Induced differentiation of SHH gene modified rat ectomesenchymal stem cells into neural-like cells
BI Shi-qi 1, LV De-ming 1, YANG Kai-yuan 1, DAI Yao 1, ZHUANG Qin 2 , HUANG Qiu-sheng 3, ZHANG Zhi-jian 1, CUI Xue-wen 4
（1. School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013; 2. Department of Hematology, Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001; 3. Department of Otorhinolaryngology, Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001; 4. Department of Orthopedics, Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001, China)
Abstract：Objective: To investigate the effect of sonic hedgehog(SHH) gene transfection on differentiation of nasal mucosa-derived ectomesenchymal stem cells(EMSCs) into neural-like cells. Methods: EMSCs were isolated, cultured and purified by tissue adherent method.SHH gene recombinant adenovirus was transfected into EMSCs(SHH-EMSCs).The stem cells markers included Connexin-43, Nestin, and Vimentin in the EMSCs and SHH-EMSCs were stained by immunofluorescence. The expression of SHH was detected by Western blotting.Then these cells were induced to neural cells.The experiments were divided into four groups:SHH-EMSCs inducing group,SHH-EMSCs normal culture group,EMSCs inducing group and EMSCs normal culture group.The morphology of the cells were observed by the inverted microscope.The expression of neural markers included GAP-43, TUBB3, MBP and GFAP were detected by immunofluorescence and WesternBlotting.In addition,the SHH-EMSCs suspension was injected into the spinal cord. Two weeks after transplantation, the survived SHH-EMSCs in the rat spinal cord and secreted SHH were examined by immunohistochemistry. Results: Immunofluorescence results showed that the staining positive rates of Connexin-43,Nestin and Vimentin of SHH-EMSCs were all about 90%.Western blotting data showed that SHH-EMSCs highly expressed SHH. Differentiated SHH-EMSCs showed typical bipolar, multipolar and pyramidal neural-like morphology after inducing for 7 days in experimental group. Immunofluorescence and Western-blotting data showed that differentiated SHH-EMSCs expressed higher GAP-43,β3-tubin, MBP and Smoothen compared with the other three groups; GFAP of cells were weakly expressed in the four groups, with no statistical significance. The experimental results in vivo showed that transplanted SHH-EMSCs have higher survival rate and could express TUBB3 and secrete SHH in the spinal cord. Conclusion: SHH gene modified EMSCs are easy to differentiate into neural-like cells and can be used as the excellent seed cells for repairing spinal cord injury.
［Key words］nasal mucosa; ectomesenchymal stem cells; sonic hedgehog; gene transfection; neurallile cells; differentiation; spinal cord injury
2018, Vol. 28 
