炎症和高渗因素对角膜上皮干细胞活性的影响及其机制的体外研究

发布时间：2018-04-11 14:52

本文选题：炎性分子 + 高渗　；参考：《济南大学》2014年硕士论文

【摘要】：目的：通过体外使用炎性分子和高渗透压处理小鼠角膜上皮干/祖细胞系（TKE2）和原代兔角膜缘干细胞，分别模拟持续性炎症和干眼高渗因素，研究其对角膜上皮干细胞活性的不同影响及其可能的机制。方法：1.体外角膜上皮干细胞模型的建立：（1）小鼠角膜上皮干/祖细胞系(TKE2)：采用含BPE和EGF的KSFM培养基培养；（2）原代兔角膜缘干细胞：新西兰大白兔6只，耳缘静脉空气栓塞处死后剪取其眼球，无菌条件下Dispase-Trypsin/EDTA消化法分离获得原代兔角膜缘干细胞，将细胞以1000个/孔的密度接种于6孔板中，采用含10%胎牛血清的DMEM/F12培养基培养。 2．炎性分子对角膜上皮干细胞活性的影响：用10ng/ml IFN-γ，IL-1β或TNF-α处理角膜上皮干细胞8天，筛选对细胞克隆形成能力影响较大的炎性分子。采用筛选出的IL-1β进行进一步研究，用不同浓度的IL-1β（0，1，5，10，20ng/ml）处理角膜上皮干细胞8天，检测其对细胞克隆形成能力及细胞生存能力的影响。 3．高渗因素对角膜上皮干细胞活性的影响：通过不同浓度的NaCl（0，10，30，60，90mM）处理角膜上皮干细胞8天，检测其对细胞克隆形成能力及细胞生存能力的影响。 4．炎性分子和高渗因素共同作用对角膜上皮干细胞克隆形成能力的影响：用10ng/ml IL-1β复合不同浓度的NaCl处理角膜上皮干细胞8天，检测两种不同因素的叠加对细胞克隆形成能力的影响。 5.炎性分子和高渗因素对角膜上皮干细胞活性影响的可能机制研究：通过研究IL-1β或NaCl处理对角膜上皮干细胞细胞凋亡和坏死、细胞周期分布、干细胞标志物表达及活性氧（ROS）水平等方面的影响来探讨炎性分子和高渗因素对角膜上皮干细胞活性影响的可能机制。结果：1．三种不同炎性分子处理TKE2细胞，发现与对照或IFN-γ处理相比，IL-β和TNF-α处理能显著影响TKE2细胞的克隆形成能力和克隆集落大小。用不同浓度IL-1β或NaCl处理TKE2细胞发现，随IL-1β和NaCl浓度的增加，TKE2细胞克隆形成率和集落直径的减小呈现明显的剂量依赖效应；且IL-1β或NaCl同样能够显著影响兔原代角膜缘干细胞的克隆形成能力。而当IL-1β与不同浓度的NaCl共同处理TKE2细胞时，其克隆形成效率和集落直径比单独用IL-1β处理时减小的更为显著。不同浓度IL-1β或NaCl处理TKE2细胞能够不同程度的影响细胞的存活，其中高浓度的NaCl对细胞的存活影响更大。 2．高浓度的NaCl（60mM）会造成显著的细胞凋亡和坏死，使细胞周期停滞在G2/M期；而IL-1β几乎不造成细胞凋亡和细胞周期分布的改变。IL-1β和NaCl可下调TKE2细胞干细胞标记物ΔNp63的表达，且NaCl可明显上调分化上皮细胞标记物involucrin的表达。IL-1β和NaCl处理均可升高TKE2细胞内ROS的水平。结论：1.炎性分子IL-1β可抑制角膜上皮干细胞的克隆形成能力。 2. NaCl模拟的高渗透压微环境下，干细胞表现为早期凋亡甚至坏死，细胞周期阻滞。 3.炎性分子和高渗因素对角膜上皮干细胞活性的影响可能与其过度活化细胞内ROS的水平相关。
[Abstract]:Objective : To study the effects of inflammatory and high osmotic pressure on corneal epithelial stem / progenitor cell line ( TKE2 ) and primary rabbit corneal limbal stem cells in vitro .

Methods : 1 . Establishment of the model of corneal epithelial stem cells in vitro : ( 1 ) Mouse corneal epithelial stem / progenitor cell line ( TKE2 ) : cultured with the culture medium containing BPE and EGF ;
( 2 ) The corneal limbal stem cells of the primary rabbit : 6 rabbits in New Zealand white rabbits , after the ear marginal venous air embolism were killed , the eyeball was cut and the corneal limbal stem cells of the primary rabbit were separated under sterile conditions . The cells were inoculated into 6 - well plates at a density of 1000 cells / well , and cultured in DMEM / F12 medium containing 10 % fetal bovine serum .

2 . The effect of inflammatory molecules on corneal epithelial stem cell activity was studied by using 10 ng / ml IFN - 纬 , IL - 1尾 or TNF - 伪 in the treatment of corneal epithelial stem cells for 8 days . The results showed that IL - 1尾 ( 0 , 1 , 5 , 10 , 20 ng / ml ) was used to treat corneal epithelial stem cells for 8 days . The effects of different concentrations of IL - 1尾 ( 0 , 1 , 5 , 10 , 20 ng / ml ) on the formation of cell clones and cell viability were examined .

3 . The effect of hyperosmotic factors on corneal epithelial stem cell activity was studied by treating corneal epithelial stem cells with different concentrations of NaCl ( 0 , 10 , 30 , 60 , 90 mM ) for 8 days .

4 . The effects of inflammatory and hyperosmotic factors on the formation of corneal epithelial stem cell clones were studied . The effects of two different factors on the formation of corneal epithelial stem cells were studied by treating corneal epithelial stem cells with 10 ng / ml IL - 1尾 compound at different concentrations for 8 days .

5 . Possible mechanisms of inflammatory and hyperosmotic factors on corneal epithelial stem cell activity : The possible mechanism of inflammatory and hyperosmotic factors on corneal epithelial stem cell activity was investigated by investigating the effects of IL - 1尾 or NaCl on corneal epithelial stem cell apoptosis and necrosis , cell cycle distribution , stem cell marker expression and reactive oxygen ( ROS ) levels .

Results : 1 . TKE2 cells were treated with three different inflammatory molecules . Compared with control or IFN - 纬 treatment , IL - 1尾 and TNF - 伪 treatment could significantly affect the clonal formation and colony size of TKE2 cells .
When IL - 1尾 and NaCl were treated with different concentrations of NaCl to deal with TKE2 cells , the colony forming efficiency and colony diameter were more significant than that of IL - 1尾 alone . Different concentrations of IL - 1尾 or NaCl treated TKE2 cells could influence the survival of the cells in different degrees , and the high concentration of NaCl had a greater effect on the survival of the cells .

2 . High concentration of NaCl ( 60 mM ) can cause significant apoptosis and necrosis , and the cell cycle arrest in G2 / M phase ;
IL - 1尾 and NaCl could down - regulate the expression of the marker 螖Np63 of TKE2 cell stem cell marker , and NaCl could significantly increase the expression of the differentiation epithelial cell marker 螖Np63 . Both IL - 1尾 and NaCl treatment could increase the level of ROS in TKE2 cells .

Conclusion : 1 . IL - 1尾 can inhibit the formation of corneal epithelial stem cells .

2 . Under the high osmotic pressure microenvironment of NaCl simulation , the stem cells showed early apoptosis or even necrosis and cell cycle arrest .

3 . The influence of inflammatory molecules and hyperosmotic factors on corneal epithelial stem cell activity may be related to the level of ROS in over - activated cells .

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R772.2

【共引文献】