分析 Cyp11a1 基因剔除對發育中小鼠視網膜細胞凋亡之影響

Abstract

由神經系統合成之不同神經類固醇，可以調控神經系統之發育以及生理上之功能。許多文獻指出，神經類固醇，例如：progesterone、estrogen，扮演保護神經細胞存活之角色。視網膜為視覺神經系統之ㄧ部分，其本身可生成神經類固醇，例如：pregnenolone，並受到類固醇之調控。DHEA/DEHAS, 17β-oestradiol 和 3α5βS 可保護受損視網膜細胞之存活。然而，pregnenolone sulfate 會更促進 NMDA 所誘發之視網膜細胞凋亡。由於 Cyp11a1 基因是參與類固醇生成之重要酵素，本篇研究利用 Cyp11a1 基因剔除小鼠探討類固醇對視網膜細胞存活之影響。 Cyp11a1 基因剔除 (KO) 鼠由於缺乏類固醇之生成，在出生後約六天就會死亡。五天大之 Cyp11a1 KO 鼠其重量為同胎之野生型 (WT) 與異合子型 (HE) 幼鼠 25%；Cyp11a1 KO 鼠之視網膜無法偵測 Cyp11a1 mRNA 之表現。另外，本實驗室建立的 SCC-Cre 轉殖小鼠是以人類 CYP11A1 啟動子驅動 Cre 重組酶之表現，於五天大之 SCC-Cre 轉殖幼鼠之視網膜內可看到明顯之 Cre 重組酶活性，主要分布於內細胞核層 (INL) 無長突細胞 (amacrine cells) 之位置，而神經節細胞核層 (GCL) 與外細胞核層 (ONL) 則有較弱之呈色，說明 CYP11A1 啟動子於五天大幼鼠視網膜有轉錄活性。利用 TUNEL assay 觀察 Cyp11a1 KO 幼鼠與 WT 幼鼠視網膜細胞之凋亡，可看見五天大幼鼠視網膜之 INL 細胞凋亡數目最多，且 Cyp11a1 KO 幼鼠視網膜細胞凋亡之程度顯著低於同胎之 WT 幼鼠，表示類固醇缺乏會導致視網膜細胞凋亡程度下降。進一步利用 western blotting 分析 WT 與 KO 幼鼠視網膜細胞凋亡之分子機制，發現 KO 幼鼠視網膜細胞之 procaspase-3 表現量高於 WT 幼鼠，表示 caspase-3 於KO 視網膜表現較低；而 Bcl-2 表現量也高於 WT 幼鼠，顯示 caspase-3 與 Bcl-2 有參與在視網膜細胞凋亡之路徑。综合上述結果，Cyp11a1 缺乏會降低發育中視網膜細胞之凋亡。而其透過之細胞凋亡路徑，有 Bcl-2 與 caspase-3 參與其中。

Neuroactive steroids can be synthesized de novo in the nervous system that influence the brain development and many neurophysiological functions. It’s recording that neurosteroids such as progesterone and estrogen could protect neuron cells from apoptosis. Retina is a target of steroids, and it also could produce neurosteroids like pregnenolone. DHEA/DEHAS, 17β-oestradiol and 3α5βS reduced the cell death in retinal excitotoxicity. However, pregnenolone sulfate increased the NMDA-induced excitotoxic retinal cell death. In this study, we utilize knockout mice disrupted with Cyp11a1, the key gene controlling steroidogenesis, to explore the role of steroids in retina apoptosis. Cyp11a1 null mice lack the synthesis of steroids and die about postnatal 6 days. By 5 day of age, Cyp11a1 null mice gained 25% less weight than their wild-type and heterozygote littermates. Expression of Cyp11a1 mRNA could not be detected in Cyp11a1 KO mice. Additionally, we generated SCC-Cre transgenic mice, in which the human CYP11A1 promoter drives Cre expression. Cre recombinase activity was detected in retina at postnatal day 5, indicating CYP11A1 promoter is functional in mouse retina. We used TUNEL assay to investigate the apoptosis in retina of WT and KO mice. On postnatal day 5, the majority of the apoptotic cells detected by TUNEL assay were observed in the inner nuclear layer of retina. The number of TUNEL positive cells in the retina of Cyp11a1 null mice was significant reduced when compared with their wild-type littermates. It indicated that apoptosis of retina was decreased in the condition of steroids deficiency. To understand the molecular pathway, we analyzed the expression of procaspase-3 and Bcl-2 in WT and KO retina. The protein levels of Bcl-2 in KO retina were significantly increased compared with WT. The protein levels of procaspase-3 in KO retina were higher than those in WT, suggesting that caspase-3 activity was reduced. Therefore, caspase-3 and Bcl2 could be involved in the steroid-mediated retinal apoptosis.