Preliminary study of muscle-derived stem cells in mice with SMDF gene transfected into Schwann-like cells

Abstract

Objective The constructed Neuregulin-1(NRG-1) type Ⅲ sensory and motor neuron-derived factor (SMDF) eukaryotic expression vector was transfected into muscle-derived stem cell (MDSC) and the MDSC were induced to differentiate into Schwann-like cells by co-culture method. It was confirmed that SMDF gene can promote the proliferation and differentiation of Schwann cells, and further verify the function of SMDF protein, which lays an experimental foundation for studying the process and molecular mechanism of SC myelination after peripheral nerve injury. Methods (1) Western blotting method was used to analyze the SMDF protein expression in brain, spinal cord, dorsal root ganglia (DRG), skeletal muscle, liver, spleen and other tissues, and identification of SMDF in DRG by immunohistochemical staining expression. (2) The complete sequence of the mouse SMDF gene coding region was obtained by PCR, and the recombinant eukaryotic expression vector pEGFP-N2-SMDF was constructed. (3) The eukaryotic expression vector pEGFP-N2-SMDF and empty plasmid pEGFP-N2 were transfected into MDSC cultured in vitro by liposome-mediated method, and they were divided into recombinant plasmid group, plasmid group and non-transformed group to detect of SMDF gene and protein expression before and after transfection. (4) MDSC transfected with SMDF gene were induced to differentiate into Schwann-like cells by cell culture medium, and identified by immunocytochemistry. Data were compared with one-way ANOVA and t test. Results After pEGFP-N2-SMDF eukaryotic expression vector was transfected into MDSC, the expression of green fluorescent protein was observed under fluorescence microscope. (2) PCR and Western-blotting results showed that the expression of SMDF mRNA and protein in the cells transfected with recombinant vector pEGFP-N2-SMDF was significantly increased. The SMDF/GAPDH values of the recombinant plasmid group were 1.7321±0.1346, compared with the empty plasmid group (0.5975±0.0084)and the untransfected plasmid group (0.4816±0.0092), the difference was statistically significant (t=4.1258, 4.3314, with P values below 0.01). (3)The positive rate of S-100β was significantly increased in MDSC transfected with SMDF gene after induction by SC conditioned medium. Conclusions The results showed that SMDF protein could be expressed in MDSC, and the expression of SMDF mRNA and protein in pEGFP-N2-SMDF cells was significantly increased. SMDF gene could promote the proliferation and differentiation of Schwann-like cells. Key words: Adult stem cells; Genes; Mice; Schwann cells; Gene transfection; Peripheral nerve injury