摘要
目的 构建高迁移率族蛋白 1(HMGB1)基因的反义真核表达载体 ,寻找胰腺癌基因治疗新途径。方法应用分子克隆技术构建HMGB1基因反义真核表达载体 pcDNA3 1/antisense HMGB1,转染胰腺癌细胞株PANC 1,通过逆转录 聚合酶链反应 (RT PCR)、免疫印迹法 (Westernblot)、噻唑蓝 (MTT)比色法检测转染 4 8h后胰腺癌细胞HMGB1基因表达和体外增殖活性的变化。结果 成功构建 pcDNA3 1/antisense HMGB1反义真核表达载体。所获反义表达载体 pcDNA3 1/antisense HMGB1转染可使PANC 1细胞HMGB1mRNA和蛋白表达水平显著降低 (P <0 0 1)。反义 pcDNA3 1/antisense HMGB1的导入能有效抑制PANC 1增殖活性 (P <0 0 1)。 结论 应用反义RNA技术阻断HMGB1基因的表达 ,能有效抑制癌细胞的体外增殖活性 。
Objective To construct and identify eukaryoti expression vector carrying human antisense high mobility group box 1 (HMGB1 cDNA), and to study its effect on the proliferation of human pancreatic cancer cell line (PANC-1). Methods HMGB1 was cloned and inserted into eukaryoti expression vector pcDNA3.1 reversely to construct pcDNA3.1/antisense-HMGB1, in which restriction enzyme analysis was used to confirm the reverse orientation of the HMGB1. The vector was transfected into PANC-1 and the positive clone was screened by G418. The HMGB1 expression of PANC-1 cells before and after transfection was detected by RT-PCR and Western blot. The proliferating activity in vitro was examined by MTT analysis. Results The pcDNA3.1/antisense-HMGB1 vector was obtained. HMGB1 expression of PANC-1 cells was blocked by antisense HMGB1. It was verified that the antisense-transfected cells showed lower HMGB1 mRNA expression. HMGB1 protein expression was also inhibited upon transfecting. Proliferation activity was suppressed dramatically by antisense HMGB1. Conclusion Constructed antisense eukaryoti expression vector pcDNA3.1/antisense-HMGB1 could suppress the HMGB1 expression and effectively inhibit the proliferation of pancreatic cancer cell.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2004年第5期562-565,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家"8 6 3"计划资助项目 (No 2 0 0 1AA2 180 5 1)
