为建立一种用SYBR Green I荧光染料检测PK-15细胞α干扰素(α-IFN)效应因子Mx1、OAS的mRNA表达水平的qPCR检测方法,通过在猪圆环病毒2型(Porcine Circo-virus Type 2,PCV2)抑制α-IFN发挥效应的信号通路中进行初步应用。根据GenBank中...为建立一种用SYBR Green I荧光染料检测PK-15细胞α干扰素(α-IFN)效应因子Mx1、OAS的mRNA表达水平的qPCR检测方法,通过在猪圆环病毒2型(Porcine Circo-virus Type 2,PCV2)抑制α-IFN发挥效应的信号通路中进行初步应用。根据GenBank中目的基因的序列,利用分子生物学软件Premier 5.0在其保守区设计并合成相应的特异性引物。利用TRIzol法提取总RNA,经Oligo d(T)15进行反转录,利用PCR扩增各段目的基因,并克隆至pMD 18-T载体,转化大肠杆菌DH 5α,经鉴定为阳性的重组质粒作为标准品模板建立SYBR Green I qPCR标准曲线和溶解曲线,并进行灵敏性、特异性和重复性试验。根据建立的实时qPCR方法,检测PCV2对α-IFN效应因子的抑制效果。对建立的PK-15细胞α-IFN效应因子SYBR Green I qPCR方法进行分析,结果表明Mx1、OAS和内参β-actin基因的Ct值与标准品稀释度在1×101～1×108 copies/μL的范围分别呈良好的线性关系。PK-15细胞在接种PCV2,并受到α-IFN刺激后Mx1、OAS的相对表达量较未接种PCV2明显降低。本试验建立了PK-15细胞α-IFN效应因子的qPCR检测方法,为在mRNA水平上对PK-15细胞α-IFN效应因子的定量分析奠定了基础,并成功地初步应用于PCV2抑制α-IFN发挥效应的信号通路研究中。展开更多
BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(X...BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(XIST)expression has been reported to be elevated in the serum of DN patients.AIM To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell(RTEC)pyroptosis in DN.METHODS A DN rat model was established through streptozotocin injection,and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST.Renal metabolic and biochemical indices were detected,and pathological changes in the renal tissue were assessed.The expression of indicators related to inflammation and pyroptosis was also detected.High glucose(HG)was used to treat HK2 cells,and cell viability and lactate dehydrogenase(LDH)activity were detected after silencing XIST.The subcellular localization and downstream mechanism of XIST were investigated.Finally,a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3(NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p(miR-15b-5p)/Toll-like receptor 4(TLR4)axis.RESULTS XIST was highly expressed in the DN models.XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury.The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells;cell viability was decreased and LDH activity was increased after HGtreatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically,XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promotingmiR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect ofsilencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury inDN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.展开更多
文摘为建立一种用SYBR Green I荧光染料检测PK-15细胞α干扰素(α-IFN)效应因子Mx1、OAS的mRNA表达水平的qPCR检测方法,通过在猪圆环病毒2型(Porcine Circo-virus Type 2,PCV2)抑制α-IFN发挥效应的信号通路中进行初步应用。根据GenBank中目的基因的序列,利用分子生物学软件Premier 5.0在其保守区设计并合成相应的特异性引物。利用TRIzol法提取总RNA,经Oligo d(T)15进行反转录,利用PCR扩增各段目的基因,并克隆至pMD 18-T载体,转化大肠杆菌DH 5α,经鉴定为阳性的重组质粒作为标准品模板建立SYBR Green I qPCR标准曲线和溶解曲线,并进行灵敏性、特异性和重复性试验。根据建立的实时qPCR方法,检测PCV2对α-IFN效应因子的抑制效果。对建立的PK-15细胞α-IFN效应因子SYBR Green I qPCR方法进行分析,结果表明Mx1、OAS和内参β-actin基因的Ct值与标准品稀释度在1×101～1×108 copies/μL的范围分别呈良好的线性关系。PK-15细胞在接种PCV2,并受到α-IFN刺激后Mx1、OAS的相对表达量较未接种PCV2明显降低。本试验建立了PK-15细胞α-IFN效应因子的qPCR检测方法,为在mRNA水平上对PK-15细胞α-IFN效应因子的定量分析奠定了基础,并成功地初步应用于PCV2抑制α-IFN发挥效应的信号通路研究中。
基金Supported by Natural Science Foundation of Shenzhen University General Hospital (SUGH2020QD011)
文摘BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(XIST)expression has been reported to be elevated in the serum of DN patients.AIM To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell(RTEC)pyroptosis in DN.METHODS A DN rat model was established through streptozotocin injection,and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST.Renal metabolic and biochemical indices were detected,and pathological changes in the renal tissue were assessed.The expression of indicators related to inflammation and pyroptosis was also detected.High glucose(HG)was used to treat HK2 cells,and cell viability and lactate dehydrogenase(LDH)activity were detected after silencing XIST.The subcellular localization and downstream mechanism of XIST were investigated.Finally,a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3(NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p(miR-15b-5p)/Toll-like receptor 4(TLR4)axis.RESULTS XIST was highly expressed in the DN models.XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury.The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells;cell viability was decreased and LDH activity was increased after HGtreatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically,XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promotingmiR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect ofsilencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury inDN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.
