兔干眼细胞模型的建立与生物学特征评价

目的:通过体外分离及培养泪腺上皮细胞,建立干眼细胞模型,分析相关炎症因子,为进一步探究干眼治疗的有效药物奠定基础。方法:体外分离培养兔泪腺上皮细胞,并采用细胞增殖实验和免疫荧光实验对原代细胞活性和纯度进行鉴定。根据脂多糖(LPS)和肿瘤坏死因子-α(TNF-α)的IC50值,采用两者的0.5倍IC50刺激兔泪腺上皮细胞,构建干眼细胞模型即LPS组和TNF-α组,并通过细胞增殖实验、ELISA和流式方法对比两种构建干眼细胞模型的相关生物学特征。结果:兔泪腺上皮原代细胞培养12h后细胞基本贴壁,48h可见细胞形态舒展,呈长三角形。兔泪腺上皮原代细胞活性为92%以上,标志角化蛋白(Pan-cytoeratin)阳性率>90%,符合实验要求。LPS和TNF-α12h的IC50分别为20μg/mL、4.996ng/mL,采用LPS(10μg/mL)和TNF-α(2.5ng/mL)干预细胞12h后:两组细胞凋亡率明显高于空白对照组(P<0.01),组间比较,TNF-α组细胞凋亡率高于LPS组(P<0.01);两组细胞上清液中IL-1β和IL-6的含量均明显高于空白对照组(P<0.01),组间比较,TNF-α组IL-1β和IL-6的含量明显高于LPS组(P<0.01)。提示TNF-α模拟干眼的炎症反应效果优于LPS。结论:本研究成功建立了一种细胞纯度较高、相对简便、快速,模型稳定的兔干眼细胞模型,为兔泪腺上皮细胞功能及干眼的基础研究提供了更稳定的体外实验模型。

Establishment of rabbit dry eye cells model and evaluation of its biological characteristics

AIM:To further explore effective drugs for dry eye treatment by isolating and culturing lacrimal gland epithelial cells in vitro,establishing a dry eye cell model and analyzing relevant inflammatory factors.METHODS:Rabbit lacrimal gland epithelial cells were in vitro isolated and cultured,and the activity and purity of primary cells were identified by cell proliferation experiment and immunofluorescence experiment.In addition,0.5 times IC50 of lipopolysaccharide LPS and TNF-αwere used respectively to stimulate rabbit lacrimal gland epithelial cells and then establish two dry eye cell models.Finally,through cell proliferation experiment,ELISA and flow cytometry,the biological characteristics of these two dry eye cell models were compared.RESULTS:After 12h of culture,the primary cells of lacrimal gland epithelial cells basically adhered to the wall of culture bottles;and 48h later,the cells stretched and almost each of them presented a shape of a long triangle.The activity of primary cells of lacrimal gland epithelium was 92%,and the positive rate of marker Pan-rkeratin was more than 90%,which accorded with the experimental requirements.The IC50 of LPS and TNF-αare 20μg/mL and 4.996ng/mL respectively.After 12h of intervention with LPS(10μg/mL)and TNF-α(2.5ng/mL),the cell activity of the two groups was significantly lower than that of control group(P<0.01);compared between these two groups,the apoptosis rate of TNF-αgroup is higher than that of LPS group(P<0.01).The levers of IL-1βand IL-6 in the cell supernatants of the two groups were significantly higher than those of the control group(P<0.01);compared between the two groups,IL-1βand IL-6 in TNF-αgroup were significantly higher than those in LPS group(P<0.01).It was suggested that TNF-αwas superior to LPS in simulating inflammatory response of dry eye.CONCLUSION:This study successfully established a relatively simple and rapid rabbit dry eye cell model with high cell purity and stability,which provided a more stable in vitro experimental model for the basic research on the function of rabbit lacrimal gland epithelial cells and dry eye.