Immortalized human corneal epithelial cells for ocular toxicity and inflammation studies.
Immortalized human corneal epithelial cells for ocular toxicity and inflammation studies.
复制标题
用于眼毒性和炎症研究的永生化人角膜上皮细胞。
DOI：
--
复制
发表时间：
1999
影响因子：
4.4
通讯作者：
A. Pfeifer
中科院分区：
文献类型：
--
作者：
E. Offord;N. Sharif;K. Macé;Y. Tromvoukis;E. Spillare;O. Avanti;W. Howe;A. Pfeifer
目的
开发具有代谢能力的人永生化角膜上皮细胞系，用于毒性和炎症研究。
方法
原代角膜上皮细胞（P-CEPI）永生化的重组猿猴病毒（SV）40 T抗原的病毒复制缺陷的逆转录病毒载体。细胞在添加牛垂体提取物的无血清培养基中生长，在第15代克隆，并且生长最好的克隆之一CEPI-17-CL 4被广泛表征为人角膜上皮的分化和代谢特征。所用方法为免疫染色、逆转录-聚合酶链反应（RT-PCR）、北方印迹分析和酶测定。
结果
CEPI-17-CL 4细胞呈典型的鹅卵石样细胞形态，传代200代以上，细胞核内均表达SV 40 T抗原。CEPI-17-CL 4细胞的免疫荧光染色对角蛋白（K）8、K18和K19以及波形蛋白呈强阳性; K3、K13和K17呈弱阳性; K4、K7和K14呈阴性。细胞因子表达（白细胞介素[IL]-1 α、IL-1 β、IL-6、IL-8、肿瘤坏死因子-α和IL-ra）、生长因子（转化生长因子[TGF]-α、表皮生长因子[EGF]、EGF受体[EGFR]、TGF-β 1、TGF-β 2和血小板衍生生长因子-β）和细胞色素P450酶（1A 1、2C、2 E1和3A 5）在CEPI-17-CL 4细胞和在活组织检查中获得的人角膜上皮样品中相似。CEPI-17-CL 4细胞代谢酶谷胱甘肽S-转移酶，醌还原酶，黄曲霉毒素醛还原酶，谷胱甘肽过氧化物酶，谷胱甘肽还原酶，超氧化物歧化酶和过氧化氢酶。
结论
CEPI-17-CL 4细胞是真正的永生细胞，并表达大量类似于原始组织的细胞因子、生长因子和代谢酶。这些特性在高传代时保持稳定，将允许对毒性、炎症和伤口愈合进行可重现的机制研究。
PURPOSE
To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies.
METHODS
Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays.
RESULTS
The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase.
CONCLUSIONS
The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.
登录
查看更多内容
影响因子：
4.4
作者：
M. Conners;R. Stoltz;S. Webb;J. Rosenberg;M. Dunn;N. Abraham;M. Laniado‐Schwartzman
通讯作者：
M. Conners;R. Stoltz;S. Webb;J. Rosenberg;M. Dunn;N. Abraham;M. Laniado‐Schwartzman
影响因子：
4.4
作者：
Christopher L. Cubitt;R. Lausch;J. Oakes
通讯作者：
Christopher L. Cubitt;R. Lausch;J. Oakes
影响因子：
4.4
作者：
S. Singhal;S. Awasthi;Satish K. Srivastava;P. Zimniak;Naseem H. Ansari;Yogesh C. Awasthi
通讯作者：
S. Singhal;S. Awasthi;Satish K. Srivastava;P. Zimniak;Naseem H. Ansari;Yogesh C. Awasthi
检测人工晶状体大疱性角膜病患者角膜中细胞因子、白细胞介素 1 α 和白细胞介素 8 的 mRNA。
影响因子：
4.4
作者：
Rosenbaum,JT;Planck,ST;Huang,XN;Rich,L;Ansel,JC
通讯作者：
Ansel,JC
人角膜上皮细胞和角膜细胞培养物中 IL-8 基因的表达。
影响因子：
4.4
作者：
Cubitt,CL;Tang,Q;Monteiro,CA;Lausch,RN;Oakes,JE
通讯作者：
Oakes,JE