An eukaryotic expression system of Congjiang pigs IFN-λ1 was constructed to obtain its expression in CHO-K1 cells and the inhibition effect of Congjiang pig IFN-λ1 on PRRSV proliferation was verified. The eukaryotic expression plasmid pEGFP-PoIFN-λ1 was constructed from the pig IFN-λ1 gene fragment and transfected into CHO-K1 cells. Expression was detected by fluorescence microscopy and Western blotting. The influence on the proliferation of PRRSV was assessed. The results of the study showed that the recombinant plasmid pEGFP-PoIFN-λ1 was constructed correctly. After transfection, green fluorescent signal was detected in CHO-K1 cells by fluorescence microscopy. Western blot analysis revealed that in cells at different time periods after transfection, porcine IFN-λ1 was expressed, with the highest expression observed 36 h after transfection. The antiviral activity of the supernatant after 36 h of transfection was determined by the micro cytopathic inhibition method, and the biological activity was 2.1×103 U/mL. Quantitative PCR was used to detect the proliferation of PRRSV, and the results showed that Congjiang pigs IFN-λ1 significantly inhibited the mRNA expression of PRRSV and viral proliferation in a dose- and time-dependent manner. This study established a Congjiang pig IFN-λ1 eukaryotic expression system, and the quantitative PCR method showed that it has a significant inhibitory effect on the proliferation of PRRSV, which lays a foundation for the future production of antiviral drugs and clinical application.
Telomerase reverse transcriptase (TERT) vectors were transfected into bone marrow mesen- chymal stem cells (BMSCs) which were then cultured and selected to establish TERT-BMSC cell lines whilst sequencing BMSCs and TERT-BMSCs via transcriptome in this study to explore their regulatory mechanism and effect on osteogenic differentiation after TERT ectopic expres- sion in sheep BMSCs. After sequencing and analysing differential genes, PI3K/Akt signalling pathway related to osteogenic differentiation was investigated. Western blot was used before and after applying the PI3K/Akt signalling pathway inhibitor LY294002 to detect protein expression levels of AKT and p-AKT. On the twenty-first day of osteogenic differentiation, RT-qPCR and Western blot were used to detect mRNA and protein expression levels of RUNX2 and OPN and alizarin red staining was utilised to analyse calcium salt deposition. Results showed that pro- tein expression levels of AKT and p-AKT were significantly up-regulated, mRNA and protein expression levels of RUNX2 and OPN increased and calcium salt deposition increased after ectopic expression of TERT. After applying LY294002, the protein expression of AKT and p-AKT was down-regulated, mRNA and protein expression levels of RUNX2 and OPN were reduced and calcium salt deposition was reduced. These results confirmed the stable integration and expression of the exogenous TERT gene in BMSCs to promote the differentiation of BMSC osteoblasts, which may be mediated by the PI3K/Akt signalling pathway.
Emerging researches in humans, pigs and mice, highlighted that estrogen plays a pivotal role in self-renewal and differentiation of bone marrow mesenchymal stem cells (BMSCs). The present study aimed at evaluating effects of 17 beta-estradiol (E2) on proliferation and apoptosis of canine-derived bone marrow mesenchymal stem cells (cBMSCs) in vitro. The results showed that E2 supplementation at the concentration of 10-11 M promoted the proliferation of cBMSCs by CCK-8 assay and RT-qPCR analysis for the proliferation-related genes, with proliferating cell nuclear antigen (PCNA), cyclin-D1 (CCND1) being up-regulated and cyclin-dependent kinase inhibitor 1B (CDKN1B) being down-regulated. Contrarily, analysis of fluorescence-activated cell sorting (FACS) and RT-qPCR demonstrated that E2 supplementation above 10-11 M had inhibitory effects on the proliferation of cBMSCs and induced apoptosis. Intriguingly, cBMSCs still possessed the capability to differentiate into osteoblasts and adipocytes with 10-11 M E2 addition. Taken together, this study determined the optimal culture condition of cBMSCs in vitro, and has important implications for further understanding the regulatory effect of E2 on the self-renewal of cBMSCs, which are helpful for the clinical application of BMSCs.