关键词： A3−, integrin alpha3 deficient cells AB8/13 is a conditionally immortalized podocyte cell line carrying a temperature-sensitive T antigen as transgene, in text and figures the abbreviation Podo was used for simplicity CRISPR/Cas9 HK2, human kidney-2 ILNEB ILNEB, interstitial lung disease, nephrotic syndrome and epidermolysis bullosa Integrin α3 Kidney Nephrotic syndrome PodoA3−, integrin α3 negative podocytes Podocyte Skin blistering A3−, integrin alpha3 deficient cells AB8/13 is a conditionally immortalized podocyte cell line carrying a temperature-sensitive T antigen as transgene, in text and figures the abbreviation Podo was used for simplicity CRISPR/Cas9 HK2, human kidney-2 ILNEB ILNEB, interstitial lung disease, nephrotic syndrome and epidermolysis bullosa Integrin α3 Kidney Nephrotic syndrome PodoA3−, integrin α3 negative podocytes Podocyte Skin blistering

来  源：   DOI：10.1016/j.mbplus.2022.100119   PDF(Pubmed)

Abstract：
Integrin α3β1 is a cell adhesion receptor widely expressed in epithelial cells. Pathogenic variants in the gene encoding the integrin α3 subunit ITGA3 lead to a syndrome including interstitial lung disease, nephrotic syndrome, and epidermolysis bullosa (ILNEB). Renal involvement mainly consists of glomerular disease caused by loss of adhesion between podocytes and the glomerular basement membrane. The aim of this study was to characterize the impact of loss of integrin α3 on human podocytes. ITGA3 was stably knocked-out in the human podocyte cell line AB8/13, designated as PodoA3-, and in human proximal tubule epithelial cell line HK2 using the targeted genome editing technique CRISPR/Cas9. Cell clones were characterized by Sanger sequencing, quantitative PCR, Western Blot and immunofluorescence staining. RNASeq of integrin α3 negative cells and controls was performed to identify differential gene expression patterns. Differentiated PodoA3- did not substantially change morphology and adhesion under standard culture conditions, but displayed significantly reduced spreading and adhesion when seed on laminin 511 in serum free medium. Gene expression studies demonstrated a distinct dysregulation of the adhesion network with downregulation of most integrin α3 interaction partners. In agreement with this, biological processes such as \"extracellular matrix organization\" and \"cell differentiation\" as well as KEGG pathways such as \"ECM-receptor interaction\", \"focal adhesion\" and the \"PI3K-Akt signaling pathway\" were significantly downregulated in human podocytes lacking the integrin α3 subunit.
摘要：
整合素α3β1是在上皮细胞中广泛表达的细胞粘附受体。编码整合素α3亚基ITGA3的基因中的致病变异导致包括间质性肺病在内的综合征,肾病综合征,和大疱性表皮松解症（ILNEB）。肾脏受累主要包括由足细胞与肾小球基底膜之间的粘附丧失引起的肾小球疾病。这项研究的目的是表征整合素α3的损失对人足细胞的影响。ITGA3在人足细胞细胞系AB8/13中稳定敲除，命名为PodoA3-，以及使用靶向基因组编辑技术CRISPR/Cas9在人类近端小管上皮细胞系HK2中。通过Sanger测序对细胞克隆进行表征，定量PCR,Westernblot和免疫荧光染色。进行整联蛋白α3阴性细胞和对照的RNASeq以鉴定差异基因表达模式。分化的PodoA3-在标准培养条件下没有实质性改变形态和粘附，但在无血清培养基中在层粘连蛋白511上播种时，播散和粘附显着降低。基因表达研究表明粘附网络的明显失调，大多数整合素α3相互作用伴侣的下调。同意这一点，生物过程，如“细胞外基质组织”和“细胞分化”，以及KEGG途径，如“ECM-受体相互作用”，在缺乏整合素α3亚基的人足细胞中，“粘着斑”和“PI3K-Akt信号通路”显着下调。
公众号