培养兔角膜基质细胞的一种新方法——悬浮基质片法

Suspended stroma culture technique,a novel method for culturing rabbit corneal stromal cells

目的寻找一种新的、简易的兔角膜基质细胞培养方法。方法去除兔角膜上皮层、后弹力层及内皮细胞层,将处理后剩下的全层基质(简称基质片)置于6孔板中,分别进行悬浮培养(悬浮基质片法)、组织块贴壁培养法培养、Ⅱ型胶原蛋白酶消化法培养。观察所培养细胞的体外生长特性,并进行波形蛋白免疫组化鉴定。结果采用悬浮基质片法可成功培养出兔角膜基质细胞,细胞具有长短不等的数个突起,呈梭形、不规则三角形或纺锤形,核椭圆、居中,胞浆清亮,未见其他细胞混杂其中。悬浮培养8～10d贴壁,贴壁后约5d基本融合。其生物学特性与组织块贴壁培养法及Ⅱ型胶原蛋白酶消化法培养的细胞一致。波形蛋白免疫组化染色显示3种方法均为阳性。结论悬浮基质片法培养角膜基质细胞,不需用酶,且具有简便、可靠、不易污染、成功率高等明显优势,为角膜基质细胞的培养提供了新的途径,值得推广。

Objective To explore a novel,simple method for culturing rabbit corneal stromal cells.Methods Fresh rabbits＇ corneas were obtained,and the epithelium,descemet＇s membrane and endothelium were removed.The stroma slices after treatment were cultured in 6-well culture plate,and treatment with suspended culture（suspended stroma culture technique）,tissue adherence culture technique,type Ⅱ collagenase digestion,respectively.The biological characteristics of the cultured cells in vitro were observed,and these cells were identified by vimentin immunostaining.Results Rabbit corneal stromal cells were successfully cultured with suspended stroma culture technique,with numbers of apophysis at different lengths,with spindle shape,irregular,triangle or fusiform.Nucleus were with ellipse shape and at center.Cytoplasm was clear and without other kinds of cells.Cells were with adherence after suspended culture for 8 days to 10 days and with fusion at 5 days later.The biological characteristics of suspended stromal culture technique were similar with those cultured by traditional culture techniques or type Ⅱ collagenase digestion.Cultured cells were positive with vimentin immunostaining by three methods.Conclusions The suspended stroma culture technique for culturing rabbit corneal stromal cells has many obvious advantages,such as simple,reliable,pollution-proof and highly successful rate.It provides a new way for culturing corneal stromal cells and needs to be popularized.