A Study On The Therapeutic Effect And Mechanism Of NeuroD On Radiation-Induced Intestinal Injury In Mice

Par1: A study on the expression and purification of Neuro D-EGFP fusion protein and its transmembrane transductionObjective: To construct the recombinant Neuro D-EGFP fusion protein and verify its capability of transmembrane transduction.Methods: To construct the prokaryotic expression vector of Neuro D-EGFP and the expression of Neuro D-EGFP fusion protein was performed in E.coli BL21(DE3), then obtain the purified fusion protein by the chromatography column. Verify the Neuro D-EGFP fusion protein’s capability of transmembrane transduction by the fluorescence microscopy and the laser scanning confocal microscope.Results: The prokaryotic-system based expression vector was constructed successfully. The Neuro D-EGFP fusion protein was obtained by inducible expression and purification in prokaryotic expression system and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Green fluorescence was observed gathered within the cells after the Neuro D-EGFP fusion protein was added in the culture medium. Five hours after intraperitoneal injection of Neuro D-EGFP, green fluorescence gathered within the cells of the intestinal villi epithelium.Conclusion: The recombinant Neuro D-EGFP fusion protein was obtained successfully by inducible expression and purification, and its capability of transmembrane transduction was verified.Par2: The therapeutic effect of Neuro D on radiation-induced intestinal injury in miceObjective: To explore the therapeutic effect of Neuro D-EGFP fusion protein on the mice of intestinal radiation injury.Methods: Two batches of C57 mice were randomly divided into 3 groups, the PBS group, the EGFP group and the Neuro D-EGFP group. Two batches were treated with 8Gy and 9Gy total body irradiation(TBI). After the whole-body exposure to radiation, the weight change and survival were recorded, the survival curve was plotted. Through the H&E staining to observe the Neuro D-EGFP fusion protein’s effect on the intestinal pathological changes of the mice after 9Gy TBI. Count the number of apoptotic intestinal crypt cells through the TUNEL, and the number of Ki-67+ intestinal crypt cells through the Ki-67 staining. To explore the effect of Neuro D-EGFP fusion protein on the intestine of mice after γ-ray radiation.Results: The weight change and survival of 8Gy TBI mice were ameliorated after Neuro D-EGFP fusion protein treated. The damage of intestinal villi and crypts of mice after 9Gy TBI were relieved. Compared with PBS and EGFP-administrated group, The Neuro D-EGFP treated mice show higher level of villus height, the crypt depth and the number of crypts(F=49.49,16.72,10.32,P<0.01), and a reduced number of apoptotic intestinal crypt cells(F=42.39,P<0.01) and a increased number of Ki-67+ intestinal crypt cells(F=33.21,P<0.01).Conclusion: Neuro D-EGFP fusion protein promoted the repair of intestinal villi and crypts of mice after radiation and reduced the number of apoptotic intestinal crypt cells. This protein also increased the number of proliferated intestinal crypt cells. Taken together, Neuro D showed the therapeutic effect on the intestinal radiation injury.Par3: The mechanism of therapeutic effect of Neuro D on radiation-induced intestinal injury in miceObjective: To explore the mechanism of Neuro D-EGFP fusion protein’s therapeutic effect on the mice of intestinal radiation injury.Methods: Microarray technology was used to detect the intestinal gene expression of mice after 9Gy total body irradiation treated by Neuro D-EGFP fusion protein. And q RT-PCR was used to verify its differential expressed genes such as Enpp7, Mbl2, Slc13a1 and Slc40a1. By filtering the target gene of Neuro D, the immunohistochemical staining was used to measure the Timp-1’s expression of intestine of mice after TBI.Results: Many genes and pathways were dysregulated by Neuro D through the microarray technology. And its result was verified by q RT-PCR. GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis indicated Neuro D differential expressed genes were involved in multiple biological processes and signaling pathways. And Neuro D could increase the intestinal expression of Timp-1 after TBI. These results preliminarily illustrated the mechanism of Neuro D-EGFP fusion protein’s therapeutic effect on the mice of intestinal radiation injury.Conclusion: Neuro D affected many genes and pathways, which finally promoted the repair of intestine of mice after radiation. Specifically, Neuro D possibly conferred radioprotection through increasing the expression of anti-apoptotic Timp-1.