实验性脉络膜新生血管发生机制及Bevacizumab（Avastin）抑制CNV的试验研究
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摘要
目的
1.建立氪激光引导的BN大鼠CNV(choroidal neovascularization,脉络膜新生血管)模型,观察4W时间内CNV的生长情况。
2.用RT-PCR方法检测VEGF(vascular endothelial growth factor,血管内皮生长因子)在试验过程中的表达情况。检测FⅧ-RAg(FactorFⅧ-Related Antigen,Ⅷ因子相关抗原)表达来对新生血管进行半定量研究,观察CNV的生长情况,研究VEGF与CNV生长的关系,从而探讨在CNV生长中的作用。
3.光凝后一周,FFA检查显示有CNV形成,玻璃体腔内注射Bevacizumab(Avastin),观察3W时间内CNV的生长情况,从而对Bevacizumab(Avastin)治疗试验性CNV的疗效做一初步的探讨。
方法
1.对5组40只BN大鼠应用氪激光(波长647nm)实验眼视网膜进行光凝,功率360mW、光斑直径50um、曝光时间0.05s,围绕视盘等距光凝8个点,创建CNV模型。
2.于光凝后3d,14d,21d,28d行FFA检查,造影早期计算CNV面积。在上述时间各处死动物8只,摘除眼球。光镜下对HE染色切片进行CNV形态学改变的评价。应用免疫组化方法,检测FⅧ-RAg表达来对新生血管进行半定量研究,观察CNV的生长情况。观察各组大鼠光凝区和正常视网膜中VEGF的表达,并用RT-PCR方法对VEGFmRNA的表达做半定量分析。分析VEGF在CNV形成中的作用。
3.对48只BN大鼠单眼进行氪激光光凝,建立CNV模型,随机分为治疗组及对照组。一周后,经FFA证实有CNV形成,药物治疗组玻璃体腔内注射1μl Avastin(25μg/1μl),对照组则在光凝眼行玻璃体腔内注射BSS1μl。注射后1W、2W、3W行FFA检查,造影早期计算CNV面积。在上述各时间各处死动物6只,摘除眼球,制作石蜡切片。免疫组化对FⅧ-Rag蛋白的表达进行半定量检测。
4.实验数据以均值±标准差表示,行方差分析和t检验等统计学处理。
结果
1. FFA光凝斑盘状荧光素渗漏,证实CNV形成。在4W时间里,FFA造影结果和FⅧ-RAg免疫组化结果显示,CNV呈持续增长(P<0.05),21d达到高峰。
2.在CNV形成的4W时间里,FⅧ-Rag蛋白表达增加(P<0.05)。VEGFmRNA的表达也显著增加。VEGF的蛋白质在正常BN大鼠视网膜神经节细胞层、内核层、色素上皮层、视网膜和脉络膜血管内皮细胞表达。试验眼光凝后,在激光损伤区,VEGF的蛋白质表达于视网膜神经节细胞层、内核层、外核层缺损区、色素上皮层、视网膜和脉络膜血管内皮细胞、CNV增殖区。
3.在BN大鼠CNV动物模型采用玻璃体腔内注射1μl Avastin(25μg/1μl)后,在1W、2W、3W时间里,与对照组相比,CNV面积及荧光渗漏明显降低(P<0.05),FⅧ-Rag蛋白表达较对照组降低(P<0.05),证实CNV的形成较对照组明显降低。
结论
1.氪激光诱导BN大鼠产生CNV模型,病理结构稳定。
2. VEGF诱导内皮细胞分化,在CNV的形成过程中起着主要作用。
3.玻璃体腔内注射Bevacizumab(Avastin)可以抑制氪激光诱导的BN大鼠CNV的形成和发展
Objective
1. This study was designed to establish an experimental CNV model in BN rats by laser infusion in order to establish the foundation for studying the mechanism of CNV during 4 weeks.
2. The total mRNA of VEGF was detected by RT-PCR. Exam FⅧ-Rag protein expression in CNV and investigate the relationship between VEGF and CNV development.
3. Study the evolution of CNV during 3 weeks with intravitreal injection of Bevacizumab(Avastin)1μl(25μg/1μl), so make a preliminary investigation of the treatment effect of Bevacizumab(Avastin)on experimental CNV.
Methods
1. Five groups of 40 BN rats was photocoagulated by krypton laser in one eye to induce CNV, the laser spots were placed separately using a setting of 50 um diameter, 0.05 second duration ,and 360 mW intensity, surrounding the optic nerve at the posterior pole.
2. Fluorescein angiography was performed from 3 day to 4 weeks after laser photocoagulation .The CNV area was measured by early phase FFA. Then the rats were sacrificed immediately, the eyes were enucleated and processed for histopathologic examination and RT-PCR. Exam the FⅧ-Rag protein expression in CNV .Observe the expression of VEGF protein in rat retinal and the lesion of CNV.
3. Tow groups of 48 BN rats was photocoagulated by krypton laser in one eye to induce CNV model in one eye, in which 1 group received intravitreal injection of Bevacizumab(Avastin)1μl(25μg/1μl)after photocoagulation one week and the other group received the same volume intravitreal injection of BSS as control. FFA examinations occurred on 1,2,3 week and the eyes were enucleated and processed for histopathologic examination. The area of CNV were measured in early phase FFA. FⅧ-Rag were investigated with immunohistochemistry and were semiquantitatively analyzed.
4. The experimental data were expressed as the mean±S.D. and statistical significance was determingd by t-test.
Results
1. Disciform fluorescein leakage was observed and confirmed the formation of CNV.The immunostaining of FⅧ-Rag and FFA show the persistent increase of the CNV development during 4 weeks.
2. During the CNV development,the expression of FⅧ-Rag and VEGF increase gradually. V EGF expression was mainly observed in the Vascular endothelial cells , the ganglion cells , the inner nuclear layers , the retinal pigment epithelial cells in normal retina and the vascular endothelial cells of normal choroids of BN rat . VEGF expressed in the ganglion cells , the inner nuclear layers , lesion of outer nuclear layers inretina after photocoagulation .
3. The area and the leakage of CNV in Bevacizumab(Avastin)treated BN rat eyes obviously reduced compared with the control group at each interval. In the treatment group,the expression of FⅧ-Rag depressed. The examination of FⅧ-Rag and FFA verified the depression of CNV compared with the control group at each interval.The density of treatment is lower than that of control group(P<0.05)
Conclusion
1. krypton laser photocoagulation can be successfully used to produce CNV experimental model in the BN rat. CNV pathologic structure possesses stability.
2. VEGF play the important role in CNV development.
3. Intravitreal Bevacizumab(Avastin)Injection can inhibit the development of CNV
1.建立氪激光引导的BN大鼠CNV(choroidal neovascularization,脉络膜新生血管)模型,观察4W时间内CNV的生长情况。
2.用RT-PCR方法检测VEGF(vascular endothelial growth factor,血管内皮生长因子)在试验过程中的表达情况。检测FⅧ-RAg(FactorFⅧ-Related Antigen,Ⅷ因子相关抗原)表达来对新生血管进行半定量研究,观察CNV的生长情况,研究VEGF与CNV生长的关系,从而探讨在CNV生长中的作用。
3.光凝后一周,FFA检查显示有CNV形成,玻璃体腔内注射Bevacizumab(Avastin),观察3W时间内CNV的生长情况,从而对Bevacizumab(Avastin)治疗试验性CNV的疗效做一初步的探讨。
方法
1.对5组40只BN大鼠应用氪激光(波长647nm)实验眼视网膜进行光凝,功率360mW、光斑直径50um、曝光时间0.05s,围绕视盘等距光凝8个点,创建CNV模型。
2.于光凝后3d,14d,21d,28d行FFA检查,造影早期计算CNV面积。在上述时间各处死动物8只,摘除眼球。光镜下对HE染色切片进行CNV形态学改变的评价。应用免疫组化方法,检测FⅧ-RAg表达来对新生血管进行半定量研究,观察CNV的生长情况。观察各组大鼠光凝区和正常视网膜中VEGF的表达,并用RT-PCR方法对VEGFmRNA的表达做半定量分析。分析VEGF在CNV形成中的作用。
3.对48只BN大鼠单眼进行氪激光光凝,建立CNV模型,随机分为治疗组及对照组。一周后,经FFA证实有CNV形成,药物治疗组玻璃体腔内注射1μl Avastin(25μg/1μl),对照组则在光凝眼行玻璃体腔内注射BSS1μl。注射后1W、2W、3W行FFA检查,造影早期计算CNV面积。在上述各时间各处死动物6只,摘除眼球,制作石蜡切片。免疫组化对FⅧ-Rag蛋白的表达进行半定量检测。
4.实验数据以均值±标准差表示,行方差分析和t检验等统计学处理。
结果
1. FFA光凝斑盘状荧光素渗漏,证实CNV形成。在4W时间里,FFA造影结果和FⅧ-RAg免疫组化结果显示,CNV呈持续增长(P<0.05),21d达到高峰。
2.在CNV形成的4W时间里,FⅧ-Rag蛋白表达增加(P<0.05)。VEGFmRNA的表达也显著增加。VEGF的蛋白质在正常BN大鼠视网膜神经节细胞层、内核层、色素上皮层、视网膜和脉络膜血管内皮细胞表达。试验眼光凝后,在激光损伤区,VEGF的蛋白质表达于视网膜神经节细胞层、内核层、外核层缺损区、色素上皮层、视网膜和脉络膜血管内皮细胞、CNV增殖区。
3.在BN大鼠CNV动物模型采用玻璃体腔内注射1μl Avastin(25μg/1μl)后,在1W、2W、3W时间里,与对照组相比,CNV面积及荧光渗漏明显降低(P<0.05),FⅧ-Rag蛋白表达较对照组降低(P<0.05),证实CNV的形成较对照组明显降低。
结论
1.氪激光诱导BN大鼠产生CNV模型,病理结构稳定。
2. VEGF诱导内皮细胞分化,在CNV的形成过程中起着主要作用。
3.玻璃体腔内注射Bevacizumab(Avastin)可以抑制氪激光诱导的BN大鼠CNV的形成和发展
Objective
1. This study was designed to establish an experimental CNV model in BN rats by laser infusion in order to establish the foundation for studying the mechanism of CNV during 4 weeks.
2. The total mRNA of VEGF was detected by RT-PCR. Exam FⅧ-Rag protein expression in CNV and investigate the relationship between VEGF and CNV development.
3. Study the evolution of CNV during 3 weeks with intravitreal injection of Bevacizumab(Avastin)1μl(25μg/1μl), so make a preliminary investigation of the treatment effect of Bevacizumab(Avastin)on experimental CNV.
Methods
1. Five groups of 40 BN rats was photocoagulated by krypton laser in one eye to induce CNV, the laser spots were placed separately using a setting of 50 um diameter, 0.05 second duration ,and 360 mW intensity, surrounding the optic nerve at the posterior pole.
2. Fluorescein angiography was performed from 3 day to 4 weeks after laser photocoagulation .The CNV area was measured by early phase FFA. Then the rats were sacrificed immediately, the eyes were enucleated and processed for histopathologic examination and RT-PCR. Exam the FⅧ-Rag protein expression in CNV .Observe the expression of VEGF protein in rat retinal and the lesion of CNV.
3. Tow groups of 48 BN rats was photocoagulated by krypton laser in one eye to induce CNV model in one eye, in which 1 group received intravitreal injection of Bevacizumab(Avastin)1μl(25μg/1μl)after photocoagulation one week and the other group received the same volume intravitreal injection of BSS as control. FFA examinations occurred on 1,2,3 week and the eyes were enucleated and processed for histopathologic examination. The area of CNV were measured in early phase FFA. FⅧ-Rag were investigated with immunohistochemistry and were semiquantitatively analyzed.
4. The experimental data were expressed as the mean±S.D. and statistical significance was determingd by t-test.
Results
1. Disciform fluorescein leakage was observed and confirmed the formation of CNV.The immunostaining of FⅧ-Rag and FFA show the persistent increase of the CNV development during 4 weeks.
2. During the CNV development,the expression of FⅧ-Rag and VEGF increase gradually. V EGF expression was mainly observed in the Vascular endothelial cells , the ganglion cells , the inner nuclear layers , the retinal pigment epithelial cells in normal retina and the vascular endothelial cells of normal choroids of BN rat . VEGF expressed in the ganglion cells , the inner nuclear layers , lesion of outer nuclear layers inretina after photocoagulation .
3. The area and the leakage of CNV in Bevacizumab(Avastin)treated BN rat eyes obviously reduced compared with the control group at each interval. In the treatment group,the expression of FⅧ-Rag depressed. The examination of FⅧ-Rag and FFA verified the depression of CNV compared with the control group at each interval.The density of treatment is lower than that of control group(P<0.05)
Conclusion
1. krypton laser photocoagulation can be successfully used to produce CNV experimental model in the BN rat. CNV pathologic structure possesses stability.
2. VEGF play the important role in CNV development.
3. Intravitreal Bevacizumab(Avastin)Injection can inhibit the development of CNV
引文
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