冷凝术后玻璃体腔碱性成纤维细胞生长因子含量的测定
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摘要
目的通过测定冷凝术后视网膜未复位或复发性孔源性视网膜脱离患者和初发性孔源性视网膜脱离患者玻璃体腔碱性成纤维细胞生长因子(bFGF)的含量,探讨冷凝与bFGF的关系,及其在增殖性玻璃体视网膜病变(PVR)形成中的作用与可能的机制,以期望减少术后PVR的发生,降低复发性视网膜脱离的发生率,提高视网膜复位手术的成功率。
方法以2009年3月～2010年7月在我科住院的62例(62眼)孔源性视网膜脱离(伴不同程度PVR)患者为研究对象,分为两组。实验组:冷凝术后视网膜未复位者或复发性孔源性视网膜脱离的患者28例;对照组:初发性孔源性视网膜脱离的患者34例。其中实验组28例均为接受巩膜扣带术并使用过冷凝视网膜未复位或再次发生视网膜脱离拟行玻璃体切除术的患者;对照组34例为首次孔源性视网膜脱离拟行玻璃体切除术的患者。两组病例均在行玻璃体切除术前,未行眼内灌注液灌注时抽取玻璃体液,离心后取上清液,采用高敏感度的“双抗体夹心法”酶联免疫吸附分析试剂盒(R&DSystems,USA)测定玻璃体液标本中bFGF的含量,试剂盒内附有纯化的人bFGF标准品。对bFGF特异的单克隆抗体粘附在ELISA检测板的微孔板底面,往包被单抗的微孔中加入样品或标准品后,bFGF与单克隆抗体结合,再与辣根过氧化物酶(Horseradish Peroxidase,HRP)标记的bFGF抗体结合,产生抗体–抗原(bFGF)–酶标抗体复合物,经过彻底洗涤后加入底物四甲基联苯胺(3,3’,5,5’-Tetramethyl benzidine,TMB)显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。采用ELISA微孔板光密度测定仪(DG5032型)测定样本在450 nm光波的光密度值(OD值),颜色越深,表示OD值越大,bFGF的含量也就越高。用bFGF标准品制备一条浓度从0～40ng/L并与其光密度值相对应的标准曲线。通过将测得的每个样品的光密度值与标准曲线相比较或根据标准曲线的直线回归方程式,计算出样品中bFGF的浓度含量。
结果1.玻璃体液bFGF的浓度含量为:实验组PVR B级:(7.99±1.41)ng/L,PVR C级:(12.18±3.01)ng/L,PVR D级:(19.45±4.10)ng/L;对照组PVR A级:(3.57±1.28)ng/L ,PVR B级:(5.51±1.07)ng/L,PVR C级:(8.32±1.45)ng/L,PVR D级:(13.55±3.08)ng/L;2.实验组与对照组相同PVR级别(PVR B级及以上)之间玻璃体腔bFGF含量差异均有统计学意义(P﹤0.05),实验组玻璃体液bFGF含量均高于对照组;3.实验组PVR B级与PVR C级玻璃体腔bFGF含量差异有统计学意义(P<0.01);实验组PVR C级与PVR D级玻璃体腔bFGF含量差异有统计学意义(P<0.05);实验组PVR严重,玻璃体液bFGF含量高;4.两组患者术后视力均有明显提高,实验组视力提高者24例(85.7%),其中视力提高2～3行者有3例(10.7%),<2行者有21例(75%),最佳矫正视力最高者为0.4;对照组视力提高者有29例(85.3%),其中提高2～3行者有5例(14.7%),<2行者有24例(70.6%),最佳矫正视力最高者为0.8;5.视网膜脱离的解剖复位率:本次手术视网膜复位率实验组25例(89.3%),对照组32例(94.1%);视网膜最终复位率实验组27例(96.4%),对照组33例(97.1%);经统计学分析,差异均无统计学意义(P>0.05)。
结论冷凝可增加玻璃体腔bFGF的含量, bFGF与PVR的严重程度密切相关。
Objective To investigate the relationship of cryocoagulation and basic fibroblast growth factor(bFGF) and observe the effect and possible mechanism of bFGF in proliferative vitreoretinopathy(PVR) by measuring the assay of bFGF in vitreous of patients with primary rhegmatogenous retinal detachment (the Control Group) and patients with recurred rhegmatogenous retinal detachment who had been undergone cryocoagulation(the Experimental Group).We expect to decrease the rate of PVR and recurred rhegmatogenous retinal detachment,and finally improve the retinal resetting rate.
Methods Analyze records of 62 patients with rhegmatogenous retinal detachment who hospitalized in our department during the period between March,2009 and July,2010. 28 cases in the experimental group who underwent recurred rhegmatogenous retinal detachment applied scleral buckling associated with Cryocoagulation. 34 cases in the control group with rhegmatogenous retinal detachment applied vitrectomy for the first time. All the cases were taken primary vitreous liquid before vitrectomy.And got supernate after centrifugate. Then a two-site enzyme-linked immunosorbent assay was applied to quantitate the level of bFGF in 34 cases’(34 eyes) vitreous of control group and 28 cases (28 eyes) of experimental group.There are purified bFGF standard substances in the kit.The monoclonal antibody which is specific to bFGF situates at the subface of the ELISA pick-up plate. Add the sample or standard substance into the micropore which peridium the monoclonal antibody.Then bFGF combines with monoclonal antibody and bFGF antibody which are tagged by Horseradish Peroxidase.Then produces antibody-antigen enzyme labelled antibody compounds.They are chromogenic after thorough washing with 3,3’,5,5’-Tetramethyl benzidine.Under the catalysis of HRP enzyme,TMB translate into blue color and finally yellow color with the effect of acid.Apply ELISA microwell plate spectrodensitometry instrument to measure the Optical Density Value of the sample under light-wave of 450nm.The deeper the color is,the larger OD value is and higher content of bFGF.Use bFGF standard sample making a standard curve whose density is from 0~40ng/L and homologous with its OD value.Compare each OD value of the sample with the standard curve or the linear regression equation and calculate the content of bFGF in the sample.
Results 1.The bFGF level in vitreous: the Experimental Group: PVR B:(7.99±1.41)ng/L,PVR C:(12.18±3.01)ng/L,PVR D:(19.45±4.10)ng/L;the Control Group: PVR A:(3.57±1.28)ng/L ,PVR B:(5.51±1.07)ng/L,PVR C:(8.32±1.45)ng/L,PVR D:(13.55±3.08)ng/L; 2.The distinction between the same PVR(above PVR B) level in Experimental Group and Control Group had statistical significance(P﹤0.05).And bFGF contents of experimental group are all higher than the control group; 3.The distinction between PVRB and PVR C, PVR C and PVR D in Experimental group has statistical significance(P=0.003<0.01, P=0.014<0.05). 4.The corrected vision when discharged from hospital:24 cases(85.7%)improved in experimental group,3 cases(10.7%)improved 2~3 lines,21 cases (75%)lower than 2 lines and the best corrected vision is 0.4;In the control group, 29 cases(85.3%)improved in experimental group,5 cases(14.7%)improved 2~3 lines,24 cases(70.6%)lower than 2 lines and the best corrected vision is 0.8. 5.The anatomical reduction rate:25 cases(89.3%)in experimerntal group and 32 cases(94.1%)in control group acquire the reattachment of retina;The final rate of the reattachment of retina:27 cases(96.4%)in experimental group and 33 cases(97.1%)in the control group;The distinction of the anatomical reduction rate between the two groups has no statistical significance(P>0.05).
Conclusions Cryocoagulation could increase the bFGF content in vitreous;bFGF has close relationship with the severity degree of PVR.
方法以2009年3月～2010年7月在我科住院的62例(62眼)孔源性视网膜脱离(伴不同程度PVR)患者为研究对象,分为两组。实验组:冷凝术后视网膜未复位者或复发性孔源性视网膜脱离的患者28例;对照组:初发性孔源性视网膜脱离的患者34例。其中实验组28例均为接受巩膜扣带术并使用过冷凝视网膜未复位或再次发生视网膜脱离拟行玻璃体切除术的患者;对照组34例为首次孔源性视网膜脱离拟行玻璃体切除术的患者。两组病例均在行玻璃体切除术前,未行眼内灌注液灌注时抽取玻璃体液,离心后取上清液,采用高敏感度的“双抗体夹心法”酶联免疫吸附分析试剂盒(R&DSystems,USA)测定玻璃体液标本中bFGF的含量,试剂盒内附有纯化的人bFGF标准品。对bFGF特异的单克隆抗体粘附在ELISA检测板的微孔板底面,往包被单抗的微孔中加入样品或标准品后,bFGF与单克隆抗体结合,再与辣根过氧化物酶(Horseradish Peroxidase,HRP)标记的bFGF抗体结合,产生抗体–抗原(bFGF)–酶标抗体复合物,经过彻底洗涤后加入底物四甲基联苯胺(3,3’,5,5’-Tetramethyl benzidine,TMB)显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。采用ELISA微孔板光密度测定仪(DG5032型)测定样本在450 nm光波的光密度值(OD值),颜色越深,表示OD值越大,bFGF的含量也就越高。用bFGF标准品制备一条浓度从0～40ng/L并与其光密度值相对应的标准曲线。通过将测得的每个样品的光密度值与标准曲线相比较或根据标准曲线的直线回归方程式,计算出样品中bFGF的浓度含量。
结果1.玻璃体液bFGF的浓度含量为:实验组PVR B级:(7.99±1.41)ng/L,PVR C级:(12.18±3.01)ng/L,PVR D级:(19.45±4.10)ng/L;对照组PVR A级:(3.57±1.28)ng/L ,PVR B级:(5.51±1.07)ng/L,PVR C级:(8.32±1.45)ng/L,PVR D级:(13.55±3.08)ng/L;2.实验组与对照组相同PVR级别(PVR B级及以上)之间玻璃体腔bFGF含量差异均有统计学意义(P﹤0.05),实验组玻璃体液bFGF含量均高于对照组;3.实验组PVR B级与PVR C级玻璃体腔bFGF含量差异有统计学意义(P<0.01);实验组PVR C级与PVR D级玻璃体腔bFGF含量差异有统计学意义(P<0.05);实验组PVR严重,玻璃体液bFGF含量高;4.两组患者术后视力均有明显提高,实验组视力提高者24例(85.7%),其中视力提高2～3行者有3例(10.7%),<2行者有21例(75%),最佳矫正视力最高者为0.4;对照组视力提高者有29例(85.3%),其中提高2～3行者有5例(14.7%),<2行者有24例(70.6%),最佳矫正视力最高者为0.8;5.视网膜脱离的解剖复位率:本次手术视网膜复位率实验组25例(89.3%),对照组32例(94.1%);视网膜最终复位率实验组27例(96.4%),对照组33例(97.1%);经统计学分析,差异均无统计学意义(P>0.05)。
结论冷凝可增加玻璃体腔bFGF的含量, bFGF与PVR的严重程度密切相关。
Objective To investigate the relationship of cryocoagulation and basic fibroblast growth factor(bFGF) and observe the effect and possible mechanism of bFGF in proliferative vitreoretinopathy(PVR) by measuring the assay of bFGF in vitreous of patients with primary rhegmatogenous retinal detachment (the Control Group) and patients with recurred rhegmatogenous retinal detachment who had been undergone cryocoagulation(the Experimental Group).We expect to decrease the rate of PVR and recurred rhegmatogenous retinal detachment,and finally improve the retinal resetting rate.
Methods Analyze records of 62 patients with rhegmatogenous retinal detachment who hospitalized in our department during the period between March,2009 and July,2010. 28 cases in the experimental group who underwent recurred rhegmatogenous retinal detachment applied scleral buckling associated with Cryocoagulation. 34 cases in the control group with rhegmatogenous retinal detachment applied vitrectomy for the first time. All the cases were taken primary vitreous liquid before vitrectomy.And got supernate after centrifugate. Then a two-site enzyme-linked immunosorbent assay was applied to quantitate the level of bFGF in 34 cases’(34 eyes) vitreous of control group and 28 cases (28 eyes) of experimental group.There are purified bFGF standard substances in the kit.The monoclonal antibody which is specific to bFGF situates at the subface of the ELISA pick-up plate. Add the sample or standard substance into the micropore which peridium the monoclonal antibody.Then bFGF combines with monoclonal antibody and bFGF antibody which are tagged by Horseradish Peroxidase.Then produces antibody-antigen enzyme labelled antibody compounds.They are chromogenic after thorough washing with 3,3’,5,5’-Tetramethyl benzidine.Under the catalysis of HRP enzyme,TMB translate into blue color and finally yellow color with the effect of acid.Apply ELISA microwell plate spectrodensitometry instrument to measure the Optical Density Value of the sample under light-wave of 450nm.The deeper the color is,the larger OD value is and higher content of bFGF.Use bFGF standard sample making a standard curve whose density is from 0~40ng/L and homologous with its OD value.Compare each OD value of the sample with the standard curve or the linear regression equation and calculate the content of bFGF in the sample.
Results 1.The bFGF level in vitreous: the Experimental Group: PVR B:(7.99±1.41)ng/L,PVR C:(12.18±3.01)ng/L,PVR D:(19.45±4.10)ng/L;the Control Group: PVR A:(3.57±1.28)ng/L ,PVR B:(5.51±1.07)ng/L,PVR C:(8.32±1.45)ng/L,PVR D:(13.55±3.08)ng/L; 2.The distinction between the same PVR(above PVR B) level in Experimental Group and Control Group had statistical significance(P﹤0.05).And bFGF contents of experimental group are all higher than the control group; 3.The distinction between PVRB and PVR C, PVR C and PVR D in Experimental group has statistical significance(P=0.003<0.01, P=0.014<0.05). 4.The corrected vision when discharged from hospital:24 cases(85.7%)improved in experimental group,3 cases(10.7%)improved 2~3 lines,21 cases (75%)lower than 2 lines and the best corrected vision is 0.4;In the control group, 29 cases(85.3%)improved in experimental group,5 cases(14.7%)improved 2~3 lines,24 cases(70.6%)lower than 2 lines and the best corrected vision is 0.8. 5.The anatomical reduction rate:25 cases(89.3%)in experimerntal group and 32 cases(94.1%)in control group acquire the reattachment of retina;The final rate of the reattachment of retina:27 cases(96.4%)in experimental group and 33 cases(97.1%)in the control group;The distinction of the anatomical reduction rate between the two groups has no statistical significance(P>0.05).
Conclusions Cryocoagulation could increase the bFGF content in vitreous;bFGF has close relationship with the severity degree of PVR.
引文
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8.Nagasaki H,Shinagawa K,Mochizuki M.Risk factors for proliferative vitreoretinopathy[J].Prog Retin Eye Res,1998,17(1):77-98.
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41.宋琛主编.手术学全集.眼科卷[M].第1版.北京:人民军医出版社,1994,543-559.
1. Pastor JC,de la Rua ER,Martin F,et al. Proliferative vitreoretinopathy: risk factors and pathobiology[J].Prog Retin Eye Res,2002,21(1):127- 144.
2.The Retinal Society Terminology Committee.The classification of retinal detachment with proliferative vitreoretinopathy[J].Ophthalmo- logy,1983,90(2):121-125.
3.Ryan SJ.Traction retinal detachment .XLIX Edward Jackson Memorial Lecture[J].AmJ Ophthalmol,1993,115(1):1-20.
4.张秀兰,吕林,高汝龙.牵引性视网膜脱离[J].国外医学.眼科分册,1993,17(6):361-364.
5.Baudouin C,Khosrzvi E,Pisella PJ,et al.Inflammation measurement and immunocharacterization of cell proliferation in an experimental model of proliferative vitreoretinopathy[J].Ophthalmic Res,1998,30(6):340- 350.
6.Pastor JC.Proliferative vitreoretinopathy:an overview[J].Surv Ophthal- mol,1998,43(1):3-18.
7.Machemer R.Proliferative Vitreoretinopathy(PVR):A personal account of its pathogenesis and treatment[J].Invest Ophthalmol Vis Sci,1988,29 (12): 1771- 1783.
8.Nagasaki H,Shinagawa K,Mochizuki M.Risk factors for proliferative vitreoretinopathy[J].Prog Retin Eye Res,1998,17(1):77-98.
9.Cowley M,Conway BP,Campochiaro PA,et al.Clinical risk factors for proliferative vitreoretinopathy[J].Arch Ophthalmol,1989,107(8):114 7-1151.
10.Girard P,Mimoun G,Karpouzas I,et al.Clinical risk factors for proliferative vitreoretinopathy after retinal detachment surgery[J]. Retina, 1994, 14(5): 417-424.
11. Sporn M, Roberts A.Ggandbook of Experimental Phaomacology Vol 95/ⅠPeptide growth factors and their recetoysⅠ[M].Berlm: Spring Verlay, 1990,3-5.
12.Gospodarowicz D.Purification of a fibroblast growth factor from bovine pituitary[J].J Biol Chem,1975,250(7):2515-2520.
13.Cassidy L, Barry P, ShawC,et al. Platelet derived growth factor and fibroblast growth factor basic levels in the vitreous of patients with vitreoretinal disorders[J]. Br J Ophthalmol,1998,82(2):181-185.
14. Liang X,Li C,Li Y,et al.Platelet-derived growth factor and basic fibroblast growth factor immunolocalized in proliferative retinal diseases[J]. Chin Med J (Engl),2000,113(2):144-147.
15.Sporn MB,Roberts AB,Wakefield LM,et al. Some recent advances in the chemistry and biology of transforming growth factor-beta[J].J Cell Biol,1987,105(3):1039-1045.
16.张蕙蓉,曹景泰.视网膜增殖性疾病玻璃体中表皮生长因子的放射受体定量测定[J].中华眼底病杂志,1996,12(2):91-93.
17.陈明,蓝平,高明宏,等.表皮生长因子、碱性成纤维细胞生长因子及5-氟尿嘧啶对培养的人视网膜色素上皮损伤愈合的体外研究[J].中华眼科杂志,1999,35(2):134-136.
18.郭长梅,惠延年,马吉献等.转化生长因子-β受体Ⅱ在增生性玻璃体视网膜病变增生膜中的表达[J].眼科学报,2003,19(4):244-247.
19.Xu X.Effects of LPA on DNA synthesis and proliferation in cultured human RPE cells[J].Invest Ophtholmol Vis Sci, 1997,38(3):671-675.
20.Guerin CJ,Hu L,Scicli G et al. Transforming growth factor beta in experimentally detached retina and periretinal membranes[J].Exp Eye Res,2001,73(6):753-764.
21.Bochaton-Piallat ML,Kapetanios AD,Donati G,et al. TGF-beta1, TGF- beta receptor II and ED-A fibronectin expression in myofibroblast of vitreoretinopathy[J].Invest Ophthalmol Vis Sci,2000,41(8):2336-2342.
22.高方,王一,陈少军,等.TNF-α、IGF、TGF-α在增殖性玻璃体视网膜疾病中的表达[J].第三军医大学学报, 2004,6 (5):429-431.
23.Connor TB Jr,Roberts AB,et al.Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye[J].J Chin Invest, 1989, 83(5): 1661-1666.
24.Carrington L,McLeod D,Boulton M. IL-10 and antibodies to TGF-β2 and PDGF inhibit RPE-mediated retinal contraction[J].Invest Ophtha- lmol Vis Sci,2000,41(5):1210-1216.
25.Psychiatry R. Progression tomalignancy in gliomas reflects inverse relation of PEDF and VEGF expression[J].J Neurol Neurosurg Psychiatr, 2004, 75(2): 201-202.
26.Eichler W,Yafai Y,Wiedemann P,et al. Angiogenesis-related factors derived from retinalglial(Muller)cell in hypoxia[J].Neuro Psychiatry Report, 2004, 15 (10):1633-1637.
27.张晨明,陈晓钟,李志敏,等.增生性玻璃体视网膜病变患者血清和玻璃体液PEDF水平的检测[J].西南军医,2006,8(2):4-6.
28.Ando A, Ueda M, Uyama M, et al. Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells byplatelet derived growth factor[J]. Br J Ophthalmol,2000,84(11):1306-1311.
29. Jin M, He S, Worpel V,et al. Promotion of adhesion and migration of RPEcells to provisional extracellular matrices by TNF-α[J].Invest Ophthalmol Vis Sci,2000,41(13):4324-4332.
30.Kenarova B,Voinov L,Apostolov C,et al. Levels of some cytokines in subretinal fluid in proliferative vitreoretinopathy and rhegmatogenous retinal detachment[J].Eur J Ophthalmol,1997,7(1):64-67.
31.Kojima S,Yamada T,Tamai M,et al.Quantitative analysis of interleuki- n-6 in vitreous from patients with proliferative vitreoretinal diseases [J].Jpn J Ophthalmol,2001,45(1):40-45.
32.Aksunger A,Or M,Okur H,et al. Role of interleukin 8 in the pathogen- nesis of proliferative vitreoretinopathy[J].Ophthalmologica, 1997,211 (4):223-225.
33.Weller M,Wiedemann P,Heimann K.Proliferative vitreoretinopathy --is it anything more than wound healing at the wrong place?[J].Int Ophthalmol, 1990,14(2):105-117.
34.Hinton DR,He S,Jin ML,et al.Novel growth factor involved in the pat- hogenesis of proliferative vitreoretinopathy [J]. Eye, 2002, 16 (4): 422- 428.
35.Wu WC,Kao YH,TSeng HY. The cell cycle distribution of cultured human retinal pigmented epithetlial cells under exposure of anti-proliferative drugs[J]. J Ocul Phamacol Ther, 2003, 19(1): 83-90.
36.Lesehey KH,Hines J,Singer JH,et al. Inhibition of growth factor effects in retinal pigment epithelial cells [J].Invest OPhthalmol Vis Sci1991,32(6):1770-1778.
37.Pena PA,Jerdan JA,Glaser BM,et al.Effects of TGF-beta and TGF-beta neutralizing antibodies on fibroblast-induced collagen gel contraction: implications for proliferative vitreoretinopathy[J].Invest OPhthalmol Vis Sei,1994,35(6):2804-2808.
38.宋学英.手术治疗增生性玻璃体视网膜病变的时机探讨[J].兰州医学院学报,2001,27(4):53-54.
39.Glaser BM,Vidaurri-Leal J,Michels R,et al.Cryotherapy during surgeryfor giant retinal tears and intravitreal dispersion of viable retinal pigment epithelial cells[J].Ophthalmology,1993,100(4):466- 470.
40.The sillicone study group. Vitrectomy with silicone oil or perfluoropr- opane gas in eyes with severe proliferative vitreoretinopathy:results of a randomized clinical trial.Silicone Study Report 2[J].Arch Ophthal- mol,1992,110(6):780-792.
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