Bevacizumab对视网膜色素上皮细胞纤维化相关因子表达的影响

Effects of Bevacizumab on fibrosis associated cytokines expression in human retinal pigment epithelial cells

目的观察Bevacizumab对人视网膜色素上皮细胞纤维化相关因子基因及蛋白表达的影响,探讨Bevacizumab在新生血管纤维化中的作用。方法用含终浓度0.25 g·L-1Bevacizumab的DMED/F12培养液培养ARPE-19细胞,按Bevacizumab处理的时间分为0 h、6 h、12 h、24 h和48 h共5组,0 h为对照组。不同时间点收集细胞及上清。用逆转录聚合酶链反应检测ARPE-19细胞中结缔组织生长因子(connective tissue growth factor,CTGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、转化生长因子-β2(transforming growth factorβ2,TGF-β2)和基质金属蛋白酶-2(matrix metalloproteinase 2,MMP-2)的mRNA相对表达量。采用ELISA检测上清中CTGF、bFGF和TGF-β2的蛋白浓度,应用免疫蛋白印迹法(Western blotting)检测MMP-2蛋白的表达。结果 Bevacizumab作用后6 h、12 h、24 h和48 h,CTGF、bFGF、TGF-β2及MMP-2的mRNA相对表达量分别为CTGF:1.694±0.160、1.994±0.221、1.733±0.057、1.397±0.075,bFGF:1.720±0.068、2.144±0.080、1.893±0.068、1.372±0.076,TGF-β2:1.356±0.070、1.737±0.515、1.576±0.098、1.439±0.050,MMP-2:1.414±0.115、1.644±0.094、1.435±0.075、1.285±0.024,与对照组相比均升高,差异均有统计学意义(均为P<0.05)。ELISA结果显示Bevacizumab作用后6 h、12 h、24 h和48 h,CTGF、bFGF、TGF-β2蛋白的表达量与对照组相比均升高,差异均有统计学意义(均为P<0.05);对照组及各时间组蛋白浓度分别为CTGF:(31.949±0.388)ng·L-1、(48.697±0.863)ng·L-1、(56.847±0.499)ng·L-1、(51.753±1.395)ng·L-1、(43.085±1.240)ng·L-1,bFGF:(26.660±1.360)ng·L-1、(46.529±3.297)ng·L-1、(55.883±1.705)ng·L-1、(47.937±1.303)ng·L-1、(36.894±2.532)ng·L-1,TGF-β2:(26.982±2.404)ng·L-1、(35.452±1.360)ng·L-1、(46.787±0.896)ng·L-1、(43.683±1.630)ng·L-1、(38.008±1.110)ng·L-1。Western blotting结果显示,Bevacizumb处理后6 h、12 h、24 h、48 h MMP-2蛋白含量升高,组间比较差异有统计学意义(F=2620.429,P=0.000)。结论 Bevacizumab能够上调人RPE细胞中CTGF、bFGF、TGF-β2及MMP-2的表达,可能有促纤维化形成的作用。

Objective To evaluate the effects of Bevacizumab on expressions of cytokines associated with fibrosis in gene and protein levels in human retinal pigment epithelial cells, and further investigate the possible role of Bevacizmnab in the process of new vascular fibrosis. Methods Human retinal pigment epithelial cells were incu- bated in DMEM/F12 medium containing 0.25 g .L-1 Bevacizumab. Cells were divided into 5 groups according to incubation period： 0 hour, 6 hours, 12 hours, 24 hours and 48 hours, 0 hour served as control. The ceils and supernatant were collected at differ- ent time points. The mRNA and protein levels of connective tissue growth factor （ CT- GF）, basic fibroblast growth factor （bFGF）, transforming growth factor-β2 （TGF-β2） and matrix metalloproteinase-2 （MMP-2）were evaluated by RT-PCR and ELISA or Western blotting, respectively. Results The mRNA levels at 6 hours, 12 hours, 24 hours and 48 hours were as following, CTGF： 1. 694 ± 0. 160, 1. 994 ± 0. 221, 1. 733 ± 0.057, 1.397±0.075; bFGF： 1.720±0.068, 2. 144 ±-0.080, 1.893 ±0.058, 1.372± 0.076; TGF-β2：1.356 ±0.070, 1.737 ±0.515, 1.576 ±0.098, 1.439 ±0.050; MMP-2： 1.414±0.115, 1.644 ±0.094, 1.435 ±0.075, 1.285 ±0.024. Compared with control group, the mRNA levels were increased at all time points after incubation with Bevaci- zumab （ all P 〈 0.05 ）. ELISA and Western blotting analysis showed that the protein lev- els of CTGF, bFGF, TGF-β2 and MMP-2 increased after treatment with Bevacizumab and the differences were statistically significant （ all P 〈 0.05 ）. The protein concentra- tions of each group were as following, CTGF： （31. 949 ± 0. 388 ）μg. L-1, （48. 697 ± 0.863）μg . L-1, （56. 847 ±0.499）μg . L-1,（51.753 ± 1.395）μg . L -1,（43.085 ± 1.240）μg.L-1; bFGF： （26.66±1.36）μg.L -1, （46.529±3.297）μg.L-1,（55.883± 1.705） μg. L-1, （47. 937 ± 1. 303） μg. L-1, （36. 894 ±2. 532） μg . L-1; TGF-β2： （26.982±2.404）μg . L-1,（35.452 ± 1.360）μg . L-1, （46.787 ±0. 896）μg . L-1, （43. 683 ± 1. 630） μg .L -1, （38. 008 ± 1. 110） μg . L-1. MMP-2 protein levels at 6 hours, 12 hours, 24 hours and 48 hours after incubation with Bevacizumab were all increased, the differences were statistically significant among different groups （ F = 2620.429, P = 0.000）. Conclusion Bevacizumab can up-regulate both mRNA and protein expres- sion of CTGF, bFGF, TGF-β2 and MMP-2 in human retinal pigment epithelial cells,which indicates hat Bevacizumab may have pro-fibrotic effects.