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背景:研究发现,骨髓间充质干细胞移植入心肌梗死区域后易发生凋亡导致移植细胞存活率较低。目的:构建caspase-3基因的小分子干扰RNA片段(small interfering RNAs,siRNAs)表达质粒,探讨其在体外对骨髓间充质干细胞中caspase-3基因表达的抑制作用。方法:根据siRNA设计原则,针对caspase-3基因设计并合成siRNAs,构建caspase-3siRNA表达载体并利用脂质体介导方法瞬时转染骨髓间充质干细胞。实验分为正常对照组,阴性序列对照组(si-ncg)和3个干扰组(si-caspase-3-1、si-caspase-3-2和si-caspase-3-3)。结果与结论:成功构建了caspase-3siRNA表达载体并转染骨髓间充质干细胞。Westernblot和RT-PCR检测转染后的骨髓间充质干细胞中caspase-3蛋白和mRNA的表达下降,以转染si-caspase-3-3的骨髓间充质干细胞下降最明显。结果提示构建的caspase-3siRNA表达质粒在体外能抑制骨髓间充质干细胞中caspase-3基因的表达。
Background: The study found that bone marrow mesenchymal stem cells prone to apoptosis after transplantation into myocardial infarction area lead to lower survival rate of transplanted cells. OBJECTIVE: To construct small interfering RNAs (siRNAs) expression plasmids for caspase-3 gene and to investigate its inhibitory effect on caspase-3 gene expression in bone marrow mesenchymal stem cells in vitro. Methods: According to the principle of siRNA design, siRNAs were designed and synthesized based on caspase-3 gene. Caspase-3siRNA expression vector was constructed and transiently transfected into bone marrow mesenchymal stem cells by liposome-mediated method. The experiment was divided into normal control group, negative sequence control group (si-ncg) and three interference groups (si-caspase-3-1, si-caspase-3-2 and si-caspase-3-3). RESULTS AND CONCLUSION: The caspase-3siRNA expression vector was successfully constructed and transfected into bone marrow mesenchymal stem cells. Western blot and RT-PCR showed that the expression of caspase-3 protein and mRNA were down-regulated in transfected MSCs, and the most significant decrease was in MSCs transfected with si-caspase-3-3. The results suggest that the constructed caspase-3siRNA expression plasmid can inhibit caspase-3 gene expression in bone marrow mesenchymal stem cells in vitro.