The therapeutic effect of NeuroD on radiation-induced intestinal injury in mice / 中华放射医学与防护杂志

Objective To evaluate the therapeutic effect of NeuroD protein on radiation-induced intestinal injuries.Methods The expression and purification of NeuroD-enhanced green fluorescent protein (EGFP) fusion protein was performed in prokaryotic expression system.The efficiency of the fusion protein transduction into cells was monitored under fluorescence microscope.C57BL/6J mice were randomly divided into four groups with 10 mice in each group:normal control group,PBS group,EGFP group,and NeuroD-EGFP group.Besides the normal control group,the other three groups of mice received 9 Gy γ-ray total body irradiation.Intestinal tissues were collected,frozen sections were prepared to monitor the distribution of NeuroD in mice intestinal tract under fluorescence microscope,and pathological sections were prepared for H&E staining to evaluate the therapeutic effect of NeuroD protein.Results The NeuroD-EGFP fusion protein was purified by Ni-NTA column and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Visible green fluorescence gathered within the cells after NeuroD-EGFP fusion protein was added in the culture medium,suggesting that NeuroD-EGFP could penetrate the cell membrane into the cells.Five hours after intraperitoneal injection of NeuroD-EGFP,visible green fluorescence gathered within the intestinal epithelial cells in villi.At 3.5 d after irradiation,NeuroD-EGFP treated mice showed significantly higher villus (F =49.49,P ＜ 0.01) and crypt depth (F =16.72,P ＜ 0.01) and more crypts per circumference (F =10.32,P ＜ 0.01) compared with PBS and EGFP groups.Conclusion NeuroD protein can accelerate the post-irradiation recovery of injured villi and crypt of intestinal tract and could be used to treat radiation-induced intestinal injuries.

Neurogenic differentiation of human dental stem cells in vitro

OBJECTIVES: The purpose of this study was to investigate the neurogenic differentiation of human dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and stem cells from apical papilla (SCAP). MATERIALS AND METHODS: After induction of neurogenic differentiation using commercial differentiation medium, expression levels of neural markers, microtubule-associated protein 2 (MAP2), class III beta-tubulin, and glial fibrillary acidic protein (GFAP) were identified using reverse transcriptase polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. RESULTS: The induced cells showed neuron-like morphologies, similar to axons, dendrites, and perikaryons, which are composed of neurons in DPSCs, PDLSCs, and SCAP. The mRNA levels of neuronal markers tended to increase in differentiated cells. The expression of MAP2 and beta-tubulin III also increased at the protein level in differentiation groups, even though GFAP was not detected via immunocytochemistry. CONCLUSION: Human dental stem cells including DPSCs, PDLSCs, and SCAP may have neurogenic differentiation capability in vitro. The presented data support the use of human dental stem cells as a possible alternative source of stem cells for therapeutic utility in the treatment of neurological diseases.

The Effects of Adipose Derived Stem Cells on Neurogenic Differentiation and Induction of Nerve Regeneration

Using adipose derived stem cells(ASCs), neurogenic differentiation was induced in a mono layered culture medium containing neuronal induction agents. Cells differentiated to the neuronal cells were observed with a inverted microscope and immunofluorecent study. We made a 15 mm long defect in the sciatic nerve of 14 rats and connected a silicone tube to the defect. Then, we mixed neuronal progenitor cells differentiated from ASCs with collagen gel and grafted them to a group of rats(experimental group) and grafted only collagen gel into another group(control group). In 4 and 8 weeks after the graft, histological observation was made. According to the result, the number and diameter of myelinated axons were significantly increased in the experimental group. In addition, the nerve conduction velocity was improved more in the experimental group and neovascularity also increased. Moreover, reaction with S100 and p75 was observed in regenerated nerves in the experimental group, suggesting that the grafted cells were differentiated into supportive cells such as Schwann's cells. In conclusion, this research proved that ASCs can multiply and differentiate into neuronal cells. If they are grafted into nerve defects, the grafted cells are differ entiated into supportive cells such as Schwann's cells and thus contribute to nerve regeneration. Accordingly, the use of adipose tissue obtained easily without the limitation of donor site can be greatly helpful in treating peripheral nerve defects.

Combination of Pdx1 and NeuroD1 results in Nkx6.1 and GLUT2 expressions in L02 cells / 中国病理生理杂志

AIM: To construct a recombinant plasmid vector containing human pancreatic duodenal homeobox1(Pdx1) and neurogenic differentiation 1(NeuroD1) genes,and to detect its effective expression in eukaryocytes and the ability to induce differentiation of hepatocytes.METHODS: Using human embryo pancreas mRNA as template,Pdx1 and NeuroD1 genes were amplified by RT-PCR and cloned into the two different multiple cloning sites(MCSA and MCSB) of plasmid pIRES.The recombinant plasmid pI/Pdx1/NeuroD1 was transfected into L02 cells.The expression of Pdx1 and NeuroD1 in transfected cells was detected by immunocytochemistry,IFA,RT-PCR and Western blotting,respectively.RESULTS: The length and sequence of cloned segments were correct.Pdx1 and NeuroD1 were expressed in eukaryocytes.Furthermore,the hepatic cells were induced to express glucose transporter 2(GLUT2) and eukaryocyte transcription factor Nkx6.1,which were functionally correlated to ? cells.CONCLUSION: pI/Pdx1/NeuroD1 plasmid is successfully constructed and expressed in human eukaryocytes,with which the cells express the eukaryocyte transcription factor and GLUT2,indicating the transfected cells functionally correlates to ? cells.The results suggest that Pdx1 and NeuroD1 genes can induce the differentiation the cells from hepatic cells to pancreatic endocrine secretion cells.