玻璃體腔注射Bevacizumab在早期糖尿病視網(wǎng)膜病變大鼠模型治療中的實驗研究
發(fā)布時間：2018-07-28 12:17
【摘要】：目的 通過觀察早期糖尿病視網(wǎng)膜病變(DR)大鼠玻璃體腔注射Bevacizumab后血視網(wǎng)膜屏障(BRB)功能、視網(wǎng)膜血管形態(tài)的變化,研究玻璃體腔注射Bevacizumab應(yīng)用于早期DR后延緩DR進展的有效性,明確Bevacizumab的劑量及合適重復(fù)給藥時間,探討預(yù)防性使用的可行性。 方法 1糖尿病(DM)大鼠模型制造：正常清潔級雄性SD大鼠80只,隨即抽取13只作為正常對照組(N組),余67只尾靜脈注射鏈脲佐菌素(STZ) 45 mg/kg誘導(dǎo)糖尿病(DM)大鼠模型。于給藥后一周剪尾采血測空腹血糖,若血糖水平16.65 mmol／L,表明DM大鼠模型建立成功。模型建立后觀察1周,然后開始計算DM病程。觀察各組大鼠的形態(tài)改變,每4周檢測1次空腹血糖及體質(zhì)量。 2 DM大鼠患有早期DR驗證：成模后10周,抽取DM大鼠及正常大鼠各2只進行視網(wǎng)膜鋪片,比較觀察正常大鼠和DM大鼠微血管形態(tài)變化,確定DM大鼠已患有早期DR。 3玻璃體腔注射Bevacizumab:在確定DM大鼠已患有DR后,DM大鼠隨機分為糖尿病對照組(DR組)、大劑量治療組(H組)及小劑量治療組(L組),L組及H組分別給予濃度為1.25mg/ml及2.5mg/ml的Bevacizumab4μ1玻璃體腔注射,均注射一次,N組及DR組未接受任何治療。 4治療效果觀察：干預(yù)治療后2周、4周及8周,進行免疫組織化學(xué)檢測分析血.管內(nèi)皮生長因子(VEGF)表達的變化,用伊文思藍(EB)法定量檢測BRB破壞程度,用EB灌注視網(wǎng)膜鋪片法觀察視網(wǎng)膜血管形態(tài)的變化,研究玻璃體腔注射Bevacizumab延緩DR進展的作用。采用SPSS17.0軟件包進行統(tǒng)計學(xué)處理,采用單因素方差分析分析數(shù)據(jù),P值0.05認為差異有統(tǒng)計學(xué)意義。 結(jié)果 1 DM大鼠模型制造：實驗組大鼠一次成模65只,未成模的大鼠用于正常對照組,成模率為97%。 2 DM大鼠患有早期DR驗證：成模后10周,視網(wǎng)膜鋪片示：正常大鼠視網(wǎng)膜血管走行大致正常,血管密度均勻,毛細血管管徑粗細一致,未見迂曲擴張及微血管瘤等病理變化,未見染料滲漏；DM大鼠視網(wǎng)膜血管變密,血管走行迂曲,可見靜脈串珠樣改變,偶見小血.管滲漏染料。DM大鼠已發(fā)生早期視網(wǎng)膜病變。 3本實驗中共50只DM大鼠接受玻璃體腔注射Bevacizumab治療,其中2只由于技術(shù)操作問題,出現(xiàn)玻璃體積血,至實驗結(jié)束,未發(fā)現(xiàn)眼內(nèi)炎的患鼠,玻璃體腔注射Bevacizumab的大鼠隨著注射時間的延長,均出現(xiàn)白內(nèi)障。 4治療效果：干預(yù)治療后,免疫組化顯示：各組大鼠視網(wǎng)膜內(nèi)VEGF各層均有表達,干預(yù)治療組VEGF表達強于N組而弱于DR組。干預(yù)治療后2周,N組、DR組、L組及H組EB滲漏量差異有統(tǒng)計學(xué)意義(P=0.000),L組及H組之間差異無統(tǒng)計學(xué)意義(P=0.364)；干預(yù)治療后4周,N組、DR組、L組及H組EB滲漏量差異有統(tǒng)計學(xué)意義(P=0.000),L組及H組之間差異有統(tǒng)計學(xué)意義(P=0.035)；干預(yù)治療后8周,N組、DR組、L組及H組EB滲漏量差異有統(tǒng)計學(xué)意義(P=0.000),L組及H組之間差異有統(tǒng)計學(xué)意義(P=0.031)。視網(wǎng)膜鋪片示：DR組：給藥后2周,視網(wǎng)膜血管明顯變密,血管走行明顯迂曲,可見視網(wǎng)膜大血管分支滲漏染料,可見微血管瘤,未見新生血管；給藥后4周及8周,視網(wǎng)膜血管迂曲擴張,管徑粗細不均,某些部位管腔狹窄甚至閉塞,走行紊亂,周邊視網(wǎng)膜小血管大片染料滲漏,可見微血管瘤、無灌注區(qū)。各干預(yù)組視網(wǎng)膜血管的密度較N組明顯增高,較DR組明顯減輕.視網(wǎng)膜血管迂曲不明顯,未見染料滲漏,未見微血管瘤及無灌注區(qū)。L組偶見小血管滲漏。 結(jié)論 (1)檢測DR大鼠視網(wǎng)膜內(nèi)EB含量,可以比較精確地發(fā)現(xiàn)大鼠視網(wǎng)膜BRB的改變,是評價DR大鼠BRB功能的有效方法。 (2)給予早期DR大鼠玻璃體腔注射Bevacizumab可以部分修復(fù)受損的BRB,延緩視網(wǎng)膜微血管病變的進展,在不引起視網(wǎng)膜毒性損傷的情況下,玻璃體腔注入適量的Bevacizumab有延緩DR進展的作用,2.5mg/0.1ml的遠期效果可能優(yōu)于1.25mg/0.1ml。 (3)玻璃體腔注射Bevacizumab修復(fù)DR大鼠BRB/及延緩視網(wǎng)膜微血管病變進展的作用與劑量有關(guān),且注射后至少8周內(nèi)效果穩(wěn)定,本實驗認為Bevacizumab重復(fù)給藥的時間可以延長至給藥后8周。
[Abstract]:objective
By observing the function of retinal barrier (BRB) after injection of Bevacizumab in the vitreous cavity of the early diabetic retinopathy (DR) rats and the changes of retinal vascular morphology, the effectiveness of the application of Bevacizumab in the vitreous cavity to postpone the progress of DR after early DR was studied, and the dosage of Bevacizumab and the appropriate time of repeated administration were determined, and the preventive effect was discussed. It is feasible to use.
Method
1 diabetic (DM) rat model was made: 80 rats of normal clean grade male SD rats, 13 rats were selected as normal control group (group N), and the remaining 67 tail veins were injected with streptozotocin (STZ) 45 mg/kg to induce diabetic (DM) rat model. The blood glucose was measured by cutting the tail and measuring the blood glucose in one week after administration. If blood glucose level was 16.65 mmol / L, the model of DM rat was built. After the establishment of the model, the rats were observed for 1 weeks, and then the DM course was calculated. Morphological changes were observed in each group, and fasting blood glucose and body weight were detected every 1 weeks every 4 weeks.
2 DM rats had early DR verification: 10 weeks after the completion of the model, 2 rats were selected from the DM rats and the normal rats, and the microvascular morphologic changes of the normal rats and the DM rats were observed and the DM rats had early DR..
3 DM rats were randomly divided into the diabetes control group (group DR), the large dose treatment group (group H) and the small dose group (group L), and the L group and the H group were given the Bevacizumab4 U 1 vitreous cavity of 1.25mg/ml and 2.5mg/ml, respectively, after the Bevacizumab: rats were determined to have DR in the DM rats, and the group and the H group were injected once, and the group and the group did not receive any treatment.
4 treatment effect observation: 2 weeks, 4 weeks and 8 weeks after intervention, the changes in the expression of vascular endothelial growth factor (VEGF) were detected by immunohistochemical staining. The damage degree of BRB was detected by the statutory amount of Evans blue (EB), and the changes of retinal vascular morphology were observed by EB perfusion retina paving method, and Bevacizumab was delayed by intravitreal injection of Bevacizumab to postpone DR advance. The SPSS17.0 software package was used for statistical analysis. The data were analyzed by one-way ANOVA. The P value of 0.05 was considered to be statistically significant.
Result
1 DM rat model: 65 rats in the experimental group were modeled at one time, and the rats in the unmodeled group were used in the normal control group with the rate of 97%..
2 DM rats had early DR verification: 10 weeks after the formation of the model, the retina spread showed that the retinal vessels in the normal rats walked roughly, the blood vessel density was uniform, the capillary diameter of the capillary was the same, the pathological changes such as tortuous dilation and microangioma were not seen, no dyestuff leakage was found, the retinal vessels of DM rats were denser, blood vessels were circuitous, venous string was visible. Pearson changes and occasional small blood. Early leakage of dyestuff.DM rat retinopathy occurred.
3 50 DM rats were treated with vitreous intravitreal injection of Bevacizumab, of which 2 were caused by vitreous hemorrhage due to the technical operation problems. To the end of the experiment, no endophthalmitis was found in the rats. The rats of the glass cavity injected with Bevacizumab were all cataract with the prolongation of the injection time.
4 treatment effect: after intervention, immunohistochemistry showed that each layer of VEGF in each group was expressed in the retina, and the expression of VEGF in the intervention group was stronger than that in the group N, but in the group DR. The difference of EB leakage of the N group, the DR group, the L group and the H group was statistically significant (P=0.000), and the difference between the L group and the H group was not statistically significant. There was significant difference in EB leakage between group N, group DR, group L and group H after 4 weeks of treatment (P=0.000), and there was significant difference between group L and H group (P=0.035), and there was a significant difference between the N group, DR group, L group and group 8 weeks after intervention. 2 weeks after the administration, the retinal vessels were obviously denser and the blood vessels were obviously tortuous. It could be seen that the branches of the retinal vessels leaked the dye, and the microangioma was seen and the neovascularization was not seen. 4 and 8 weeks after the administration, the retinal vessels were dilated, the diameter of the tube was unevenly, the lumen in some parts of the tube narrowed or even obliterated, and the small vessels in the peripheral retina were large. The density of retinal vessels in each intervention group was significantly higher than that in the N group, and the retinal vessels were less obvious than those in the DR group. No dyestuff leakage was found, no microangioma and.L group in the non perfusion area were found to see small vascular leakage.
conclusion
(1) detecting the content of EB in the retina of DR rats can accurately detect the change of BRB in the retina of rats. It is an effective method to evaluate the BRB function of DR rats.
(2) the injection of Bevacizumab in the vitreous cavity of early DR rats can partially repair the damaged BRB and delay the progression of retinal microvascular lesions. In the case of no retinal toxicity, the injection of appropriate amount of Bevacizumab in the vitreous cavity can delay the progress of DR, and the long-term effect of 2.5mg/ 0.1ml may be better than 1.25mg/0.1ml..
(3) the effect of intravitreal injection of Bevacizumab on the repair of BRB/ in DR rats and to delay the progression of retinal microvascular lesions is related to the dose, and the effect is stable at least 8 weeks after injection. This experiment suggests that the time of Bevacizumab repeated administration can be prolonged to 8 weeks after the Administration.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R774.1
本文編號：2150115
[Abstract]:objective
By observing the function of retinal barrier (BRB) after injection of Bevacizumab in the vitreous cavity of the early diabetic retinopathy (DR) rats and the changes of retinal vascular morphology, the effectiveness of the application of Bevacizumab in the vitreous cavity to postpone the progress of DR after early DR was studied, and the dosage of Bevacizumab and the appropriate time of repeated administration were determined, and the preventive effect was discussed. It is feasible to use.
Method
1 diabetic (DM) rat model was made: 80 rats of normal clean grade male SD rats, 13 rats were selected as normal control group (group N), and the remaining 67 tail veins were injected with streptozotocin (STZ) 45 mg/kg to induce diabetic (DM) rat model. The blood glucose was measured by cutting the tail and measuring the blood glucose in one week after administration. If blood glucose level was 16.65 mmol / L, the model of DM rat was built. After the establishment of the model, the rats were observed for 1 weeks, and then the DM course was calculated. Morphological changes were observed in each group, and fasting blood glucose and body weight were detected every 1 weeks every 4 weeks.
2 DM rats had early DR verification: 10 weeks after the completion of the model, 2 rats were selected from the DM rats and the normal rats, and the microvascular morphologic changes of the normal rats and the DM rats were observed and the DM rats had early DR..
3 DM rats were randomly divided into the diabetes control group (group DR), the large dose treatment group (group H) and the small dose group (group L), and the L group and the H group were given the Bevacizumab4 U 1 vitreous cavity of 1.25mg/ml and 2.5mg/ml, respectively, after the Bevacizumab: rats were determined to have DR in the DM rats, and the group and the H group were injected once, and the group and the group did not receive any treatment.
4 treatment effect observation: 2 weeks, 4 weeks and 8 weeks after intervention, the changes in the expression of vascular endothelial growth factor (VEGF) were detected by immunohistochemical staining. The damage degree of BRB was detected by the statutory amount of Evans blue (EB), and the changes of retinal vascular morphology were observed by EB perfusion retina paving method, and Bevacizumab was delayed by intravitreal injection of Bevacizumab to postpone DR advance. The SPSS17.0 software package was used for statistical analysis. The data were analyzed by one-way ANOVA. The P value of 0.05 was considered to be statistically significant.
Result
1 DM rat model: 65 rats in the experimental group were modeled at one time, and the rats in the unmodeled group were used in the normal control group with the rate of 97%..
2 DM rats had early DR verification: 10 weeks after the formation of the model, the retina spread showed that the retinal vessels in the normal rats walked roughly, the blood vessel density was uniform, the capillary diameter of the capillary was the same, the pathological changes such as tortuous dilation and microangioma were not seen, no dyestuff leakage was found, the retinal vessels of DM rats were denser, blood vessels were circuitous, venous string was visible. Pearson changes and occasional small blood. Early leakage of dyestuff.DM rat retinopathy occurred.
3 50 DM rats were treated with vitreous intravitreal injection of Bevacizumab, of which 2 were caused by vitreous hemorrhage due to the technical operation problems. To the end of the experiment, no endophthalmitis was found in the rats. The rats of the glass cavity injected with Bevacizumab were all cataract with the prolongation of the injection time.
4 treatment effect: after intervention, immunohistochemistry showed that each layer of VEGF in each group was expressed in the retina, and the expression of VEGF in the intervention group was stronger than that in the group N, but in the group DR. The difference of EB leakage of the N group, the DR group, the L group and the H group was statistically significant (P=0.000), and the difference between the L group and the H group was not statistically significant. There was significant difference in EB leakage between group N, group DR, group L and group H after 4 weeks of treatment (P=0.000), and there was significant difference between group L and H group (P=0.035), and there was a significant difference between the N group, DR group, L group and group 8 weeks after intervention. 2 weeks after the administration, the retinal vessels were obviously denser and the blood vessels were obviously tortuous. It could be seen that the branches of the retinal vessels leaked the dye, and the microangioma was seen and the neovascularization was not seen. 4 and 8 weeks after the administration, the retinal vessels were dilated, the diameter of the tube was unevenly, the lumen in some parts of the tube narrowed or even obliterated, and the small vessels in the peripheral retina were large. The density of retinal vessels in each intervention group was significantly higher than that in the N group, and the retinal vessels were less obvious than those in the DR group. No dyestuff leakage was found, no microangioma and.L group in the non perfusion area were found to see small vascular leakage.
conclusion
(1) detecting the content of EB in the retina of DR rats can accurately detect the change of BRB in the retina of rats. It is an effective method to evaluate the BRB function of DR rats.
(2) the injection of Bevacizumab in the vitreous cavity of early DR rats can partially repair the damaged BRB and delay the progression of retinal microvascular lesions. In the case of no retinal toxicity, the injection of appropriate amount of Bevacizumab in the vitreous cavity can delay the progress of DR, and the long-term effect of 2.5mg/ 0.1ml may be better than 1.25mg/0.1ml..
(3) the effect of intravitreal injection of Bevacizumab on the repair of BRB/ in DR rats and to delay the progression of retinal microvascular lesions is related to the dose, and the effect is stable at least 8 weeks after injection. This experiment suggests that the time of Bevacizumab repeated administration can be prolonged to 8 weeks after the Administration.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R774.1
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