1.Effect of cardiotrophin-1 mediated by recombinant adenovirus on the neural differentiation of bone marrow mesenchymal stem cells of rats
Shasha LI ; Changhui LANG ; Longying PENG ; Xiaomei SHU
Chinese Journal of Applied Clinical Pediatrics 2015;30(15):1176-1179
Objective To investigate the effect of cardiotrophin-1 (CT-1) mediated by recombinant adenovirus (Adv) on the neural differentiation of bone marrow mesenchymal stem cells (rBMSCs) of rats.Methods The rBMSCs were isolated and cultured by attachment method.The surface marker protein was identified by flow cytometry.The rBMSCs were divided into 4 groups:control group,AdV enhanced green fluorescence protein (Adv-EGFP) + induction group(empty virus group),CT-1 group and induction group.The cells were transfected by Adv in the corresponding multiplicity of infections.Each group was induced by neural induction medium except for control group.The cells morphological changes were observed by microscope and the expressions of Nestin,neuronal nuclei (NeuN) and glial fibrillary acidic protein(GFAP) were detected respectively by cellular immunofluorescence at 5 h,3 d and 7 d after induction.Results After induction treatment,neuron-like cells morphological changes were observed in all the groups except for control group,among which the most obvious change was found in CT-1 group.The positive rate of Nestin was the highest at 5 h after induction.Positive rate of CT-1 group[(86.31 ± 4.27)%] was higher than any other groups,the differences were statistically significant (all P < 0.001);after which its positive rate gradually declined,the positive rate of CT-1 group changed more obviously than other groups,and the differences were statistically significant (all P < 0.001).The difference in Nestin positive rate between the empty virus group and induction group was not statistically significant (all P > 0.05) at various time points.The NeuN and GFAP could be observed in CT-1 group at 5 h after induction.Then the positive rate of NeuN and GFAP increased gradually,and climbed to the highest point [(64.41 ± 3.65)%,(47.14 ± 4.29)%] on 7 d after induction.Positive rate of NeuN and GFAP in CT-1 group at various time points were higher than that of other groups,and the differences were statistically significant (all P <0.001).The difference of NeuN and GFAP positive rate between the empty virus group and induction group was not statistically significant (all P > 0.05).Conclusion CT-1 could promote the neural differentiation of rBMSCs.
2.The study of 5-Hydroxytryptamine 2B receptor-nanodisc self-assembling and its ligand binding activity
Shuyan DAI ; Fang PENG ; Jun LI ; Lingzhi QU ; Longying JIANG ; Xiaojuan CHEN ; Ming GUO ; Meiying SHAO ; Yongheng CHEN
Journal of Chinese Physician 2017;19(7):984-987
Objective To explore the application of nanodisc in functional and drug discovery research of G protein-coupled receptor (GPCR).Methods The purified recombinant 5-Hydroxytryptamine 2B receptor (5-HT2BR) was reconstituted into nanodisc complex.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclution chromatography were performed to evaluate the reconstitution reaction,followed by the use of surface plasmon resonance to validate the ligand-binding activity of 5-HT2BR after reconstitution.Results 5-HT2B R was effectively self-assembled into nanodisc while maintained its binding activity toward the antagonist SB204741.Conclusions The presented study provided potential application of 5-HT2B R-nanodisc for the development of subtype-selective drugs against 5-HT2B R and the fundamental of utilizing nanodisc for GPCR structural and functional studies as well as drug discovery.