| Diabetes mellitus is a kind of metabolic disease, which is characterized by insulin resistance and relative insulin deficiency and elevated blood glucose. The insulin-dependent diabetes can not be cured currently. At present, insulin injection in vitro is the main treatment for Diabetes and its complication. Islet or β cell transplantation can afford therapeutic options for diabetic patients. However, this therapy has been hampered by the shortage of donor islets. The usage of porcine islet cells is currently viewed as the most promising alternative, as there is a plentiful supply of porcine islet cells. Moreover, porcine and human insulin are highly conserved, and normal porcine physiological glucose levels are similar to those in humans. Many studies showed that β cell mass derived from pancreatic stem cells was an idel candidate. However, pancreatic stem cells are rare and have a finite proliferation lifespan, known as replicative senescence. Wnt3 a is a canonical Wnt signal pathway, which plays a vital role during embryo development, oxidative stress, mitochondrial membrane potential, stem cell proliferation and differentiation. In this study, we investigated the molecular mechanism of Wnt3 a on the proliferation of porcine pancreatic stem cell. The results showed that stably transfected Wnt3 a inhibited the apoptosis of porcine pancreatic stem cell through activating canonical Wnt signal. We established Dox-inducible Wnt3 a expression system, Wnt3 a was regulated in time- and dose-dependent manner. Wnt3 a expression was induced by Dox and the cells′ proliferation potential was promoted. Further, we constructed porcine pancreatic stem cell being transfected GFP-LC3 stably, which is an ideal cell model for evaluating autophagic activity in vitro. The proliferation potential of porcine pancreatic stem cells was enhanced by autophagy. The study elucidated that Wnt3 a may regulate porcine pancreatic stem cell self-renewal and anti-apoptosis through Wnt/β-catenin signaling payhway.1. The stably transfected Wnt3 a porcine pancreatic stem cells promoted cell proliferation and inhibited cell apoptosisFirstly, we construct eukaryotic expression vector of pIRES2-AcGFP-Wnt3 a. Secondly, we transfected p IRES2-Ac GFP-Wnt3 a into porcine pancreatic stem cells and screened by antibiotic pressure. After identification, we successfully obtained the stably transfected Wnt3 a porcine pancreatic stem cells increased the expression level of pluripotent and proliferative markers analysed by QRT-PCR and Western blotting assay. Meanwhile, the proliferative of potentiality of pancreatic stem cells proliferative was enhanced after Wnt3 a transfection analyzed by the growth cruve and cell cycle, Brd U assay. The results showed that canonical Wnt/β-catenin was activated by Wnt3 a in porcine pancreatic stem cell analyzed by Dual luiferase report assay, immunofluorescent staining and western blotting. Canonical Wnt signaling pathway can resist mitochondrial membrane potential decrease caused by reactive oxygen stress, but Dkk1 addition has a rescue effect. In addition, Wnt3 a inhibit porcine pancreatic stem cell apoptosis and Dkk1 could reverse Wnt3 a inhibitory effect analyzed by Annexin V detection. The results indicated that stably transfected Wnt3 a promote proliferation and inhibit apoptosis of porcie pancreatice stem cell through activating canonical Wnt/β-catenin signaling.2. Establishment of Dox inducible overexpression Wnt3 a regulate on porcine pancreatic stem cells promotion proliferationWnt3a gene was subcloned to pCDNA4T/O and inducible expression p CDNA4T/O-Wnt3 a vector was successfully constructed. We established Dox-inducible Wnt3 a expression porcine pancreatic stem cells through cotransducting p CDNA4T/O-Wnt3 a and p CDNA6T/R and screening by blasticitin and zeocin. The Wnt3 a and GFP expression were strictly regulated by Dox in time- and dose-dependent manner detected by immunofluorexcence, QRT-PCR, flow cytometry and Westen blotting assay. Further more, we found that the Wnt3a-expressing porcine PSCs induced by Dox exhibited a higher proliferative potential analyzed by Brd U and cell cycle analysis. After Dox stimulation, the expression of PCNA, c-Myc and active β-catenin were increased, but down-regualted after Dkk1 addition. These results revealed that the promotion of PSCs proliferation capability could be caused by activation of Wnt signaling pathway and successive downstream target genes’ transcription related to proliferation after Dox treatment. In conclusion, we established porcine PSCs line that dynamically express Wnt3 a and found Wnt3 a could promote the PSCs cell proliferative potential. This inducible expression system thus provides an important tool for further studying on the mechanism of porcine PSCs self-renewal and differentiation.3. Autophagy promoted porcine pancreatic stem cells proliferationThe lentivirus vector was transduced into 293 T cells for obtanining the lentivirus liquid. We established stably transfected LC3 porcine pancreatic stem cells after GFP-LC3 lentivirus transfection and puromycin screening through detection by PCR, Western blotting and flow cytometry assay. The stably transfected LC3 porcine pancreatic stem cells express mesenchymal stem cell marker(CD90 and Vimentin), related proliferation marker(PCNA, c-Myc) and autophagic markrer(p62) detected by immunofluorescence. We still found that serum free medium stimulated autophage and chloriquine could rescue this effect. Finally, we found that activated autophagy accelerated cell proliferation analyzed by cell cycle and Brd U incorporation assay, autophagy occurs may determine the fate of porcine pancreatic stem cell self-renewal and differentiation. The study propose an ideal cell model and technical platform for further elucidating the mechanism of pancreatic stem cell self-renewal and development regualated by Wnt3 a and autophagy. |
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