大鼠Tenon''''s囊成纤维细胞体外培养及生长特性的研究

目的 探讨建立体外大鼠Tenon’s囊成纤维细胞的培养方法，并对体外培养的Tenon’s囊成纤维细胞的生长特性进行观察。方法 选用健康年幼的SD大鼠40只，采用断颈法将其处死，摘除眼球，无菌条件下，分离剪下Tenon’s囊组织，采用植块法对Tenon’s囊成纤维细胞进行体外培养；通过细胞形态学观察和细胞免疫组化染色技术对其细胞性质进行鉴定；通过细胞记数和MTT法对其生长特性进行研究。结果 原代培养的大鼠Tenon’s囊组织块接种后，约48h即可见细胞由组织块中爬出，培养8～10d，细胞汇合程度大约可达70％左右；培养14～16d，细胞完全汇合；在细胞汇合程度达到70％-80％时，可以进行细胞传代，传代细胞接种后4—5h大部分细胞贴壁，10-12d左右传代细胞完全汇合。光镜下观察，可见Tenon’s囊成纤维细胞，在汇合程度低时细胞呈细长梭形外观，汇合程度高时细胞可呈多边形改变。免疫组化染色可见Tenon’s囊成纤维细胞角蛋白染色阴性，波蛋白染色阳性。细胞生长曲线显示，在2—8d细胞处于对数生长期。结论 体外成功地培养了大鼠Tenon’S囊成纤维细胞，这不仅为青光眼滤过泡瘢痕化防治的基础研究提供了一种新的细胞来源，同时为抗结膜瘢痕化药物的研究和开发提供了一种新的简便实用的细胞来源途径。

Study on the culture of rat Tenon''''s fibroblast in vitro and its growth character

Objective To investigate the methodology of the culture of rats Tenon's fibroblast in vitro and then observe its growth character.Methods Forty young SD rats adopted in the experiment were sacrificed by breaking neck.The eyes were enucleated integrated with conjunctival tissue and conjunctival tissue was then dissected from the eye ball under asepsis conditions.The pieces of Tenon's capsule were taken from the anterior conjunctiva of those eyes.The tissue was minced and placed in a 25 mm culture dish containing Dulbecco's modified Eagle's medium(DMEM) for incubating.The fibroblast was identified by cell morphology and immunohistochemistry.The growth character of fibroblast was observed by cell counting and methylthiazolyl tetrazolium. Results During the primary cell culture,the fibroblast crept from the pieces of rat Tenon's capsule tissue on days 2 or so after it was planted,and 70% percent of fibroblast confluenced on day 8-10,all fibroblast confluenced on days 14-16.The cells may be passaged when the primary cultures of the fibrobast reached 70%-80% confluent stage.Most cells could adhere to the wall of dish and reached the confluent monolayer stage on day 10-12 after the fibroblasts were transferred to a new culture dish for 4-5 hours.The fibroblast would turn out to be fusiform if the confluent degree was low,and they would become polygon under the condition that the confluent degree was high by light microscope examination.The staining of cytokeratin of fibroblast was negative and that of vimentin of fibroblast was positive by immunohistochemistry.Cell curve showed that the cells in the passage culture growed exponentially during day 2-8. Conclusion The cultured rat Tenon's fibroblast provided a new source for study of preventing and treating the filtering bleb scarring after glaucoma filtration surgery and simultaneously for researching and developing a new anticonjunctival scar drug.