RESUMO
Objective To establish PKD2 gene recombinant lentivirus and to investigate its restorative effects on polycystin-2 and Wnt/β-catenin signaling pathways in Pkd2-null cell lines.Methods From August 2016 to February 2017,PKD2 gene was cleaved from the pcDNA3.1-TM-PKD2 plasmid and was inserted into the pLV-sfGFP 2A Puro by restriction enzymes Xba Ⅰ and Xho Ⅰ.The recombinant pLV-sfGFP-PKD2 plasmid was sequenced to verify a correct construction.Then we obtained recombinant lentiviruses by co-transfecting 293T cells with recombinant plasmid and packaging plasmids.B3/D3 (Pkd2 +/-) and B2/E8 (Pkd2-/-) cell lines were used to evaluate the effectiveness of lentivirus,they were divided into experimental group,control group,blank group and treated with pLV-sfGFP-PKD2 virus,pLV-sfGFP virus or culture media,respectively.The expression of PC2 was detected by Western blotting and immunofluorescence staining.Finally the cell proliferation was evaluated by detecting of proliferating cell nuclear antigen.The changes of Wnt/β-catenin signaling pathway were evaluated by real-time quantitative PCR.Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-sfGFP-PKD2 was constructed successfully.After infected with pLV-sfGFP-PKD2 virus,the expression of PC2 in the experimental group B2 and E8 cells(0.668 ±0.013,0.763 ±0.021) was restored to the normal level,compared with control group B3 and D3 cells,respectively (0.687 ± 0.015,P =0.164;0.776 ± 0.008,P =0.409).The proliferative activity in experimental group B2 cells(0.573 ±0.010) was significantly lower than that in control group B2 cells (0.848 ±0.031,P ＜0.01),and was returned to the level of blank group B3 cells (0.585 ±0.017,P =0.369).Reexpression of PKD2 in experimental group B2 cells also reduced the expression of Wnt7a,β-catenin,back to blank group B3 cells' level(0.037 ±0.005 vs.0.037 ±0.004,P=0.969;0.554 ±0.008 vs.0.571 ±0.013,P =0.64).Conclusions The recombinant pLV-sfGFP-PKD2 lentivirus has been constructed successfully.The lentivirus could rectify the absence of PC2 in PKD2-null cell lines,by which the hyperactivated Wnt/β-catenin signaling pathway and the abnormal cell proliferation caused by PC2 deficiency could be also restored to normal levels.
RESUMO
PKHD1 ( polycystic kidney and hepatic disease 1), the causal gene of human autosomal recessive polycystic kidney disease(ARPKD), is located on chromosome 6p12.2 and covers a genomic region of ～500 kb. PKHD1 is among the largest human genes, with a minimum of 86 exons from which multiple transcripts may be generated by alternative splicing. The longest continuous open reading frame consists of 12 222 bp, encoding a 4 074 amino acid protein, designated as fibrocystin/polyductin (FPC). FPC is predicted to be a receptor like protein, with single transmembrane domain and a short cytoplasmic tail. The expression of mouse FPC can be detected in various duct-containing organs. In mouse embryogenesis, FPC appears in developing neural tube, bronchi, and the primordial gut as early as the day of E9.5. In fetal human kidneys, high level of FPC expression is present in ureteric bud and its expression continues throughout the process of renal tubuobranching. In adult human kidneys, FPC mainly expresses in the epithelia of renal collecting ducts. FPC is subcellularly localized to the primary cilia and concentrated on the basal bodies in renal epithelial cells. The detail function of FPC is still unrevealed, most recent studies demonstrate that FPC, as a receptor, may transduce cell signal by interacting with a trp superfamily TRPP2 (PKD2), which is a causal gene for ADPKD, and mediate intracellular calcium homeostasis to regulate differentiation, proliferation, migration and polarity of various duct/tubular epithelia, in turn, to modulate the formation of all physiologic ducts, tubules and tracts.
