Mer-/-视网膜色素上皮细胞在吞噬光感受器外节盘膜中的基因表达谱的改变

目的：研究在视网膜色素上皮细胞（RPE）吞噬光感受器外节盘膜（POS）时，由于RPE的Mer基因敲出（Mer-/-）后，所导致的基因表达谱的改变。方法：将RPE从Mer-/-小鼠（实验组）和野生型小鼠（对照组）中分离出后，并培养至第3代。在实验组和对照组中，将提取的POS加入含有20nMGas6和Protein S的培养液中，以激活Mer受体通道所介导的RPE特异性吞噬POS。在刺激吞噬后3小时和12小时终止实验，在两组中分别通过显微镜评价RPE吞噬POS的能力，通过基因表达谱芯片筛选出差异表达的基因。并且，在相同的实验条件下，在实验组和对照组中，分别重复3次吞噬实验，采用QPCR，对于其中5个差异表达的基因进行确认。结果：显微镜下观察到不论在3小时还是12小时，与对照组相比，实验组中内吞的POS颗粒少。在基因表达谱芯片中发现，反应3小时和反应12小时，实验组中分别有38个和45个已知基因上调，68个和59个已知基因下调。与对照组相比，实验组中RPE内吞POS能力的下降与基因表达谱中差异表达的基因相一致，比如，发现信号转导（WNT,MAPK）、吞噬（Vav3, Hsd11b1）、细胞骨架成份（Myo7a）和代谢等方面相关的基因在不同的时间点均有不同的表达谱改变。QPCR结果发现Vav3、Hsd11b1、Myo7a、Rtn2等基因的表达变化与基因芯片结果相一致。结论：Mer-/-基因敲出后的RPE在吞噬过程中随时间变化而出现的不同的基因表达谱的改变，给研究Mer受体通道介导的RPE吞噬POS的分子机制提供了新的研究方向和线索，特别有利于MerTK基因突变所致的人类视网膜色素变性患者发病机制的研究。

Gene expression profile changes caused by the dysfunction of Mer during retinal pigment epithelium phagocytosis

PurposeTo identify changes in gene expression profiles caused by Mer gene knockout (Mer-/-) during phagocytosis of photoreceptor outer segments (POS) in retina pigment epithelium (RPE).MethodsRPE from both Mer-/- and wild-type (WT) mice were isolated and cultured to the 3rd passage. POS were subjected to culture medium with 20 nM Gas6 and Protein S to activate specific Mer-mediated phagocytosis. RPE phagocytosis was evaluated by phagocytosis assays and differential gene expression identified by microarray at 3 and 12 hours; the 0-hour time point served as the control. Three independent samples for each Mer-/- or WT RPE were subjected to the same protocol of microarray. Five genes were confirmed by real-time quantitative PCR (QPCR).ResultsThe Mer-/- RPE had less internalized POS than WT RPE after both 3 and 12 hours in phagocytosis assay. Compared to WT RPE and the 0-hour control, 38 and 45 different known genes were increased and 68 and 59 known genes were decreased in Mer-/- RPE after 3 and 12 hours, respectively. Abnormal POS phagocytosis in Mer-/- RPE was associated with significant gene expression changes in, for example, signal transduction (WNT,MAPK), phagocytosis (Vav3,Hsd11b1), cytoskeleton components (Myo7a), and metabolism, in a time-specific manner. QPCR results showed Vav3, Hsd11b1, Myo7a, Rtn2 and Itga8 in those independent samples were consistent with microarray.ConclusionsGene expression profiles modulated in a time-specific manner in Mer-/- RPE indicate a possible internalization mechanism for abnormal POS phagocytosis, which gives insight into the mechanism of retinitis pigmentosa caused by the mutation of MerTK in humans.