Cyp11a1 於小鼠視網膜之表現與Cyp11a1 基因剔除小鼠視網膜發育之探討

Abstract

Recent studies demonstrate that neurosteroids have many functions in the nervous system, including involvement in anxiety, memory and cognition, and neuron survival and degeneration. The first step in de-novo synthesis of steroids is the conversion of cholesterol to pregnenolone by Cytochrome P450 side chain cleavage enzyme (P450scc) on the mitochondrial inner membrane. P450scc is encoded by the Cyp11a1 gene and expressed in brain. However, the presence of P450scc is very low, and therefore it is difficult to detect in vivo. We have produced an antibody against a 22-amino acid peptide of P450scc to analyze its expression and location using immunohistochemistry. Our results show that P450scc is expressed in the hippocampus, amygdala, lateral septal nucleus, nucleus accumbens, and magnocellular preoptic nucleus. Pre-immune serum has no fluorescence response. Additionally, We generated SCC-Cre transgenic mice, in which the human CYP11A1 promoter drives Cre recombination allowing for detection of Cre recombinase activity in retina, illustrate that Cre recombinase activity is most strongly expressed in inner nuclear layer (INL) and ganglion cell layer (GCL). The outer nuclear cell layer (ONL) and inner plexiform layer (IPL) show weak activity. Thus, the CYP11A1 promoter may have transcriptional activity in retina. To confirm the location of P450scc within the retina, we performed immunohistochemistry and observed immunoreactivity against P450scc in the ONL, INL, IPL and GCL. The above findings suggest that neurosteroid production via P450scc may affect survival and degeneration of neurons in many locations. To further investigate the potential effect of Cyp11a1 in retinal development, we generated Cyp11a1+/+ wild type and Cyp11a1-/- knock-out mice. Retina of postnatal five days pups have not developed completely, which mainly performed nuclear layer. Immunohistochemistry studies against P450scc, however, reveal that there are no significant differences in IX fluorescence responses between Cyp11a1-/- and Cyp11a1+/+mice. Finally, we observed the morphology of retinal cells in postnatal 3-5 days Cyp11a1-/- and Cyp11a1+/+ mice by Hematoxylin and Eosin-Y staining. Again, no significant morphological differences were noted between the wild type and knock-out mice. Cyp11a1 appears critical for mice development, as knock-outs do not survive past 5-6 days; as such, we couldn’t investigate the role of Cyp11a1 gene in late retinal development. We interpret the above findings as Cyp11a1 expresses in the nervous system, such as brain and retina, and it dose not obviously involved in early retinal development.