摘要
目的:探讨β1整合素功能性过表达对角膜上皮细胞凋亡的影响及机制,为角膜细胞移植提供理论依据。方法:构建β1整合素-绿色荧光蛋白(GFP)融合基因并转染兔角膜上皮细胞。观察融合基因在角膜上皮细胞的表达及对各细胞外基质蛋白的黏附能力。检测β1整合素功能性转染对角膜上皮细胞凋亡及丝裂素活化蛋白激酶(mitogen-activated protein kinase,MAP激酶)磷酸化的影响。结果:β1整合素-GFP融合基因成功转染至兔角膜上皮细胞并过表达;β1整合素过表达能够明显增加角膜上皮细胞对各细胞外基质蛋白的黏附力并抑制角膜上皮细胞凋亡及促使MAP激酶磷酸化。结论:β1整合素过表达能有效抑制角膜上皮细胞凋亡,MAP激酶磷酸化可能在这一过程中起重要作用。
AIM： To investigate the effect of β1-integrin overexpression on the apoptosis of rabbit corneal epithelial cells and the related mechanism.
METHODS： The plasmid expressing β1-integrin-GFP fusion protein was constructed by polymerase chain reaction （PCR）, and this plasmid （β1 group） or the empty vector （mock group） was transfected into rabbit corneal epithelial cells, respectively. The expression of β1-integrin-GFP fusion gene was confirmed by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The adhesion of transfected cells to extracellular matrix （ECM） proteins was determined by adhesion assay. The apoptosis of rabbit corneal epithelial cells was assayed by Hoechst 33342 staining and DNA ladder. The phosphorylation of mitogen-activated protein （. MAP） kinase was examined by Western blot.
RESULTS： Rabbit corneal epithelial cells overexpressing β1-integrin-GFP fusion gene were successfully established. Compared with mock group, β1-integrin transfection significantly promoted the adhesive of rabbit corneal epithelial cells to ECM proteins such as laminin, fibronectin, collagen I and collagen IV. β1-integrin overexpression inhibited apoptosis and induced MAP kinase phosphorylation in rabbit corneal epithelial cells （P〈0.05）. CONCLUSION ： These data suggest that overexpression of β1-integrin confers resistance to apoptosis in rabbit corneal epithelial cells, and MAP kinase pathway may play an important role in this process.
出处
《国际眼科杂志》
CAS
2008年第8期1495-1499,共5页
International Eye Science
基金
Supported by S&T Research Project of Liaoning Provincial Department of Education(No.20061000)~~
