目的 探讨G蛋白信号转导调节子5(RGS5)参与实验性脉络膜新生血管(CNV)生成的可能机制.方法 对照实验研究.采用532 nm激光,对88只(176只眼)雄性棕色挪威(BN)大鼠诱导实验性CNV.其中40只大鼠按光凝后时间分组:光凝后1 d、3 d组各6只大鼠,光凝后7 d组7只大鼠,光凝后14 d组21只大鼠;48只大鼠在光凝后两个时间段按处理因素分组:光凝后7 d的生理盐水组和Avastin注射组各6只大鼠;光凝后14 d的生理盐水组和Avastin注射组各18只大鼠.另选择未进行任何干预的19只大鼠作为正常对照组.分别采用免疫荧光染色法、免疫印迹法和逆转录聚合酶链反应(RT-PCR)法检测大鼠视网膜和脉络膜组织中血管内皮生长因子(VEGF)和RGS5蛋白及其mRNA表达灰度值;组织病理学切片和脉络膜铺片观察CNV厚度和面积.组间RGS5蛋白和mRNA表达、VEGF蛋白和mRNA表达灰度值比较,均采用Student's t检验.结果 (1)RGS5和VEGF蛋白表达水平:光凝后14 d组大鼠RGS5蛋白表达水平达到高峰,灰度值为0.899±0.057,但与正常对照组(0.820±0.032)差异无统计学意义(t=2.079,P>0.05);光凝后7 d组大鼠VEGF蛋白表达水平最高,灰度值为0.600±0.031,较正常对照组(0.382±0.036)明显升高(t=7.959,P<0.01).(2)RGS5和VEGF的mRNA表达水平:光凝后1 d和3 d组大鼠RGS5 mRNA表达灰度值分别为0.763±0.035、0.725±0.054,较正常对照组(0.886±0.047)明显下降(t=3.646,3.888;均P<0.05),光凝后14 d组达到高峰,但与正常对照组差异无统计学意义(t=2.194,P>0.05);光凝后7 d组大鼠VEGF mRNA表达水平达到高峰,灰度值为0.855±0.029,较正常对照组(0.274±0.039)明显升高(t=20.709,P<0.01).提示在大鼠CNV形成过程中,RGS5蛋白和mRNA表达高峰期均晚于VEGF蛋白和mRNA表达高峰期.(3)CNV厚度和面积变化:光凝后14 d大鼠CNV形成,CNV厚度为(81.513±10.091)μm,CNV面积为(35 867.
Objective To observe the role and possible mechanism of regulators of C-protein signaling 5 ( RGS5) in the development of experimental choroidal neovascularization (CNV). Methods It was an experimental study. A total of 88 male Brown Norway ( BN) rats underwent the 532 run photocoagulation to set up the CNV model. Forty of them were sort by time after the photocoagulation as follows: 1 d (n=6), 3 d (n=6), 7 d (n=7) and 14 d (n=21), then 48 of them were divided by treatment factor; intravitreal injection of physiological saline or Avastin. Nineteen of them served as the controls. The expression of RGS5 and VEGF were examined by immunofluorescence, western blot and reverse transcription polymerase chain reaction ( RT-PCR). The thickness and area of CNV were qualified by histopathological sections and choroidal flatmounts. All of the results were analyzed by Student's I-test. Results (1) The expression of RGS5 and VEGF proteins: there was no statistical significance in gray-scale value of RGS5 between 14 d group (0. 899 ±0. 057) and control group (0. 820 ±0.032), although RGS5 expression reached a peak at 14 days (t =2.079,P〉0.05). There was statistical significance in gray-scale value of VEGF between 7 d group (0. 600 ± 0. 031) and control group (0. 382 ± 0. 036) , and VEGF expression reached a peak at 7 days (t = 7. 959, P 〈 0. 01). (2) The expression of RGS5 and VEGF mRNAs: the RGS5 mRNA expression decreased at 1 day (0.763 ±0.035) and 3 days (0.725 ±0.054) in comparison with the control (0.886 ±0.047, t =3.646, 3.888; P〈0.05, 0.05), and reached a peak at 14 days after photocoagulation (t = 2.194,P〉0.05). There was statistical significance in gray-scale value of VEGF between 7 d group (0. 855 ±0.029) and control group (0. 274 ±0. 039), and VEGF expression reached a peak at 7 days after photocoagulation (( = 20.709, P 〈 0.01). (3) The changes of thickness and area of CNV: the thickness and