| BackgroudNueral tube defects (NTD) had a high incidence rate, China is a higher incidence country. Myelomeningocele(MMC) is the most common NTD in live born baby, which can lead to lifelong physical disabilities, including paraplegia, urinary and fecal incontinence, hydrocephaly, sexual dysfunction, skeletal deformations and mental impairment. Retinoic acid can induce pregnent SD rats produce MMC fetal rats, which can simulate the human neural tube closure disorders with various diseases, is a good model for research of human MMC.MMC is the main cause of congenital neurogenic bladder dysfunction (NBD) in children. There had fewer research focused on spinal cord damaged by MMC in the past. Spinal cord injury (SCI) related experements mainly used the method of transected spinal cord. And further research found that HGF/C-Met and PAs expression changed in spinal cord in such a model, and these changes in cytokine expression have a neutoprotective effects. MMC fetal rats induced by retinoic acid lead to spinal cord injury related research had not been reported until present. In this study, we use retinoic acid induced MMC fetal rats and research the cytokine expression in the spinal cord.Objective:To assessed HGF/C-Met and PAs cytokines expression in spinal cord by Real Time-PCR with different damaged degree of MMC fetal rats, to understand these cytokines expression changes with different damage and gestational age.Methods:All experimental protocols were approved by the Clinical Pharmacology Base Committee of Fudan University and the Pediatric professional group Ethical Committee.(Approved material attached page) SD pregnant rats were gavage fed on9am at E10. Experimental groups of pregnant rats were gavage fed with all-trans retionoic acid dose of60mg/kg, dissolved in2ml olive oil; control group pregnant SD rats were fed only2ml olive oil. Fetus were harvested at embryo16,18,20days (E16, E18, E20) by cesarean section. Different groups were divided according to the sacrococcygeal skin and the lamina arch of closed. MMC-defect group fetal rats have a damaged sacrococcygeal skin and the lamina arch is not closed; MMC-SBO group fetal have a complete sacrococcygeal skin but the lamina arch is not closed; MMC-no-defect group fetal rats have a complete sacrococcygeal skin and the lamina arch is closed. The conrtol group fetal rats have a normal shape. There are total12groups and3-6fetal rats each group.Real Time-PCR assess of cytokines such as HGF in different groups:Dissecting microscope was used to dissect the fetal lesion spinal cord. RNA was extracted. HGF, its receptor C-Met, tPA, uPA and β-actin were underwent reverse transcription and PCR ampliation. The data of each group was got by power (2,-ΔΔct).The data was analysed by SPSS17.0ANOVA and LSD for statistical analysis.Results:19pregnant rats accepted the retinoic acid fed in experimental group,176fetus were harvested totally, of which53MMC-defect rats(30.1%),10MMC-SBO rats(5.7%),87MMC-no-defect rats(49.4%),7rats(4.0%)associated with other malformations,19stillbirth or absorption rats(10.8%). Control group of4pregnant mice, produced a total of55fetal mice, no obvious appearance of anomalies.HGF expression in the MMC-defect group is higher than the other three groups, and an increased trend over time; HGF expression in the MMC-SBO group is higher than MMC-no-defect group and control group at E18, E20; HGF expression showed no significant difference between MMC-SBO and control group; and no significant difference over time in the MMC-SBO group, MMC-no-defect and control group.C-Met expression in the MMC-defect group is higher than the other three groups, and show increased trend over time; C-Met expression show no significantly difference in the MMC-SBO group, MMC-no-defect group and control group at E16, E18. The former two groups expression is higher than control group at E20; C-Met expression showed no significant difference over time in control group, but expressed higher significantly in MMC-SBO and MMC-no-defect group.t-PA expression showed no significant difference between MMC-defect group and MMC-SBO group; and showed no significant difference between MMC-no-defect group and control group; and the former two groups expression is higher than the latter two groups. tPA expression showed no significantly different over time at each group.u-PA expression in the MMC-defect group is higher than the other three groups, and show increased trend over time; u-PA expression in the MMC-SBO group is higher than MMC-no-defect group and control group at E18、E20, and show increased trend over time in the MMC-SBO group; u-PA expression showed no significant difference between MMC-no-defect and control group; and showed no significant difference over time in the MMC-no-defect and control group.Conclutions:Retinoic acid gavage fed pregnant SD rats at dose of60mg/kg at E10can induce different degree of neural dysraphism fetal rats:including MMC defect rats, MMC spinal bifida occulta rats and no obvious defect rats, and a certain percentage of associated with other malformations and stillbirth and absorption fetus.HGF and its acceptor C-Met, uPA and tPA mRNA expression is higher in MMC-defect group than MMC-no-defect group and control group.HGF and its acceptor C-Met, uPA expression is higher in MMC-defect group than MMC-SBO group. Maybe these cytokines expression have a relation with neural tube closure and spinal cord exposed to amniotic fluid. |
PDF Full Text Request