The Radiation Effect On The Gene Expression Spectrum Of IECs And Intestinal Lamina Propria Fibroblasts Of Mouse

Background: In the previous study of C60(OH)24-SLN-E for radiation preotection in mice, it has been found that the γ-H2 AX foci in intestinal lamina propria were more than that in intestinal villus epithelial cells at the 21 th day after 6 Gy 60 Co g ray exposure. The experimental phenomena implied the difference of DNA repair potential between the two kinds of cell.Objective: To investigate the molecular mechanism of the different of DNA repair capability between the intestinal lamina propria and villus epithelial cells, and to find new molecular target for the treatment of intestinal radiation sickness.Methods:(1) The primary cells of IECs and intestinal lamina propria fibroblasts were cultured and subcultured in vitro. Each kind of cells divided into two groups, the experimental cell was irradiated by 6 Gy X-ray and the control cell was non-irradiated.(2) At 6 hours after irradiation, the γ-H2 AX protein was evaluated by western blot assay.(3) At 6 hours after irradiation,total RNA from each sample was isolated and the c DNA labelled with probe was hybridized to the mouse gene chip, after which the chip was washed and scanned, the gene expression differential spectrum was assessed by GO analysis and PATHWAY analysis.Results:(1) The primary cells of intestinal villus epithelial cells and lamina propria fibroblasts have been cultured successfully.(2) The γ-H2 AX protein in the two kinds of cells were increased after X-ray irradiated and repaired six hours, however, there was no significant difference between IECs and lamina propria fibroblasts.(3)There were 9 different expression genes in the DSB NHEJ and HR repair pathway between the two kinds of non-irradiated cell, the differential expressed genes POLM,XRCC5,FEN1, XRCC2,POLD3,POLD2,EME1, RAD54 B and BRCA2 of IECs were significantly more than that of lamina propria fibroblasts. After irradiated and recovered 6 hours, the RAD54 B gene was down-regulated in lamina propria fibroblasts, more further, the POLM,XRCC5, FEN1, XRCC2,POLD3,POLD2, EME1, RAD54 B,BRCA2, RAD51 and RPA2 genes in the IECs were differentially expressed higher than that in lamina propria fibroblasts too. Besides the DSB NHEJ and HR repair pathway,there were more differentially higher expressed genes related to cell cycle pathway and apoptosis pathway in the IECs compared with lamina propria fibroblasts.Conclusion: The primary mouse IECs and lamina propria fibroblasts have been successfully cultured for the study of gene expression differential spectrum. Due to the higher expression of genes related to NHEJ and HR repair pathway, the DNA DSB repair capacity of IECs was larger than that of lamina propria fibroblasts in the irradiated mice. Among of these genes, the down regulation of RAD54 B gene in the irradiated intestinal lamina propria fibroblasts may be the key role in the delayed repairing of DSB.