结膜松弛症球结膜成纤维细胞的原代培养及形态学观察

Primary cell culture and morphological observation of bulbar conjunctival fibroblasts in conjunctivochalasis

目的:观察原代培养的结膜松弛症(CCH)球结膜成纤维细胞生长状况及形态变化,确定最佳传代时间以获得稳定、一致的CCH球结膜成纤维细胞。方法:采用组织块贴壁法获得CCH原代球结膜成纤维细胞,胰蛋白酶差速消化法进行成纤维细胞纯化,倒置显微镜下观察并记录不同时期成纤维细胞的生长状况及形态变化,免疫荧光细胞化学染色行成纤维细胞鉴定。结果:CCH结膜组织贴壁24h即可见少量细胞从组织块周围爬出,第2~7d为细胞生长对数期,细胞生长快、增殖旺盛,轮廓清晰,分布均匀,数目增多,细胞核清晰;第9~15d细胞生长进入平台期,组织块逐渐老化失去活性,细胞增长缓慢,排列疏松,体积变大,形状扁平,细胞浆内见大量颗粒状物质和小泡产生,部分细胞从培养瓶底脱落,细胞之间出现较大空隙。传代纯化后细胞的大小、形态基本一致,经鉴定为成纤维细胞,呈长梭形、扁平星状或多突的纺锤形,中间宽大,有卵圆形细胞核,两头相对细小,伴向外伸出2~3个长短不一的细长突起。结论:采用组织块贴壁法可成功获得原代CCH球结膜成纤维细胞,当细胞生长至第8d时行消化、传代可获得稳定、一致的CCH球结膜成纤维细胞。

·AIM:To observe the growth status and morphological changes of primary cultured bulbar conjunctival fibroblasts in different stages of conjunctivochalasis(CCH),and to determine the best passage time,so as to obtain stable and consistent CCH bulbar conjunctival fibroblasts.·METHODS:CCH primary bulbar conjunctival fibroblasts were obtained by tissue block adhesion method.The fibroblasts were purified by trypsin differential digestion method.The growth status and morphological changes of fibroblasts in different periods were observed and recorded under inverted microscope.The fibroblasts were identified by immunofluorescence cytochemical staining.·RESULTS:After 24h of CCH conjunctival tissue adherent to the wall,a small number of cells would be seen crawling out around the tissue blocks.The logarithmic phase of cell growth was from the 2-7d.The cells grew fast,with vigorously proliferation,clear outline,uniform distribution,increas in numbers and clear nuclei.From the 9-15d,the cell growth entered the plateau stage,the tissue blocks gradually aged and lost activity.The cells grew slowly,arranged loosely,the volume became larger,the shape became flat,and a large number of granular substances and vesicles were produced in the cytoplasm.Some cells fell off from the bottom of the culture bottle,and large gaps appeared between the cells.After subculture and purification,the size and morphology of fibroblasts were basically the same.Through cell identification,fibroblasts were long spindle,flat star or multi-process spindle,wide in the middle,oval nucleus,relatively small at both ends,with 2-3 slender processes of different lengths extending outward.·CONCLUSION:Primary CCH bulbar conjunctival fibroblasts can be successfully obtained by tissue block adhesion method.When the cells grow to the 8d,they can be digested and passaged to obtain stable and consistent CCH conjunctival fibroblasts.