玻璃体腔注射Bevacizumab在早期糖尿病视网膜病变大鼠模型治疗中的实验研究
发布时间：2018-07-28 12:17
【摘要】：目的 通过观察早期糖尿病视网膜病变(DR)大鼠玻璃体腔注射Bevacizumab后血视网膜屏障(BRB)功能、视网膜血管形态的变化,研究玻璃体腔注射Bevacizumab应用于早期DR后延缓DR进展的有效性,明确Bevacizumab的剂量及合适重复给药时间,探讨预防性使用的可行性。 方法 1糖尿病(DM)大鼠模型制造：正常清洁级雄性SD大鼠80只,随即抽取13只作为正常对照组(N组),余67只尾静脉注射链脲佐菌素(STZ) 45 mg/kg诱导糖尿病(DM)大鼠模型。于给药后一周剪尾采血测空腹血糖,若血糖水平16.65 mmol／L,表明DM大鼠模型建立成功。模型建立后观察1周,然后开始计算DM病程。观察各组大鼠的形态改变,每4周检测1次空腹血糖及体质量。 2 DM大鼠患有早期DR验证：成模后10周,抽取DM大鼠及正常大鼠各2只进行视网膜铺片,比较观察正常大鼠和DM大鼠微血管形态变化,确定DM大鼠已患有早期DR。 3玻璃体腔注射Bevacizumab:在确定DM大鼠已患有DR后,DM大鼠随机分为糖尿病对照组(DR组)、大剂量治疗组(H组)及小剂量治疗组(L组),L组及H组分别给予浓度为1.25mg/ml及2.5mg/ml的Bevacizumab4μ1玻璃体腔注射,均注射一次,N组及DR组未接受任何治疗。 4治疗效果观察：干预治疗后2周、4周及8周,进行免疫组织化学检测分析血.管内皮生长因子(VEGF)表达的变化,用伊文思蓝(EB)法定量检测BRB破坏程度,用EB灌注视网膜铺片法观察视网膜血管形态的变化,研究玻璃体腔注射Bevacizumab延缓DR进展的作用。采用SPSS17.0软件包进行统计学处理,采用单因素方差分析分析数据,P值0.05认为差异有统计学意义。 结果 1 DM大鼠模型制造：实验组大鼠一次成模65只,未成模的大鼠用于正常对照组,成模率为97%。 2 DM大鼠患有早期DR验证：成模后10周,视网膜铺片示：正常大鼠视网膜血管走行大致正常,血管密度均匀,毛细血管管径粗细一致,未见迂曲扩张及微血管瘤等病理变化,未见染料渗漏；DM大鼠视网膜血管变密,血管走行迂曲,可见静脉串珠样改变,偶见小血.管渗漏染料。DM大鼠已发生早期视网膜病变。 3本实验中共50只DM大鼠接受玻璃体腔注射Bevacizumab治疗,其中2只由于技术操作问题,出现玻璃体积血,至实验结束,未发现眼内炎的患鼠,玻璃体腔注射Bevacizumab的大鼠随着注射时间的延长,均出现白内障。 4治疗效果：干预治疗后,免疫组化显示：各组大鼠视网膜内VEGF各层均有表达,干预治疗组VEGF表达强于N组而弱于DR组。干预治疗后2周,N组、DR组、L组及H组EB渗漏量差异有统计学意义(P=0.000),L组及H组之间差异无统计学意义(P=0.364)；干预治疗后4周,N组、DR组、L组及H组EB渗漏量差异有统计学意义(P=0.000),L组及H组之间差异有统计学意义(P=0.035)；干预治疗后8周,N组、DR组、L组及H组EB渗漏量差异有统计学意义(P=0.000),L组及H组之间差异有统计学意义(P=0.031)。视网膜铺片示：DR组：给药后2周,视网膜血管明显变密,血管走行明显迂曲,可见视网膜大血管分支渗漏染料,可见微血管瘤,未见新生血管；给药后4周及8周,视网膜血管迂曲扩张,管径粗细不均,某些部位管腔狭窄甚至闭塞,走行紊乱,周边视网膜小血管大片染料渗漏,可见微血管瘤、无灌注区。各干预组视网膜血管的密度较N组明显增高,较DR组明显减轻.视网膜血管迂曲不明显,未见染料渗漏,未见微血管瘤及无灌注区。L组偶见小血管渗漏。 结论 (1)检测DR大鼠视网膜内EB含量,可以比较精确地发现大鼠视网膜BRB的改变,是评价DR大鼠BRB功能的有效方法。 (2)给予早期DR大鼠玻璃体腔注射Bevacizumab可以部分修复受损的BRB,延缓视网膜微血管病变的进展,在不引起视网膜毒性损伤的情况下,玻璃体腔注入适量的Bevacizumab有延缓DR进展的作用,2.5mg/0.1ml的远期效果可能优于1.25mg/0.1ml。 (3)玻璃体腔注射Bevacizumab修复DR大鼠BRB/及延缓视网膜微血管病变进展的作用与剂量有关,且注射后至少8周内效果稳定,本实验认为Bevacizumab重复给药的时间可以延长至给药后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.
【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：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.
【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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