Serum-free corneal organ culture medium (SFM) but not conventional minimal essential organ culture medium (MEM) protects human corneal endothelial cells from apoptotic and necrotic cell death
Serum-free corneal organ culture medium (SFM) but not conventional minimal essential organ culture medium (MEM) protects human corneal endothelial cells from apoptotic and necrotic cell death
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DOI：
10.1136/bjo.2010.183418
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发表时间：
2011-01-01
影响因子：
4.1
通讯作者：
Engelmann, Katrin
中科院分区：
文献类型：
--
作者：
Jaeckel, Thekla;Knels, Lilla;Engelmann, Katrin
目的探讨器官培养液对人角膜内皮细胞存活的影响。方法用5种不同的培养液培养人角膜内皮细胞株HCEC-12：人角膜内皮细胞(HCEC)生长液(HCEC)、标准最低基本角膜器官培养液(MEM)+2%胎牛血清(FCS)、MEM+5%FCS和人源化无内皮细胞培养液(SFM)(加和不加抗生素)。用0.5mU·mol/L星形孢子素处理部分细胞，通过线粒体膜极化状态(活体JC-1染色)、Yo-Pro-1和碘化丙啶染色、亚G1DNA含量测定细胞核碎裂、caspase-3、caspase-8、Bcl2相关X蛋白(BAX)和B细胞淋巴瘤2(Bcl2)的免疫细胞化学检测细胞的凋亡迹象。Western blotting检测裂解caspase-3和裂解多聚(ADP-核糖)聚合酶(PARP)。结果未经处理的对照MEM细胞凋亡率明显高于F99HCEC和SFM。星状孢子素处理在所有受试培养物中都不同程度地诱导了细胞凋亡。在MEM培养的细胞中，caspase-3表达增强，caspase-8、Bax、Bcl-2和PARP表达增强，亚G1期DNA含量增加，碘化丙啶和Yo-Pro-1阳性细胞增多，线粒体膜去极化。MEM组各项参数均显著高于F99HCEC和SFM组。结论SFM在促进人脐静脉内皮细胞存活方面优于MEM。
Aim To evaluate the influence of organ culture media on corneal endothelial cell survival.Methods The human corneal endothelial cell line HCEC-12 was cultured in five different media: human corneal endothelial cell (HCEC) growth medium (F99(HCEC)), standard minimal essential corneal organ culture medium (MEM) + 2% fetal calf serum (FCS), MEM + 5% FCS, and humanised, endothelial serum-free medium (SFM) (with and without antibiotics). A portion of the cells was treated with 0.5 mu mol/l staurosporine and examined for signs of apoptosis by assessing mitochondrial membrane polarisation state (intravital JC-1 staining), by YO-PRO-1 and propidium iodide staining, by determining fragmentation of nuclei by sub-G1 DNA content, by immunocytochemistry for cleaved caspase-3, cleaved caspase-8, Bcl2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2), and by western blotting for cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP).Results The number of apoptotic cells in untreated control cultures was significantly higher in MEM compared with F99HCEC and SFM. Staurosporine treatment induced apoptosis in all tested cultures to varying degrees. Cells cultured in MEM showed stronger staining for cleaved caspase-3, cleaved caspase-8, Bax, Bcl-2 and cleaved PARP, increased sub-G1 DNA content, more propidium iodide- and YO-PRO-1-positive cells, and more mitochondria with depolarised membranes. All parameters were significantly higher in MEM compared with F99HCEC and SFM. SFM cultures were significantly less susceptible to cell stress.Conclusion SFM is superior to MEM in promoting HCEC survival.