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目的:探讨长链非编码RNA KCNQ1DN在尿源干细胞干性调控中的作用。方法:收集健康成人(n=6)尿液,提取尿源干细胞并传代培养,分别收集P3、P5和P7代尿源干细胞,并应用实时荧光定量PCR(RTPCR)检测KCNQ1DN基因表达水平。构建靶向干扰KCNQ1DN的慢病毒载体LV3-shKCNQ1DN,转染P2代尿源干细胞并培养96小时后荧光显微镜观察感染效率,RT-PCR检测KCNQ1DN的表达,CCK8法检测细胞的增殖,RT-PCR和Western blotting检测干性相关转录因子c-Myc、Nanog、Rex1的mRNA及蛋白的表达。结果:随着尿源干细胞传代代数的增加,KCNQ1DN的表达量逐渐升高(P<0.05)。shKCNQ1DN转染尿源干细胞后KCNQ1DN的表达显著降低(P<0.01),敲低尿源干细胞中KCNQ1DN的表达能够促进尿源干细胞的增殖并上调干性相关转录因子c-Myc、Nanog和Rex1mRNA及蛋白的表达。结论:随着尿源干细胞的生长,长链非编码RNA KCNQ1DN的表达逐步被激活,并对尿源干细胞的干性起到负性调控作用。
Objective: To investigate the role of long-chain non-coding RNA KCNQ1DN in the regulation of urinary stem cells. Methods: Urine of healthy adult (n = 6) was collected. Urinary stem cells were collected and subcultured. Urine stem cells of P3, P5 and P7 were harvested respectively. The expression of KCNQ1DN was detected by real-time fluorescence quantitative PCR (RTPCR). The lentiviral vector LV3-shKCNQ1DN targeting to KCNQ1DN was constructed and transfected into P2 generation of urinary stem cells and cultured for 96 hours, the infection efficiency was observed by fluorescence microscope, the expression of KCNQ1DN was detected by RT-PCR, the proliferation of cells was detected by CCK8, RT-PCR and Western blotting was used to detect the mRNA and protein expression of dry-related transcription factors c-Myc, Nanog and Rex1. Results: The expression of KCNQ1DN increased gradually with the passage of urinary-derived stem cells (P <0.05). The expression of KCNQ1DN was significantly decreased after transfection of urinary stem cells with shKCNQ1DN (P <0.01). The knockdown of KCNQ1DN in urinary stem cells could promote the proliferation of urinary stem cells and up-regulate the expression of c-Myc, Nanog and Rex1 mRNA and protein expression. CONCLUSION: With the growth of urinary stem cells, the expression of long-chain non-coding RNA KCNQ1DN is gradually activated and negatively regulates the dryness of urinary stem cells.