ABSTRACT
Objective To construct the Hut78 cell line with EZH2 gene knocked into by CRISPR/Cas9 system. Methods The EZH2 expression vector pMD-18T-EZH2 with homologous arm and the sgRNA expression vector pSpCas9 (BB)-2A-Puro-sgRNA, which could cut the double stranded genomic DNA, were constructed, and the two vectors were co-transfected into Hut78 cells. Then the expression of EZH2 mRNA was detected by qPCR, and the expressions of EZH2 and H3K27me3 proteins were detected by Western blot assay. Results The pMD-18T-EZH2 and pSpCas9(BB)-2A-Puro-sgRNA recombinant vectors were confirmed by DNA sequencing. When Hut78 cells were transfected with the two recombinant plasmid, qPCR results showed that the expression of EZH2 mRNA was significantly increased, and Western blot analysis showed that the expressions of EZH2 and H3K27me3 proteins were significantly increased. Conclusion EZH2 gene is successfully knocked into Hut78 cells by CRISPR/Cas9 system.
ABSTRACT
Objective To investigate the protective effect of insulin glargine in myocardium of diabetic rats. Methods Male Wistar rats were randomly divided into normal control group (NC), diabetes mellitus group After 6 weeks, we weighed rats and calculated the heart body weight ratio (H/B), Immunohistochemical technique was used to estimate the expression of transforming growth factor beta 1 (TGF-β_1) and the type-Ⅲ collagen (collagen Ⅲ). Myocardial pathologic changes were observed under expression of TGF-β_1 and collagen Ⅲ of DM group and DI group were significantly higher than those in NC group (P<0.05); the levels of H/B and the expression of TGF-β_1 and collagen Ⅲ of DI group were lower than myofibrils were arranged disorderly, mitochondria increased, with swelling and degeneration, while the changes of myocardial ultrastructure were obviously lightened after treatment with insulin glargine. Conclusion Insulin glargine may partly suppress the increased expression of TGF-β_1 and collagen Ⅲ in myocardial of diabetic rats, and it may decrease significantly the myocardial injury of diabetic rats.