激肽原1和类胰岛素生长因子结合蛋白6作为增生性玻璃体视网膜病变生物标志物的研究

目的 评估增生性玻璃体视网膜病变(PVR)特异性蛋白质激肽原l和类胰岛素生长因子结合蛋白6(GFBP-6)作为生物标志物的可能性.方法 对照试验研究.收集24例PVR手术前患者的玻璃体和血清,玻璃体样本分为轻度组(PVR-B级)和重度组(PVR-C、D级),以20名健康人血清和8只捐献眼球的玻璃体作为对照.同时收集15例玻璃体切除联合注气患者、8例玻璃体切除联合硅油填充术后6个月痊愈患者及8例巩膜环扎加压术后未愈患者的血清样本.应用免疫印迹法和酶联免疫吸附试验(ELISA)检测血清样本中激肽原1和IGFBP-6表达水平.对不同分级间的PVR患者玻璃体和血清中激肽原1和IGFBP-6含量比较,采用样本均数t检验;术前与术后6个月PVR患者血清中激肽原1和IGFBP-6含量比较,采用配对t检验;而巩膜环扎术后未愈、硅油填充眼患者与健康人血清激肽原1和IGFBP-6比较,采用单因素方差分析和进一步两两比较t检验.结果 免疫印迹法检测结果显示,24例PVR患者术前玻璃体样本均呈现激肽原1阳性条带,其中22例患者的IGFBP-6呈阳性,且术前血清中可检测到两种蛋白质;对照组的玻璃体和血清中均未检测出激肽原1和IGFBP-6.ELISA法检测玻璃体中蛋白激肽原1含量,显示轻度PVR患者为(237.5±32.1)μg/L,重度PVR患者为(281.0±63.0)μg/L,差异有统计学意义(t=5.44,P<0.05).ELISA法检测血清中蛋白激肽原1含量为(443.3±190.1)μg/L,高于其在玻璃体的含量,差异有统计学意义(t=5.27,P<0.05);15例玻璃体切除联合气体填充患者术后6个月血清中蛋白激肽原1含量为(81.9±18.6)μg/L,与术前(443.3±190.1)μg/L比较,差异有统计学意义(t=5.26,P<0.05);巩膜环扎术后未愈患者血清中激肽原1含量为(116.8±45.1)μg/L,健康人含量为(57.9±14.1)μg/L,硅油填充眼患者含量为(51.6±14.1)μg/L,三组间含量比较,差异有统计学意义(F=4.57,P<0.05);进一步两两比较,巩膜环扎术后未愈患者血清中激肽原1含量明显高于健康人组和硅油填充眼组,差异均有统计学意义(t=3.95,4.34;均P<0.05).ELISA法检测玻璃体中IGFBP-6含量,显示轻度PVR患者为(283.9±69.9)ng/L,重度PVR为(352.9±64.4)ng/L,差异有统计学意义(t=5.08,P<0.05);ELISA法检测血清中IGFBP-6含量为(185.3±34.9)ng/L,低于玻璃体内含量,差异有统计学意义(t=7.95,P<0.05);玻璃体切除联合气体填充术后6个月患者血清中IGFBP-6含量为(65.4±31.8)ng/L,较术前含量下降,差异有统计学意义(t=11.10,P<0.05);巩膜环扎术后未愈患者血清中IGFBP-6含量为(109.2±6.6)ng/L,硅油填充眼患者含量为(62.5±3.3)ng/L,健康人含量为(76.1±17.3)ng/L,三组间差异有统计学意义(F=4.68,P<0.05);进一步两两比较,差异也有统计学意义(t=3.16,2.77;均P<0.05).结论 PVR患者与健康人玻璃体和血清中激肽原1和IGFBP-6含量有较明显差别,且随PVR严重程度其表达水平有变化.激肽原1和IGFBP-6有可能作为PVR的生物标志物,但肯定性的结论有待于进一步的临床大样本验证.

Kininogen-1 and insulin-like growth factor binding protein-6 as serum biomarkers for proliferative vitreoretinopathy

Objective To evaluated the predictive potential of kininogen-1 and insulin-like growth factor binding protein-6 (IGFBP-6) for PVR. Methods Vitreous and serum samples were obtained from 24 PVR patients. Vitreous from 8 donated normal eyes, and serum samples from 20 healthy volunteers served as control Patients who underwent vitrecto my with C3F8 gas tamponade (n = 15) and silicone tamponade ( n = 8 ) and patients who experienced recurrent retinal detachment after scleral buckling surgery ( n = 8) were recruited for serum tests as well Western blot analysis was employed to detect the presence of kininogen-1 and IGFBP-6. The protein concentration was measured by using enzyme linked immunosorbent assay (ELISA) analysis. All date were analyzed with the SPSS 3.0 for Windows ( only-way analysis of variance and t test). Results Western blot analysis displayed that except that IGFBP-6 was absent in 2 PVR vitreous, both kininogen-1 and IGFBP-6 were otherwise found in all PVR vitreous and serum samples. Neither kininogen-1 nor IGFBP-6 can be detected in normal vitreous or serum samples. Protein expression was more intensive in severe PVR vitreous than in mild PVR vitreous, which was confirmed by a significantly higher concentration of each protein in sever PVR vitreous. The ELISA outcomes documented that kininogen-1 concentration in vitreous were significantly higher in severe PVR patients than those in mild PVR (281. 0 ± 63. 0 & 237. 5 ± 32. 1) μg/L( t = 5.44 ,P < 0. 05). Kininogen-1 was about 2 times higher in serum than in vitreous(443. 3 ± 190. 1) μg/L(t =5. 27,P <0. 05). At 6 months after vitrectomy with gas tamponade in 15 patients, their kininogen-1 level in serum was significantly lower than that of preoperation (81.9 ± 18. 6 & 443. 3 ±190. l)μg/L(t=5. 26,P<0. 05) and encircling failure group was (116. 8 ±45. l)μg/L, it was higher than that of normal and silicone tamponde groups (t = 3. 95,4. 34; P < 0. 05). Similarly, IGFBP-6 concentration in vitreous were significantly higher in severe PVR patients than those in mild PVR ( 352. 9 ± 64.4 & 283. 9 ±69. 9)ng/L (t =5.08,P <0. 05)and its level in serum was (185. 3 ±34. 9)ng/L and lower than that of in vitreous (t = 7.95, P < 0. 05 ) . At 6 months after vitrectomy with gas tamponade in 15 patients, their IGFBP-6 level in serum decreased comparing that of preoperation( 65.4 ± 31. 8 ) ng/L( t = 11. 10,P<0. 05)and encircling failure group was(109. 2 ±6. 6)ng/L,it was higher than that of normal and silicone tamponde groups(t = 3. 16,2.77 ;P <0. 05). Conclusions Kininogen-1 and IGFBP-6 are presented in serum and vitreous in PVR patients. The strength of protein expression is related to the severity of PVR. These results suggested that kininogen-1 and IGFBP-6 can be biomarkers for severe PVR.