慢病毒载体介导RNA干扰LOX表达对人阴道壁成纤维细胞弹性蛋白、基质金属蛋白酶2表达的影响

Effect of lentivirus-mediated LOX RNA interference on expression of elastin and matrix metalloproteinase-2 in human vaginal wall fibroblasts

目的探讨人阴道壁成纤维细胞中赖氨酸氧化酶(lysine oxidase,LOX)的表达在盆腔器官脱垂发生中的可能机制。方法选取绝经前、后无症状对照组的阴道前壁组织,用酶消化+组织块培养法分离培养成纤维细胞,细胞免疫荧光法对细胞进行鉴定。针对LOX基因,设计并构建4个慢病毒干扰载体(LOX-RNAi-LV),分别转染HEK-293T细胞,Real-Time PCR检测LOX的表达情况,筛选出其中1个干扰效率最高的LOX-RNAi-LV,将其包装成慢病毒并测定效价,将慢病毒感染人阴道壁细胞,Real-Time PCR检测细胞中弹性蛋白和基质金属蛋白酶-2(MMP-2)的mRNA表达水平。结果培养出绝经前、后无症状对照组的阴道前壁成纤维细胞各4例,成功筛选出了具有较高干扰效率的LOX-RNAi-LV并成功包装入慢病毒,感染慢病毒的人阴道前壁成纤维细胞,弹性蛋白表达均降低,MMP-2表达均增高,差异有统计学意义。结论 LOX-RNAi-LV慢病毒感染后的人成纤维细胞弹性蛋白表达降低,MMP-2表达增高,因此推测LOX对弹性蛋白和基质金属蛋白酶2的表达有调节作用。

Objective To investigate the effect of lentivirus-mediated lysine oxidase （LOX） RNA interference on expression of elastic protein and matrix metalloproteinase-2 （MMP-2） in human vaginal wall fibroblasts. Methods Anterior vaginal tissue samples were collected from healthy women before and after menopause, the fibroblasts were isolated from vagina tissue by enzymes digestion. The cultured cells were identified using immunofluorescence method. Four lentivirus RNAi vectors of LOX （LOX-RNAi-LV） were constructed and transfected to HEK-293T cells to screening the most efficient LOX-RNAi-LV. The screened LOX-RNAi-LV was transfected to human vaginal tissue-derived fibroblasts cells. The mRNA expression of elastin and MMP-2 was detected by RT-PCR, Results The LOX-RNAi-LV with high efficiency was successfully constructed and transfected to fibroblasts isolated from anterior vaginal wall. The mRNA expression of elastin was decreased and MMP-2 was increased, compared to those before transfection. Conclusion Results indicate that LOX may have a regulatory effect on the expression of elastin and matrix metalloproteinase 2 in fibroblasts of vagina tissue, and may be involved in pelvic organ prolapse.