玻璃體腔重復注射bevacizumab對糖尿病視網(wǎng)膜病變大鼠視網(wǎng)膜的毒性觀察
發(fā)布時間：2018-08-02 14:15
【摘要】：目的 觀察玻璃體腔重復注射抗血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)單克隆抗體bevacizumab(商品名Avastin)對糖尿病視網(wǎng)膜病變(diabetic retinopathy,DR)大鼠視網(wǎng)膜的毒性作用,探討bevacizumab在治療DR時可能有的毒性作用,為DR和其他視網(wǎng)膜新生血管性疾病的臨床防治提供基礎理論依據(jù)。 方法 1.健康成年雄性SD大鼠40只,隨機分成正常對照(A)組和糖尿病組,分別為10、30只。鏈尿佐菌素(streptozotocin,STZ)尾靜脈注射制作糖尿病大鼠模型。溴化乙錠(Evans blue,EB)染色視網(wǎng)膜鋪片證實DR模型成功后,30只糖尿病大鼠中,隨機選取10只作為DR組(B組),不作任何處理；其余20只大鼠左眼為實驗組(C組),玻璃體腔注射25 mg/ml的bevacizumab 3μ1,共注射3次,每次間隔10d；右眼為實驗對照組(D組),不給予任何處理。 2.于C組末次干預后20天,采用閃光視網(wǎng)膜電圖(Flicker Electroretinogram,F-ERG)對各組進行視網(wǎng)膜功能檢測。EB染色視網(wǎng)膜鋪片方法,熒光顯微鏡觀察各組視網(wǎng)膜血管的變化情況；蘇木精-伊紅(HE)染色,光學顯微鏡下觀察視網(wǎng)膜形態(tài)學變化；免疫組化SP法觀察視網(wǎng)膜各層Thy-1及VEGF陽性染色情況。 3.所有數(shù)據(jù)應用SPSS13.0統(tǒng)計分析軟件包進行統(tǒng)計學分析,計量資料以均數(shù)±標準差(x±s)表示,經(jīng)正態(tài)性檢驗及方差齊性檢驗,對不同處理組間的數(shù)據(jù)采用單因素方差分析,并用SNK檢驗進行組間比較。以P0.05作為差異有統(tǒng)計學意義的標準。 結(jié)果 1.F-ERG檢測 檢查顯示,A、B、C、D 4組暗適應a、b波潛伏期,暗適應b波振幅及振蕩電位Ops總振幅比較,差異均有統(tǒng)計學意義(F=33.165,36.162,19.955,23.243:P值均=0.000)；進一步用SNK法進行組間兩兩比較,B組,D組的暗適應a波潛伏期、暗適應b波振幅、振蕩電位Ops總振幅與A組,C組比較,差異均有統(tǒng)計學意義(P0.05)；A組,C組暗適應b波潛伏期與B組,與D組比較,差異均有統(tǒng)計學意義(P0.05)；B組與D組的暗適應b波潛伏期比較,差異也有統(tǒng)計學意義(P0.05)。 2.EB染色視網(wǎng)膜鋪片 A組大鼠視網(wǎng)膜血管走行良好,視網(wǎng)膜淺層大血管及深層血管網(wǎng)均清晰可見,未見視網(wǎng)膜微血管瘤和視網(wǎng)膜新生血管形成。B組大鼠視網(wǎng)膜血管走行迂曲、擴張,可見微動脈瘤,血管呈串珠樣改變,血管周圍可見高熒光滲漏,周邊視網(wǎng)膜可見大片無灌注區(qū)。C組玻璃體腔重復注射bevacizumab3次后,視網(wǎng)膜血管走形規(guī)則、變細。D組大鼠視網(wǎng)膜可見微血管瘤,血管周圍高熒光滲漏較B組減少。 3.HE染色 A組大鼠視網(wǎng)膜層次結(jié)構(gòu)整齊分明,RGCs排列整齊；內(nèi)、外核層細胞核染色深紫,均勻；光感受器細胞內(nèi)外節(jié)形態(tài)規(guī)整；視網(wǎng)膜色素上皮(RPE)細胞核呈淡紫色,扁橢圓形,邊界清晰。B組大鼠視網(wǎng)膜各層細胞結(jié)構(gòu)排列紊亂,內(nèi)界膜不完整；視網(wǎng)膜內(nèi)血管擴張,血管內(nèi)皮細胞增殖；RGCs、周細胞等各層細胞數(shù)減少。C組玻璃體腔重復注射bevacizumab3次后,RGCs數(shù)量明顯低于正常,光感受器細胞也低于正常。D組視網(wǎng)膜內(nèi)界膜增厚,局部不完整,未見明顯擴張血管,各層細胞排列不整齊。 4.免疫組織化學染色 Thy-1陽性染色主要位于神經(jīng)節(jié)細胞層(ganglion cell layer,GCL),及少數(shù)位于內(nèi)叢狀層(inner plexiform layer,IPL)、內(nèi)核層(inner nuclear layer,INL)。A組Thy-1表達呈強陽性,GCL染色明顯,神經(jīng)纖維層(nerval fiber layer,NFL)、INL輕度染色；B組Thy-1表達呈弱陽性,主要位于GCL,; C組Thy-1染色明顯減弱,GCL陽性表達稀少；D組Thy-1表達呈弱陽性,陽性染色主要位于GCL,也可見于INL。A、B、C、D四組間染色區(qū)Thy-1的平均IOD差異有統(tǒng)計學意義(F=25.819, P=0.000),進一步用SNK法進行組間兩兩比較,C組與其他三組之間差異有統(tǒng)計學意義(P0.05),B組與A組之間差異有統(tǒng)計學意義(P0.05),D組與B組之間差異也有統(tǒng)計學意義(P0.05)。 VEGF陽性表達主要定位于GCL,少量位于NFL, INL及RPE層。A組VEGF表達呈弱陽性,主要位于GCL; B組VEGF表達呈強陽性,主要位于GCL、N FLINL也有表達；C組VEGF表達明顯減少,主要位于GCL；D組VEGF表達呈弱陽性,主要位于GCL和INL層,RPE層也有表達。染色區(qū)域VEGF的平均IOD值在A、B、C、D四組之間差異有統(tǒng)計學意義(F=45.243, P=0.000),進一步用SNK法進行組間兩兩比較,B組與其他三組之間差異有統(tǒng)計學意義(P0.05)。 結(jié)論 1、玻璃體腔重復注射bevacizumab治療DR,對RGCs有一定毒性作用。 2、玻璃體腔重復注射bevacizumab治療DR,對視網(wǎng)膜血管造成一定損傷,使視網(wǎng)膜的血管變細。 3、玻璃體腔重復注射bevacizumab治療DR,對對側(cè)眼有一定的影響。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R774.1
本文編號：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R774.1
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