【作者】 李艳；

【导师】 周国民； 陈祖林；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 水平细胞和无长突细胞是视网膜中间神经元,分别位于INL两侧。目前,研究表明水平细胞和无长突细胞在发育形成时紧密关联；而且脊椎动物水平细胞具有重要意义,体现在有其独特的发育过程,如双向迁移方式、可塑性高和发育晚期分裂以及成熟水平细胞能重新进入细胞周期等特点,这些独特性挑战了当前人们对神经发育模式和癌症生物学的认识。最近,研究又证实鱼类水平细胞能直接感光,打破了传统认为水平细胞间接影响感光功能的局限性。但是,关于成年哺乳动物INL损伤和再生的相关研究却少见。本课题旨在建立INL变性模型；观察Muller细胞是否具有干细胞特性,包括活化、去分化、增殖及再生出新的水平细胞等,以期扩大对水平细胞的认识；最后,探讨内源性明胶酶在本模型的视网膜变性与再生中的作用。主要的研究方法和内容如下：1、建立INL变性模型。①经SD大鼠玻璃体腔注射不同剂量的哇巴因,HE组织学观察哇巴因损伤视网膜的部位是否在INL,以及确定合适的剂量。②采用HE染色、透射电镜和TUNEL方法检测不同时间点INL损伤程度及细胞死亡方式,③运用免疫荧光法确定哇巴因损伤的细胞类型,western-blot半定量calbindinD-28k蛋白量变化。2、MUller细胞具有干细胞特性。①经免疫荧光法单标GS表达变化、以及双标GS/GFAP和GS/nestin用来检测Muller细胞是否被活化；Western-blot检测GS和GFAP蛋白表达量。②透射电镜显示INL是否存在有丝分裂核形态。③采用免疫荧光法标记Brdu与PCNA标记以检测INL增殖细胞,并分别与GS双标,显示MUller细胞增殖情况。④经免疫双标Brdu/Proxl和Brdu/calbindin D-28k,以分析新的神经元再生；并填加生长因子SHH,观察它对再生的影响。3、MMPs,尤其MMP9在哇巴因致INL神经元死亡和Muller细胞增殖的作用。①MMP2／9在哇巴因致视网膜变性中的表达和活性。采用明胶酶谱和原位酶谱检测MMP2／9的活性,运用Western-blot和免疫组化检测MMP9表达。②采用明胶酶谱和原位酶谱法检测MMP9i是否能抑制内源性MMP9活性。③MMP9i在哇巴因致视网膜INL神经元死亡的作用。指标是INL厚度(DAPI染色)、calbindin D-28k和syntaxin免疫标记阳性细胞数量；Western-blot检测calbindin D-28K蛋白表达量。④经DAPI染色,观察视网膜中凋亡样细胞核数量和Western-blot检测caspase3表达量。⑤MMP9i在哇巴因致视网膜INL变性后Muller细胞增殖的作用。应用免疫双标法,检测GS和GFAP在视网膜的共表达情况,Western-blot检测GFAP蛋白表达量；采用Brdu和PCNA标记物分别与GS行免疫双标,显示Mtiller细胞增殖情况。主要的研究结果和结论如下：1、大鼠水平细胞和无长突细胞对哇巴因敏感当哇巴因剂量达到2.75nmol,玻璃体注射后3d时,HE组织学观察可见SD大鼠视网膜INL开始变薄,细胞数量下降；至剂量高至22nmol时,INL厚度显著变薄,其它层次无明显变化。选用22nmol剂量,HE染色显示哇巴因致视网膜变性的病理进程,结果如下：在给药后6h、12h和1d时,INL可见许多浓缩的凋亡样细胞核,细胞间隙变大；2d时,INL凋亡样细胞核减少,仅剩1-2层细胞；4d至28d时,INL厚度再无明显变化,这表明2d是ouabain引起视网膜INL明显损伤的时间点。透射电镜显示给药后7h和1d时,在INL水平细胞位置均有凋亡和坏死特征的细胞。TUNEL染色显示INL中TUNEL阳性细胞数量最多,给药后12h达到峰值。免疫荧光染色经统计分析显示,2dpi组calbindin D-28k阳性水平细胞数量明显减少,syntaxin阳性无长突细胞次之,PKCa双极细胞以及rhodopsin感光细胞等变化不明显。2、成年哺乳动物水平细胞和无长突细胞的再生哇巴因经玻璃体单次注射后7d内,视网膜GS(Muller细胞标记物)表达(?)过性下降、Nestin(神经干细胞标记物)表达先上调后下降,表明Muller细胞被激活并且去分化成神经干细胞。同时Muller细胞掺入brdu和表达PCNA,表明Muller细胞发生增殖,并在2d时能力最强。延长观察时间至15d、21d和35d,部分Brdu阳性细胞分别表达proxl和calbindin D-28k(标记所有水平细胞和小部分无长突细胞),而且双标阳性细胞位于水平细胞和无长突细胞的位置,表明在无外源性生长因子辅助刺激下,大鼠视网膜水平细胞和无长突细胞再生成功。SHH-N不能促进这两种细胞再生。。3、MMP9减缓哇巴因致视网膜INL神经元非凋亡性死亡和促进Muller细胞增殖不同剂量哇巴因诱导视网膜急性表达MMP9并且有活性,于22nmol剂量时MMP9活性最强；同一剂量(22nmol)哇巴因作用下,在0-4d检测视网膜MMP9的活性,结果显示出先增加后降低的趋势,在2d时最强；原位酶谱显示2d时,实验组视网膜在原位有明胶酶活性,结合免疫组化结果提示MMP9可能来源于Muller细胞、INL神经元和GCL细胞。但在7-21d时,MMP2在损伤的视网膜具有活性。MMP9i和哇巴因联用后2d时,结果表明MMP9i能抑制大部分内源性MMP9活性。MMP9i的作用之一是加剧了INL厚度变薄、calbindin D-28k和syntaxin免疫阳性数量减少；而减少INL中凋亡样细胞核,下调caspase3蛋白表达。综合这些变化,表明在2d时,内源性MMP9能诱导凋亡,但延缓哇巴因致视网膜INL神经元的非凋亡性死亡。另一作用是MMP9i下调GFAP在视网膜的表达,并且在给药后4d时效果明显；同时发现2d时MMP9i抑制Muller细胞的增殖能力,表明内源性MMP9能促进哇巴因致视网膜INL变性后的Muller增殖。更多还原

【Abstract】 Retina is an organ forming the vision. Horizontal cells (HCs) are, together with amacrine cells, retinal interneurons that lie within the inner nuclear layer (INL).They play a role in color vision formation. Vertebrate retinal HCs have recently been shown to exhibit a variety of unique biological properties, as compared with other nerve cells, that challenge many long-standing assumptions in the fields of neural development and cancer biology. These features include their unusual migratory behavior, their unique morphological plasticity, and their propensity to divide at a relatively late stage during development. At adulthood, fully differentiated mouse HCs have the ability to re-enter cell cycle and to give rise to metastatic retinoblastoma. In addition, the latest study showed intrinsic light response of retinal HCs of teleosts. There results suggest their significance. However, few papers have reported on HCs damage and regeneration since less attention is paid to HCs. In our study, we establish the animal model of INL degeneration, in SD rats and observe stem cell potential of Muller cell,including horizontal cells regeneration, which may broaden our understanding of HCs.; explore the roles of gelatinases.The methods are as following:1 Establishment of INL, especially HCs degeneration animal model.①INL degeneration was induced by intravitreal injection of the different doses of ouabain into Sprague-Dawley rats.②The degree of INL damage and cell death pathway were investigated by HE staining, TUNEL and transmission electron microscopy.③The types of ouabain-injured retinal cells were identified by immunoflurorescent staining of specific antigens of retinal cells and synapses and western-blot semi-quantitive analysis of calbindin D-28k protein.2 INL degeneration induced stem cell potential of Muller cell.①Muller cell activation was detected by immunostaining, single and double, of GS, GFAP and Nestin. The change of GS and GFAP protein was assessed by western-blot.②the mitosis in INL was observed by transmission electron microscopy.③Double staining of GS/brdu and GS/PCNA was applied to analyze the number of proliferating Mullercells in ouabain-induced retinal degeneration.④Double staining of brdu/prox1 and brdu/calbindin D-28k were used to detect new HCs and ACs regeneration in adult rats. The effect of SHH-N on retinal regeneration was assessed by the same indicator.3 Endogenous MMPs, especially MMP9 functions in neuronal death and Muller cell proliferation.①The expression and activity of MMP9 in ouabain-treated retina. Gelatin zymography and in situ zymography were used to detect MMP2/9 activity; western-blot assays and immunohistochemistry were used to analyse MMP9 expression.②The effect of MMP9 inhibitor on endogenous MMP9 activity was assessed by gelatin zymography and in situ zymography.③The influence of MMP9 inhibitor on ouabain-induced retinal damage. The indicators were INL thickness, the number of calbindin D-28k and syntaxin immnuopositive cells, respectively, and the change of calbindin D-28k protein by western-blot.④The effect of MMP9 inhibitor on INL apoptosis was determined by the number of apoptosis-like nuclear by DAPI staining and the change of caspase 3 protein by western-blot.⑤The effect of MMP9 inhibitor on Muller cell proliferation. Double immunostaining of GS/GFAP, GS/brdu and GS/PCNA were used to detect Muller cell activation and proliferation. GFAP protein was analyzed by western-blot.Results and conclusions:1 Rat HCs and ACs were sensitive to ouabain.At 3 dpi (day post injection), ouabain induced a rapid and obvious cell loss in the INL at all doses≥2.75 nmol, with little effect on GCL and ONL. The effect was dose- and time-dependent; at 22nmol dose and 2dpi, the damage was the severest. TUNEL staining showed there were many apoptotic nuclear in INL; at 12hpi, the number of TUNEL positive cells reached a peak. Immunostaining showed calbindin D-28k and syntaxin positive cells were lost, with horizontal cells the most susceptible.2 HCs and ACs were regenerated from adult mammalian retina.After ouabain treatment, actived Muller cells dedifferentiated into stem cells via downregulation of GS expression (mature Mullercells marker) and upregulation of Nestin expression (neuronal stem cell marker), and proliferated through brdu incorporation and upregulation of PCNA, with 2dpi the most pronounced. At 15dpi,21dpi and 35dpi(single ouabain injection), a portion of brdu positive cells expressed prox1 and calbindin D-28k, respectively (marker for all HCs and a fraction of amacrine cell), and these double immunopositive cells were located in horizontal cell and amacrine cell layer. These suggest HCs and amacrine cells regeneration from adult mammalian retina. SHH-N did not promote the two types of cell regeneration.3 MMP9 protected retinal interneurons from ouabain-induced non-apoptotic cell death and enhanced Muller cell proliferation.Ouabain induced acute MMP9 expression and resulted in an increase in MMP9 activity. At the highest dose (22nmol) and 2dpi, MMP9 active was the strongest. Muller cells and neurons in INL and cells in GCL upregulated MMP9 protein and activity. While, there was MMP2 activity in retinas at 7-21 days after ouabain treatment. At 2dpi, MMP9 inhibitor blocked most of endogenous MMP9 activity. The effect of MMP9 inhibitor on ouabain-induced retinal damage was to accelerate the change through the decrease in INL thickness and the number of calbindin D-28k and syntaxin immunopositive cells in INL. However, it resulted in the decrease in the number of apoptotic-like nuclear in INL and caspase 3 protein. Therefore, endogenous MMP9 may protect retinal interneurons from ouabain-induced non-apoptotic cell death. With respect to Muller cell reaction, the inhibitor inhibited Muller cell activation through downregulation of GFAP in NF layer and proliferation via the decrease of brdu incorporation and PCNA expression in Muller cell.更多还原