玻璃体腔重复注射bevacizumab对糖尿病视网膜病变大鼠视网膜的毒性观察
发布时间：2018-08-02 14:15
【摘要】：目的 观察玻璃体腔重复注射抗血管内皮生长因子(vascular endothelial growth factor,VEGF)单克隆抗体bevacizumab(商品名Avastin)对糖尿病视网膜病变(diabetic retinopathy,DR)大鼠视网膜的毒性作用,探讨bevacizumab在治疗DR时可能有的毒性作用,为DR和其他视网膜新生血管性疾病的临床防治提供基础理论依据。 方法 1.健康成年雄性SD大鼠40只,随机分成正常对照(A)组和糖尿病组,分别为10、30只。链尿佐菌素(streptozotocin,STZ)尾静脉注射制作糖尿病大鼠模型。溴化乙锭(Evans blue,EB)染色视网膜铺片证实DR模型成功后,30只糖尿病大鼠中,随机选取10只作为DR组(B组),不作任何处理；其余20只大鼠左眼为实验组(C组),玻璃体腔注射25 mg/ml的bevacizumab 3μ1,共注射3次,每次间隔10d；右眼为实验对照组(D组),不给予任何处理。 2.于C组末次干预后20天,采用闪光视网膜电图(Flicker Electroretinogram,F-ERG)对各组进行视网膜功能检测。EB染色视网膜铺片方法,荧光显微镜观察各组视网膜血管的变化情况；苏木精-伊红(HE)染色,光学显微镜下观察视网膜形态学变化；免疫组化SP法观察视网膜各层Thy-1及VEGF阳性染色情况。 3.所有数据应用SPSS13.0统计分析软件包进行统计学分析,计量资料以均数±标准差(x±s)表示,经正态性检验及方差齐性检验,对不同处理组间的数据采用单因素方差分析,并用SNK检验进行组间比较。以P0.05作为差异有统计学意义的标准。 结果 1.F-ERG检测 检查显示,A、B、C、D 4组暗适应a、b波潜伏期,暗适应b波振幅及振荡电位Ops总振幅比较,差异均有统计学意义(F=33.165,36.162,19.955,23.243:P值均=0.000)；进一步用SNK法进行组间两两比较,B组,D组的暗适应a波潜伏期、暗适应b波振幅、振荡电位Ops总振幅与A组,C组比较,差异均有统计学意义(P0.05)；A组,C组暗适应b波潜伏期与B组,与D组比较,差异均有统计学意义(P0.05)；B组与D组的暗适应b波潜伏期比较,差异也有统计学意义(P0.05)。 2.EB染色视网膜铺片 A组大鼠视网膜血管走行良好,视网膜浅层大血管及深层血管网均清晰可见,未见视网膜微血管瘤和视网膜新生血管形成。B组大鼠视网膜血管走行迂曲、扩张,可见微动脉瘤,血管呈串珠样改变,血管周围可见高荧光渗漏,周边视网膜可见大片无灌注区。C组玻璃体腔重复注射bevacizumab3次后,视网膜血管走形规则、变细。D组大鼠视网膜可见微血管瘤,血管周围高荧光渗漏较B组减少。 3.HE染色 A组大鼠视网膜层次结构整齐分明,RGCs排列整齐；内、外核层细胞核染色深紫,均匀；光感受器细胞内外节形态规整；视网膜色素上皮(RPE)细胞核呈淡紫色,扁椭圆形,边界清晰。B组大鼠视网膜各层细胞结构排列紊乱,内界膜不完整；视网膜内血管扩张,血管内皮细胞增殖；RGCs、周细胞等各层细胞数减少。C组玻璃体腔重复注射bevacizumab3次后,RGCs数量明显低于正常,光感受器细胞也低于正常。D组视网膜内界膜增厚,局部不完整,未见明显扩张血管,各层细胞排列不整齐。 4.免疫组织化学染色 Thy-1阳性染色主要位于神经节细胞层(ganglion cell layer,GCL),及少数位于内丛状层(inner plexiform layer,IPL)、内核层(inner nuclear layer,INL)。A组Thy-1表达呈强阳性,GCL染色明显,神经纤维层(nerval fiber layer,NFL)、INL轻度染色；B组Thy-1表达呈弱阳性,主要位于GCL,; C组Thy-1染色明显减弱,GCL阳性表达稀少；D组Thy-1表达呈弱阳性,阳性染色主要位于GCL,也可见于INL。A、B、C、D四组间染色区Thy-1的平均IOD差异有统计学意义(F=25.819, P=0.000),进一步用SNK法进行组间两两比较,C组与其他三组之间差异有统计学意义(P0.05),B组与A组之间差异有统计学意义(P0.05),D组与B组之间差异也有统计学意义(P0.05)。 VEGF阳性表达主要定位于GCL,少量位于NFL, INL及RPE层。A组VEGF表达呈弱阳性,主要位于GCL; B组VEGF表达呈强阳性,主要位于GCL、N FLINL也有表达；C组VEGF表达明显减少,主要位于GCL；D组VEGF表达呈弱阳性,主要位于GCL和INL层,RPE层也有表达。染色区域VEGF的平均IOD值在A、B、C、D四组之间差异有统计学意义(F=45.243, P=0.000),进一步用SNK法进行组间两两比较,B组与其他三组之间差异有统计学意义(P0.05)。 结论 1、玻璃体腔重复注射bevacizumab治疗DR,对RGCs有一定毒性作用。 2、玻璃体腔重复注射bevacizumab治疗DR,对视网膜血管造成一定损伤,使视网膜的血管变细。 3、玻璃体腔重复注射bevacizumab治疗DR,对对侧眼有一定的影响。
[Abstract]:objective
To observe the toxicity of vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab (commodity name Avastin) to the retina of diabetic retinopathy (diabetic retinopathy, DR) in rats, and to explore the toxicity of bevacizumab in the treatment of DR. Provide a theoretical basis for clinical prevention and treatment of neovascular neovascularization.
Method
1. healthy adult male SD rats were randomly divided into the normal control (A) group and the diabetic group. The diabetic rat model was made by injection of 10,30 only. Streptozotocin (STZ) was injected into the tail vein of the diabetic rats. After the Evans blue (EB) staining retina spread was used to confirm the DR model success, 10 rats were randomly selected as DR. Group (group B) without any treatment; the left eye of the other 20 rats was the experimental group (group C), the vitreous cavity was injected with 25 mg/ml bevacizumab 3 mu 1, and the total injection was 3 times, each interval was 10d, the right eye was the experimental control group (group D), and no treatment was given.
2. at the last 20 days after the end of the C group, Flicker Electroretinogram (F-ERG) was used to detect retinal function by.EB staining of retina, and the changes of retinal vessels were observed by fluorescence microscope; the color of hematoxylin eosin (HE) and morphological changes of retina under optical microscope were observed. The positive staining of Thy-1 and VEGF in each layer of retina was observed by histochemical SP assay.
3. all data were statistically analyzed with SPSS13.0 statistical analysis software package, and the measurement data were expressed with mean standard deviation (x + s). Through normal test and variance homogeneity test, the data between different processing groups were analyzed by single factor analysis of variance and SNK test was used to compare the data between groups. P0.05 was used as the standard of statistical significance.
Result
1.F-ERG detection
The examination showed that the 4 groups of A, B, C and D were dark adapted to a, b wave incubation period, and the difference was statistically significant (F=33.165,36.162,19.955,23.243:P value =0.000) in the amplitude of B wave and the total amplitude of oscillatory potential Ops (F=33.165,36.162,19.955,23.243:P value =0.000). Compared with group A and group C, the difference was statistically significant (P0.05); in group A, the latent period of B wave in C group and B group were statistically significant (P0.05), and the difference was statistically significant compared with that of D group.
2.EB staining retina sheet
The retinal vessels of the A group were good, the large vessels of the shallow layer of the retina and the deep vascular network were clearly visible. There was no retinal microangioma and retinal neovascularization in group.B. The retinal vessels in the group of retina of the retina were found to be circuitous, dilatation, microaneurysm, blood vessels showing a bead like change, high fluorescence leakage around the blood vessels, and the peripheral retina visible. After bevacizumab3 repeated injection of the vitreous cavity in.C group, the retinal blood vessels were in shape, and microangioma was seen in the retina of group.D, and the high fluorescence leakage around the blood vessel was less than that of the B group.
3.HE staining
The retina structure of rats in group A was neatly distinct, and the RGCs arrangement was neatly arranged; inside, the nucleus staining of the outer nucleus was deep purple and uniform; the morphology of the cells inside and outside of the photoreceptor cells was regular; the nucleus of the retinal pigment epithelium (RPE) was lilac, oblong, and the boundary was clear in group.B, the structure of each layer of the retina membrane was disorganized and the inner boundary membrane was incomplete; Vascular endothelial cell proliferation and vascular dilatation in the omentum, RGCs, pericytes and other layers of cells reduced.C after bevacizumab3 repeated injection of glass cavity, the number of RGCs was significantly lower than normal, the photoreceptor cells were also lower than the normal.D group of the retinal inner boundary membrane thickening, local incomplete, no obvious dilated blood vessels, the layers of irregular cells.
4. immunohistochemical staining
Thy-1 positive staining mainly lies in the ganglion cell layer (ganglion cell layer, GCL), and a few in the inner plexiform layer (inner plexiform layer, IPL), and the kernel layer (inner nuclear layer) is strongly positive. At GCL, the Thy-1 staining of the C group was obviously weakened and the positive expression of GCL was scarce; the Thy-1 expression in the D group was weakly positive, the positive staining was mainly located in the GCL, and the average difference between the four groups of INL.A, B, C and D was statistically significant. The difference between the group and the other three groups was further compared. The difference between group B and group A was statistically significant (P 0.05), and the difference between group D and group B was statistically significant (P 0.05).
The positive expression of VEGF is mainly located in GCL, a small amount of VEGF in NFL, INL and RPE layer.A is weakly positive, mainly in GCL, B group VEGF expression is strongly positive, mainly located in GCL, N also expressed. The average IOD value of the domain VEGF was statistically significant between the four groups of A, B, C and D (F=45.243, P=0.000). The difference between the group 22 and the other three groups was statistically significant (P0.05).
conclusion
1, repeated injection of bevacizumab into vitreous cavity for treatment of DR has a certain toxic effect on RGCs.
2, repeated injection of bevacizumab into vitreous cavity to treat DR can cause certain damage to retinal vessels and make the retinal vessels thinner.
3, repeated injection of bevacizumab into vitreous cavity for DR has certain effect on contralateral eye.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1
本文编号：2159698
[Abstract]:objective
To observe the toxicity of vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab (commodity name Avastin) to the retina of diabetic retinopathy (diabetic retinopathy, DR) in rats, and to explore the toxicity of bevacizumab in the treatment of DR. Provide a theoretical basis for clinical prevention and treatment of neovascular neovascularization.
Method
1. healthy adult male SD rats were randomly divided into the normal control (A) group and the diabetic group. The diabetic rat model was made by injection of 10,30 only. Streptozotocin (STZ) was injected into the tail vein of the diabetic rats. After the Evans blue (EB) staining retina spread was used to confirm the DR model success, 10 rats were randomly selected as DR. Group (group B) without any treatment; the left eye of the other 20 rats was the experimental group (group C), the vitreous cavity was injected with 25 mg/ml bevacizumab 3 mu 1, and the total injection was 3 times, each interval was 10d, the right eye was the experimental control group (group D), and no treatment was given.
2. at the last 20 days after the end of the C group, Flicker Electroretinogram (F-ERG) was used to detect retinal function by.EB staining of retina, and the changes of retinal vessels were observed by fluorescence microscope; the color of hematoxylin eosin (HE) and morphological changes of retina under optical microscope were observed. The positive staining of Thy-1 and VEGF in each layer of retina was observed by histochemical SP assay.
3. all data were statistically analyzed with SPSS13.0 statistical analysis software package, and the measurement data were expressed with mean standard deviation (x + s). Through normal test and variance homogeneity test, the data between different processing groups were analyzed by single factor analysis of variance and SNK test was used to compare the data between groups. P0.05 was used as the standard of statistical significance.
Result
1.F-ERG detection
The examination showed that the 4 groups of A, B, C and D were dark adapted to a, b wave incubation period, and the difference was statistically significant (F=33.165,36.162,19.955,23.243:P value =0.000) in the amplitude of B wave and the total amplitude of oscillatory potential Ops (F=33.165,36.162,19.955,23.243:P value =0.000). Compared with group A and group C, the difference was statistically significant (P0.05); in group A, the latent period of B wave in C group and B group were statistically significant (P0.05), and the difference was statistically significant compared with that of D group.
2.EB staining retina sheet
The retinal vessels of the A group were good, the large vessels of the shallow layer of the retina and the deep vascular network were clearly visible. There was no retinal microangioma and retinal neovascularization in group.B. The retinal vessels in the group of retina of the retina were found to be circuitous, dilatation, microaneurysm, blood vessels showing a bead like change, high fluorescence leakage around the blood vessels, and the peripheral retina visible. After bevacizumab3 repeated injection of the vitreous cavity in.C group, the retinal blood vessels were in shape, and microangioma was seen in the retina of group.D, and the high fluorescence leakage around the blood vessel was less than that of the B group.
3.HE staining
The retina structure of rats in group A was neatly distinct, and the RGCs arrangement was neatly arranged; inside, the nucleus staining of the outer nucleus was deep purple and uniform; the morphology of the cells inside and outside of the photoreceptor cells was regular; the nucleus of the retinal pigment epithelium (RPE) was lilac, oblong, and the boundary was clear in group.B, the structure of each layer of the retina membrane was disorganized and the inner boundary membrane was incomplete; Vascular endothelial cell proliferation and vascular dilatation in the omentum, RGCs, pericytes and other layers of cells reduced.C after bevacizumab3 repeated injection of glass cavity, the number of RGCs was significantly lower than normal, the photoreceptor cells were also lower than the normal.D group of the retinal inner boundary membrane thickening, local incomplete, no obvious dilated blood vessels, the layers of irregular cells.
4. immunohistochemical staining
Thy-1 positive staining mainly lies in the ganglion cell layer (ganglion cell layer, GCL), and a few in the inner plexiform layer (inner plexiform layer, IPL), and the kernel layer (inner nuclear layer) is strongly positive. At GCL, the Thy-1 staining of the C group was obviously weakened and the positive expression of GCL was scarce; the Thy-1 expression in the D group was weakly positive, the positive staining was mainly located in the GCL, and the average difference between the four groups of INL.A, B, C and D was statistically significant. The difference between the group and the other three groups was further compared. The difference between group B and group A was statistically significant (P 0.05), and the difference between group D and group B was statistically significant (P 0.05).
The positive expression of VEGF is mainly located in GCL, a small amount of VEGF in NFL, INL and RPE layer.A is weakly positive, mainly in GCL, B group VEGF expression is strongly positive, mainly located in GCL, N also expressed. The average IOD value of the domain VEGF was statistically significant between the four groups of A, B, C and D (F=45.243, P=0.000). The difference between the group 22 and the other three groups was statistically significant (P0.05).
conclusion
1, repeated injection of bevacizumab into vitreous cavity for treatment of DR has a certain toxic effect on RGCs.
2, repeated injection of bevacizumab into vitreous cavity to treat DR can cause certain damage to retinal vessels and make the retinal vessels thinner.
3, repeated injection of bevacizumab into vitreous cavity for DR has certain effect on contralateral eye.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1
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