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目的:利用小干扰RNA(siRNA)抑制人阴茎海绵体平滑肌细胞间隙连接蛋白connexin43(Cx43)的表达和检测细胞间间隙连接通讯功能,探讨该技术在阴茎海绵体平滑肌细胞间隙连接和阴茎勃起功能研究中的应用。方法:利用Ambion公司设计软件,构建靶向人Cx43基因的siRNA重组质粒,转染人阴茎海绵体平滑肌细胞48h后,逆转录-聚合酶链反应(RT-PCR)和Western印迹检测Cx43基因和蛋白的相对表达水平、划痕标记荧光染料传输技术检测细胞间间隙通讯功能,并分别与siRNA阴性对照、空白对照组比较。结果:酶切和测序证实siRNA真核表达载体构建成功。siRNA重组质粒转染细胞后的Cx43 mRNA和蛋白相对表达水平分别为(0.45±0.08)%、(0.56±0.06)%,与siRNA阴性对照组(0.72±0.04)%、(0.80±0.08)%和空白对照组(0.74±0.09)%、(0.77±0.11)%相比,差异均有显著性(P(0.05);转染后的细胞间间隙连接通讯功能也显著降低。结论:siRNA能有效抑制人阴茎海绵体平滑肌细胞Cx43的表达和阻断间隙连接介导的间隙连接通讯功能。
OBJECTIVE: To study the effect of small interfering RNA (siRNA) on the expression of connexin43 (Cx43) in human corpus cavernosum smooth muscle cells and the detection of intercellular gap junctional intercellular communication, and to explore the gap junction and penile erectile function in the corpus cavernosum smooth muscle cells In the application. Methods: The recombinant plasmid targeting human Cx43 gene was constructed by Ambion software and transfected into human corpus cavernosum smooth muscle cells for 48 h. Cx43 gene and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting The relative expression levels of the two siRNAs were detected by fluorescence labeled dye transfer assay. The results were compared with the siRNA negative control and the blank control group respectively. Results: The eukaryotic expression vector was successfully constructed by restriction enzyme digestion and sequencing. The relative expression levels of Cx43 mRNA and protein in siRNA transfected cells were (0.45 ± 0.08)% and (0.56 ± 0.06)%, respectively, which were significantly lower than those in siRNA negative control group (0.72 ± 0.04%, 0.80 ± 0.08)% and Compared with control group (0.74 ± 0.09)% and (0.77 ± 0.11)%, the difference was significant (P (0.05)). The transfection of intercellular gap junctional intercellular communication function was also significantly reduced.Conclusion: siRNA can effectively inhibit Cx43 expression in human corpus cavernosum smooth muscle cells and the gap junction-mediated gap junctional communication function.