic activity was detected by FCM. The sequencing of the recombinant molecules (r Caspases 3) confirmed that its small subunit preceded its large subunit. After the pancreatic carcinoma cells were transfected with the pcDNA3.1(+)/r Caspases 3 by liposomes, an 894 bp strap was observed by means of RT PCR. No strap was found in control groups. A transparent hypodiploid karyotype peak was revealed by FCM. The above data indicate that the gene of r Caspase 3 has been constructed successfully, r Caspase 3 has apoptotic activity and can be used as target gene in gene therapy for pancreatic carcinoma.