碱性成纤维细胞生长因子对紫外线诱导豚鼠巩膜细胞凋亡的影响及其与凋亡抑制基因bcl-2的相关性研究

Effect of Basic Fibroblast Growth Factor(bFGF) on UV-induced Apoptosis of Scleral Cells in Guinea Pigs and Correlation with Apoptosis Inhibition Gene B-cell Lymphoma-Leukemia 2(Bcl-2)

目的探讨碱性成纤维细胞生长因子（basic fibroblast growth factor．bFGF）对紫外线（ultraviolet，UVA）诱导的巩膜细胞凋亡的影响及其对凋亡抑制的发生机制。方法用组织块培养法培养豚鼠后极部巩膜细胞。利用紫外线诱导细胞凋亡模型，将培养的巩膜细胞分为对照组（无UVA光照）、单纯UVA组（仅暴露于UAA照射），并于UVA照射前加不同剂量的bFGF（UVA＋10μg／LbFGF组，UVA＋20μg／LbFGF组，UVA＋40μg／LbFGF组）。以流式细胞仪检测细胞凋亡，以RT—PCR法和Western—blot技术测定B细胞淋巴瘤-白血病-2基因（B—cell lymphoma／leukemia2,bcl-2）的表达。结果组织培养后1-2周开始有细胞长出，3-4周融合，免疫荧光鉴定细胞为巩膜成纤维细胞。流式细胞仪检测显示，bFGF能够抑制UVA诱导的巩膜细胞凋亡，对巩膜细胞有保护作用，且bFGF浓度高于20μg／L时抑制作用更加显著；RT—PCR法、Western—blot检测显示，UVA能够引起凋亡抑制基因bcl-2表达下调，但bFGF能够抑制UVA的下调作用，且随着bFGF浓度的升高其抑制效果逐渐增强，巩膜细胞bcl-2基因的表达也逐渐增加。结论外源性bFGF能够通过上调凋亡抑制基因bcl-2的表达而抑制巩膜细胞的凋亡。

Objective To investigate the effect of basic fibroblast growth factor （bFGF） on UVA （uhraviolet）-induced apoptosis of scleral cells and its mechanism of inhibition of apoptosis. Methods With the use of UV-induced apoptosis model, the cultured scleral cells were divided into control group （no UVA light）, UVA group, 10 μg/L bFGF preceded irradiation group, 20 μg/L bFGF preceded irradiation group and 40 μg/L bFGF preceded irradiation group. Flow cytometry was employed to detect cell apoptosis, RT-PCR and Western-blot were used to detect the expression of B-cell lymphomaleukemia-2 gene （B-cell lymphoma-leukemia 2, bcl-2）. Results The cells began to grow out of the sclera after 1-2 weeks, and grew to confluent after 3-4 weeks. Scleral fibroblast cells were identified by immunofluorescence. Flow cytometry analysis showed that bFGF had a protective effect on the scleral cells through inhibiting apoptosis of the sclera induced by UVA , and the inhibition of which was more obvious when the concentrations was higher than 20 μg/L; RT-PCR and Western-blot showed that UVA could cause apoptosis gene bcl-2 down-regulation, while bFGF could inhibit the downward effect of UVA, and with the bFGF concentration increased the inhibitory effect gradually increased, the expression of bcl-2 gene of sclera was also increased gradually. Conclusion Exogenous bFGF can inhibit apoptosis of sclera by up-regulating the expression of bcl-2 gene.