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6 RGC的鉴定方法
61 Thy1抗体标记 Barnstable等发现Thy1抗原是啮齿动物许多细胞表面的一种糖蛋白,Thy11抗原仅表达在大鼠RGC上,是其RGC特异性的标记物[13]。因此这种抗原的单克隆抗体——FITCThy1抗体可用来标记大鼠RGC。
62 根据胞体直径直接鉴定 Guenther等借助FITCThy11(克隆型为OX7)染色,建立了一种以体积标准(细胞直径>13 μm)来鉴定RGC的方法,未标记细胞的胞体直径为36～124 μm,标记细胞(即RGC)为84～230 μm。所有直径>13 μm的细胞都是RGC[1]。该方法的优点是不受染料的干扰,可用于研究RGC的电生理以及药物对其功能和离子通道特征的影响等领域等。但此标准仅适用于体外培养<36 h,因为>36 h其他Thy1抗体阴性的细胞也可具有同样大小。
63 电生理测定和静息电位RGC测量 Taschenberger等通过Thy1阳性标记结合电生理测定,如电压激发的Na+流大小来鉴定RGC[26]。王宗华等通过视网膜切片膜片钳技术记录到RGC的静息电位为-70 mV,并用去极化电流诱发RGC产生3种动作电位类型，再结合细胞内组织学染色,鉴定出3种不同的RGC[27]。
64 神经纤维蛋白抗体标记 Straznicky等发现抗神经纤维蛋白三体(SMI32)能标记人视网膜中大神经节细胞体和树突,对小神经节细胞亦有较弱的标记,应用此法可以区分RGC的大小、树突分布[28]。
65 免疫细胞化学双标记法 Thy11抗原虽是RGC的特异性标记,但Taschenberger等发现在体外培养中,特别是培养1周后,一类成纤维细胞也表达Thy11抗原,同时也可见于T淋巴细胞和胚胎肌细胞上,使其易与RGC混淆[26]。而作为神经元的RGC则表达成熟神经元的标记物——抗大鼠微管相关蛋白2(MAP2)。所以有学者采用Thy11单克隆抗体联合MAP2多克隆抗体对RGC进行双标记[3]。
66 P物质法 应用直接细胞内染色标记法标记RGC细胞内的P物质(SPIR),可区分两类不同大小的RGC。研究表明,SP样免疫反应物质在大多数脊椎动物视网膜定位于无长突细胞、移位无长突细胞或神经节细胞[29]。故此标记方法并不是特异性的。
7 RGC的纯化
以羊抗鼠IgG(1∶100,PBS液稀释)室温包被细胞培养皿,24 h后用DMEM培养液漂洗3次,继以羊抗鼠Thy11单克隆抗体(1∶150,PBS液稀释)室温下包被,>2 h再用培养液漂洗3次。在包被好的培养皿内加入混合RGC单细胞悬液,37 ℃条件下孵育30 min,使RGC与Thy11单克隆抗体充分结合，后去除悬液,反复漂洗清除未粘附的细胞,再用0125%胰蛋白酶消化收集粘附的RGC,最后进行纯化培养[1516]。另有学者根据RGC胞体直径大于其他视网膜细胞的原理,通过15 μm的尼龙筛网纯化RGC[1]。
8 展望
体外培养细胞一般多努力使细胞纯化,以便于观察单一细胞群体的作用。目前体外混合培养可使RGC存活约4 周,但因杂有其他细胞不利于对RGC的干预研究;而纯化培养的RGC只能存活48 h[2],对于后续研究时间太短;另外与其他细胞共培养虽可延长纯化RGC的存活时间[1823],但对干预方面的研究有无影响尚待进一步证实。因此寻求一种简便易行且存活时间较长的RGC纯化培养方法,对深入探讨RGC损伤后的细胞学和分子生物学机制具有十分重要的意义。
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