重组型Caspase-3基因的构建及其在胰腺癌细胞中凋亡活性的观察

Construction of Recombinant Caspase-3 Gene and the Observation of It's Apoptotic Activity in Pancreatic Carcinoma

目的 通过对半胱氨酸蛋白酶 3 (Caspase 3 )大、小亚基的重组 ,构建pcDNA3 .1(+ ) /r Caspase 3真核表达质粒 ,并探讨r Caspase 3基因诱导细胞凋亡的可能性 ,以寻求肿瘤基因治疗的新途径。 方法 采用分子生物学方法克隆Caspase 3的大、小亚基 ,并在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pcDNA3 .1(+ ) /r Caspase 3真核表达质粒 ;脂质体瞬时转染人胰腺癌细胞PC Ⅱ ,RT PCR检测r Caspase 3mRNA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 Caspase 3的大、小亚基被完整克隆 ,pcDNA3 .1(+ ) /r Caspase 3真核表达质粒测序结果证实小亚基位于大亚基之前 ;RT PCR扩增出 894bp大小片段 ,流式细胞检测可见明显的凋亡峰出现。 结论 构建的r Caspase 3其mRNA可在胰腺癌细胞中表达并自催化诱导细胞凋亡 ,可作为胰腺癌基因治疗的目的基因。

Objective To explore the new gene therapy method for tumor, the recombinant Caspase 3 gene (r caspase 3) eukaryotic expression plasmid was constructed by molecular biologic method. Methods The eukaryotic expression plasmid pcDNA3.1(+)/r Caspase 3 was constructed by rearrangement of the large subunit and small subunit of Caspase 3 and it was transfected into pancreatic carcinoma cells(PC Ⅱ). After being transfected, the expression of r Caspase 3 mRNA in pancreatic carcinoma cells was detected by RT PCR and it's apoptotic activity was detected by FCM. Results The sequence of r Caspase 3 showed that the recombinant molecules (r Caspase 3) now had its' small subunit preceding its' large subunit. After pancreatic carcinoma cells being transfected with the pcDNA3.1(+)/r Caspase 3 by liposomes, a 894 bp strap was observed by RT PCR. No strap was found in control groups. A transparent hypodiploid karyotype peak was found by FCM.Conclusion The plasmid of pcDNA3.1(+)/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.