Study On Animal Model Of Neural Tube Defects Induced By PMX And Its Mechanisms

Objective:This study aims to investigate the effects of PMX on the development of NTDs and explore its pathogenesis by use of a high-thoughput genechip technology, array-comparative genomic hybridization (aCGH) to detect the copy number variations (CNV) in PMX-induced NTDs and the genome-wide expression microarray to detect gene expression profiles in NTD embryos and select the potential morbific genes to NTDs.Method:Animal model of neural tube defects (NTDs) associated with folate dysmetabolism was induced by intraperitoneal injection of pemetrexeddisodium (PMX) into C57BL/6J pregnant mice. Healthy adult female C57BL/6J mice (7～8weeks old) were randomly divided into two groups for conception. The pregnant mice in experiment group and control group were treated by intraperitoneal injection of PMX with the optimal dose and normal saline with the same dose, respectively on gestation day (GD)7.5. The pregnant mice were sacrificed on GD11.5in both groups and the characteristics of the fetuses were carefully observed under a dissecting microscope. Some abnormal fetuses were fixed, sliced and stained to observe the type and morphologies of neural tube defects under microscope. We used high performance liquid chromatography (HPLC) method to detect the serum levels of folate acid and its metabolic products in pregnant mice treated by PMX in different time point. Enzyme-linked immunosorbent assay (ELISA) was used to determine the embryotic levels of folic acid on GD11.5of pregnant mice treated by PMX and,the DHFR activity in PMX treated embryos was tested by Dihydrofolate Reductase Assay Kit. Besides, RT-PCR was used to detect the gene expressions of key enzymes in folic acid metabolic pathway. Furthermore, high-thoughput genechip technology was applied to screen differentially expressed genes of embryo between normal mice and NTD mice. GO analysis was used to analyse the differentially expressed genes. We also used Real time-PCR to validate the differentially expressed genes, including Ptch1and Endog. Finally, oxidative damages in murine embryonic stem cells caused by PMX were measured in vitro and single cell gel electrophoresis was used to determine the DNA damages induce by PMX.Results1. PMX caused significant embryo malformations in pregnant mice which presented obvious hyperemia in uterus. Absorptive fetus, hypoevolutism and NTDs of embryos were found under anatomy scope. HE staining showed that the embryonic hindbrain was not closed.2. After intraperitoneal injection of PMX into pregnant mice on GD7.5, serum levels of folate acid increased significantly at4hour and12hour and reduced to normal at24hour and96hour. No significant changes of5-FoTHF were found. But levels of5-MeTHF, SAM and SAH decreased significantly and began to recover at24hour, but still significantly lower than normal at96hour (P<0.05). Besides, folate levels in embryotic tissues were also significantly reduced by PMX treatment. DHFR activity in embryo was reduced to the lowest level at4-8hour, which is only15%of DHFR activity in normal embryo. After then, DHFR activity began to recover, but was still lower than that in normal embryo at24hour. RT-PCT showed that the mRNA expressions of5,10-methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) were significantly decreased by PMX treatment (p<0.05)3. Array comparative genomic hybridization,(aCGH) assay selected313CNV fragments, including101mutated genes.165differentially expressed genes were screened by the high-throughput genechip technology in NTD mice, including96up-regulated genes and69down-regulated genes. GO analysis showed that the differentially expressed genes involved in various metabolisms including cellular metabolic process, development process, signal transduction, transcription, material transportation, stress response, ion transportation, cell apoptosis, cell proliferation, cell differentiation, cell adhesion, cell cycle, et al. The results of RT-PCR was in accordance with that of the genechip technology (p<0.05).4. Results of single cell gel electrophoresis showed that PMX caused direct DNA damages in murine embryonic stem cells in a dose-dependent manner. As the concentration of PMX increased, DNA trailing become more obvious, leading to obvious oxidative stress injuries in murine embryonic stem cells.Conclusion1. Animal model of NTDs was successfully established by PMX in C57BL/6J mice, which is an model similar to human NTDs.2. Anti-folate drug, PMX can inhibit folate and its related metabolisms in pregnant mice and induce CNV in NTDs.3. Up-regulation of Ptch1and down-regulation of Endog both play pivotal roles in the development of NTDs and these two genes may be the candidate genes for PMX-induced NTDs.4. PMX caused DNA damages in murine embryonic stem cells, oxidative stress injuries of mice and apoptosis of embryonic stem cells, leading to abnormal closure of neural tube and ultimately NTDs.