DAT对豚鼠形觉剥夺性近视眼视网膜色素上皮bFGF表达作用的研究
发布时间：2018-08-05 19:38
【摘要】： 目的：建立豚鼠形觉剥夺性近视眼动物模型,观察多巴胺转运蛋白、多巴胺及碱性成纤维细胞生长因子在视网膜色素上皮中的表达。 方法：随机选取三周龄豚鼠30只,分为3组, A组：未行干预作为正常对照组；B组：右眼采用半透明自制眼罩遮盖2周；C组：右眼采用半透明自制眼罩遮盖1周,去遮盖1周。对各组进行视网膜检影测屈光度、A超测量眼轴及角膜曲率计测量角膜曲率。采用免疫组织化学法检测多巴胺转运蛋白在视网膜的表达定位,RT-PCR检测多巴胺转运蛋白mRNA的表达,Western blot法检测多巴胺转运蛋白及碱性成纤维细胞生长因子蛋白的表达及高效液相色谱-电化学法检测多巴胺的含量。 结果：平均屈光度A组为+0.917±0.303 D,B组为-3.541±0.579 D,C组为+0.750±0.223 D；B组与A组差异有统计学意义(P0.05),其余各组差异无统计学意义(P0.05)。眼轴长度A组为6.663±0.176mm,B组为8.045±0.318 mm,C组为6.767±0.118 mm;B组与A组比较差异有统计学意义(P0.05),其余各组差异无统计学意义(P0.05)。各组实验眼角膜曲率差异无统计学意义(F=0.583,P0.05)。多巴胺转运蛋白主要表达于视网膜色素上皮层,脉络膜中无表达;在B组呈强阳性表达,在其余各组呈阳性表达；B组多巴胺转运蛋白免疫反应增强主要表现在视网膜色素上皮。豚鼠形觉剥夺性近视眼视网膜色素上皮多巴胺转运蛋白及多巴胺较正常眼表达上调(P0.05),碱性成纤维细胞生长因子较正常眼表达下调(P0.05)。多巴胺转运蛋白的表达水平与免疫组织化学的反应强度呈密切正相关(r=0.893,P0.05)；多巴胺转运蛋白的表达水平与多巴胺含量呈密切正相关(r=0.902,P0.05)；多巴胺转运蛋白与碱性成纤维细胞生长因子的表达水平呈密切负相关(r=-0.976,P0.05)。 结论：1.多巴胺转运蛋白、多巴胺及碱性成纤维细胞生长因子在豚鼠视网膜色素上皮均有表达；多巴胺转运蛋白在脉络膜中无表达。2.豚鼠形觉剥夺性近视眼视网膜色素上皮多巴胺转运蛋白表达上调,碱性成纤维细胞生长因子表达下调；多巴胺转运蛋白与碱性成纤维细胞生长因子的表达水平呈密切负相关。 目的：观察外源性多巴胺转运蛋白反义寡核苷酸作用下,豚鼠形觉剥夺性近视眼视网膜色素上皮多巴胺转运蛋白及碱性成纤维细胞生长因子的表达改变,探讨多巴胺转运蛋白对豚鼠形觉剥夺性近视眼视网膜色素上皮碱性成纤维细胞生长因子表达的调控。 方法：随机选取三周龄豚鼠50只,分为5组, A组：未行干预作为正常对照组；B组：右眼采用半透明自制眼罩遮盖2周；C组：右眼采用半透明自制眼罩遮盖2周,同时予玻璃体腔内注射多巴胺转运蛋白反义寡核苷酸,浓度为0.02nmol／ul;D组：右眼采用半透明自制眼罩遮盖2周,同时予玻璃体腔内注射多巴胺转运蛋白核苷酸,浓度为0.02nmol／ul;E组：右眼采用半透明自制眼罩遮盖2周,同时予玻璃体腔内注射等量生理盐水作为阴性对照。对各组进行视网膜检影测屈光度、A超测量眼轴及角膜曲率计测量角膜曲率。采用RT-PCR检测多巴胺转运蛋白mRNA的表达；Western blot法检测多巴胺转运蛋白及碱性成纤维细胞生长因子蛋白的表达及高效液相色谱-电化学法检测多巴胺的含量；并采用TUNEL染色,观察多巴胺转运蛋白反义寡核苷酸对视网膜有无毒性作用。 结果：平均屈光度A组为+0.917±0.303 D,B组为-3.542±0.579 D,C组为-0.125±0.628 D,D组为-3.583±0.665 D,E组为-3.792±1.111D；B组、C组、D组及E组与A组比较差异有统计学意义(P0.05),C组与B组差异有统计学意义(P0.05),D组、E组与B组比较差异无统计学意义(P0.05)。A组实验眼眼轴长度为6.625±0.108mm,B组为8.052±0.317 mm,C组为6.702±0.140 mm,D组为7.755±0.120 mm,E组为7.893±0.249 mm；B组、C组、D组及E组与A组比较差异有统计学意义(P0.05),C组与B组差异有统计学意义(P0.05),D组、E组与B组比较差异无统计学意义(P0.05)。各组实验眼角膜曲率差异无统计学意义(F=0.305,P0.05)。多巴胺转运蛋白反义寡核苷酸可使豚鼠视网膜色素上皮多巴胺转运蛋白mRNA及蛋白表达下调(P0.05),同时可使碱性成纤维细胞生长因子较正常眼表达上调(P0.05)。多巴胺转运蛋白与碱性成纤维细胞生长因子蛋白的表达水平呈密切负相关(r=-0.973,P0.05)。 结论：1.多巴胺转运蛋白反义寡核苷酸可抑制豚鼠由于形觉剥夺所引起的近视眼以及相应的眼轴延长,同时可引起视网膜色素上皮中的多巴胺转运蛋白表达下调及碱性成纤维细胞生长因子表达上调。2.多巴胺转运蛋白可能通过调控视网膜色素上皮碱性成纤维细胞生长因子的表达,从而影响豚鼠形觉剥夺性近视眼的形成。
[Abstract]:Objective: to establish a guinea pig model of form deprivation myopia, and to observe the expression of dopamine transporter, dopamine and basic fibroblast growth factor in retinal pigment epithelium.
Methods: 30 three week old guinea pigs were randomly divided into 3 groups, group A, group B: the right eye was covered with translucent homemade eye mask for 2 weeks, and group C: the right eye was covered with translucent homemade mask for 1 weeks and covered for 1 weeks. The retinometry and refraction of retina, A eye axis and corneal curvature meter in each group were measured. The expression of dopamine transporter in the retina was detected by immunohistochemical method. The expression of dopamine transporter mRNA was detected by RT-PCR. The expression of dopamine transporter and basic fibroblast growth factor protein was detected by Western blot method and the content of dopamine was detected by high performance liquid chromatography electrochemical method.
Results: the average diopter A group was +0.917 + 0.303 D, B group was -3.541 + 0.579 D, C group was +0.750 + 0.223 D, the difference between B group and A group was statistically significant (P0.05), the other groups had no statistical significance. The axial length of the group was 6.663 +, 8.045 + 0.318, 6.767 + 0.118. Significance (P0.05), the other groups were not statistically significant (P0.05). There was no significant difference in corneal curvature between the groups (F=0.583, P0.05). The dopamine transporter was mainly expressed in the retinal pigment epithelium and the choroid was not expressed; in the B group, the positive expression was positive, and the other groups were positive in the other groups; the B group was immune to dopamine transporter protein immunization. The reaction enhanced mainly in the retinal pigment epithelium. The dopamine transporter and dopamine in the retinal pigment epithelium of the guinea pig were up to up (P0.05), and the basic fibroblast growth factor was down down (P0.05). The expression of dopamine transporter was stronger than that of immunohistochemistry. The level was closely related (r=0.893, P0.05); the expression level of dopamine transporter was closely related to the content of dopamine (r=0.902, P0.05), and the expression level of dopamine transporter was negatively correlated with the expression level of basic fibroblast growth factor (r=-0.976, P0.05).
Conclusion: 1. dopamine transporter, dopamine and basic fibroblast growth factor are expressed in the retinal pigment epithelium of guinea pigs. Dopamine transporter in choroidal membrane does not express the expression of dopamine transporter in the retinal pigment epithelium of.2. guinea-pig form deprived myopia, and the expression of basic fibroblast growth factor is expressed. The expression of dopamine transporter was negatively correlated with the expression of basic fibroblast growth factor.
Objective: To observe the changes in the expression of dopamine transporter and basic fibroblast growth factor in the retinal pigment epithelium of guinea pigs under the effect of exogenous dopamine transporter antisense oligodeoxynucleotides, and to explore the effect of dopamine transporter on the basic fibroblasts of pigmented retinal pigment epithelium in guinea pigs. Regulation of growth factor expression.
Methods: a total of 50 three week old guinea pigs were randomly selected and divided into 5 groups, group A, group B: the right eye was covered with a translucent homemade eye mask for 2 weeks; group C: the right eye was covered with a translucent homemade eye mask for 2 weeks, and the intravitreal injection of dopamine transshipment egg white antisense oligonucleotides, the concentration of 0.02nmol / UL; D group. The right eye was covered with a translucent homemade eye mask for 2 weeks and injected dopamine transporter nucleotides in the vitreous cavity for the concentration of 0.02nmol / UL. Group E: the right eye was covered with a translucent homemade eye mask for 2 weeks, and the intraocular injection of equal amount of saline was given as negative contrast. The retinometry and A ultrasonic test of each group were performed. The expression of dopamine transporter mRNA was detected by RT-PCR, and the expression of dopamine transporter and basic fibroblast growth factor protein was detected by Western blot method and the content of dopamine was detected by high performance liquid chromatography electrochemistry method by Western blot method, and the dopamine transport was observed by TUNEL staining. Protein antisense oligonucleotides have no toxic effects on the retina.
Results: the average diopter A group was +0.917 + 0.303 D, B group was -3.542 + 0.579 D, C group was -0.125 + 0.628 D, D group was -3.583 0.665 D. 05) the axial length of the experimental eye in the.A group was 6.625 + 0.108mm, the group B was 8.052 + 0.317 mm, the group C was 6.702 + 0.140 mm, the D group was 7.755 + 0.120 mm, the E group was 7.893 + 0.249 mm, the C group, the group and the group had statistical significance. .05). There was no significant difference in corneal curvature between each group (F=0.305, P0.05). Dopamine transporter antisense oligonucleotides could reduce the expression of dopamine transporter mRNA and protein (P0.05) in the pigmented epithelium of the guinea pig retina (P0.05), and increase the expression of basic fibroblast growth factor (P0.05) with normal eye (P0.05). The expression level of basic fibroblast growth factor was negatively correlated (r=-0.973, P0.05).
Conclusion: 1. dopamine transporter antisense oligonucleotides can inhibit the myopia caused by form deprivation and the corresponding ocular axis extension, and can induce down regulation of dopamine transporter expression in the retinal pigment epithelium and the regulation of.2. dopamine transporter in the expression of basic fibroblast growth factor may be regulated by the regulation of dopamine transporter. The expression of basic fibroblast growth factor in retinal pigment epithelium affects the formation of form deprivation myopia in guinea pigs.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R778
[Abstract]:Objective: to establish a guinea pig model of form deprivation myopia, and to observe the expression of dopamine transporter, dopamine and basic fibroblast growth factor in retinal pigment epithelium.
Methods: 30 three week old guinea pigs were randomly divided into 3 groups, group A, group B: the right eye was covered with translucent homemade eye mask for 2 weeks, and group C: the right eye was covered with translucent homemade mask for 1 weeks and covered for 1 weeks. The retinometry and refraction of retina, A eye axis and corneal curvature meter in each group were measured. The expression of dopamine transporter in the retina was detected by immunohistochemical method. The expression of dopamine transporter mRNA was detected by RT-PCR. The expression of dopamine transporter and basic fibroblast growth factor protein was detected by Western blot method and the content of dopamine was detected by high performance liquid chromatography electrochemical method.
Results: the average diopter A group was +0.917 + 0.303 D, B group was -3.541 + 0.579 D, C group was +0.750 + 0.223 D, the difference between B group and A group was statistically significant (P0.05), the other groups had no statistical significance. The axial length of the group was 6.663 +, 8.045 + 0.318, 6.767 + 0.118. Significance (P0.05), the other groups were not statistically significant (P0.05). There was no significant difference in corneal curvature between the groups (F=0.583, P0.05). The dopamine transporter was mainly expressed in the retinal pigment epithelium and the choroid was not expressed; in the B group, the positive expression was positive, and the other groups were positive in the other groups; the B group was immune to dopamine transporter protein immunization. The reaction enhanced mainly in the retinal pigment epithelium. The dopamine transporter and dopamine in the retinal pigment epithelium of the guinea pig were up to up (P0.05), and the basic fibroblast growth factor was down down (P0.05). The expression of dopamine transporter was stronger than that of immunohistochemistry. The level was closely related (r=0.893, P0.05); the expression level of dopamine transporter was closely related to the content of dopamine (r=0.902, P0.05), and the expression level of dopamine transporter was negatively correlated with the expression level of basic fibroblast growth factor (r=-0.976, P0.05).
Conclusion: 1. dopamine transporter, dopamine and basic fibroblast growth factor are expressed in the retinal pigment epithelium of guinea pigs. Dopamine transporter in choroidal membrane does not express the expression of dopamine transporter in the retinal pigment epithelium of.2. guinea-pig form deprived myopia, and the expression of basic fibroblast growth factor is expressed. The expression of dopamine transporter was negatively correlated with the expression of basic fibroblast growth factor.
Objective: To observe the changes in the expression of dopamine transporter and basic fibroblast growth factor in the retinal pigment epithelium of guinea pigs under the effect of exogenous dopamine transporter antisense oligodeoxynucleotides, and to explore the effect of dopamine transporter on the basic fibroblasts of pigmented retinal pigment epithelium in guinea pigs. Regulation of growth factor expression.
Methods: a total of 50 three week old guinea pigs were randomly selected and divided into 5 groups, group A, group B: the right eye was covered with a translucent homemade eye mask for 2 weeks; group C: the right eye was covered with a translucent homemade eye mask for 2 weeks, and the intravitreal injection of dopamine transshipment egg white antisense oligonucleotides, the concentration of 0.02nmol / UL; D group. The right eye was covered with a translucent homemade eye mask for 2 weeks and injected dopamine transporter nucleotides in the vitreous cavity for the concentration of 0.02nmol / UL. Group E: the right eye was covered with a translucent homemade eye mask for 2 weeks, and the intraocular injection of equal amount of saline was given as negative contrast. The retinometry and A ultrasonic test of each group were performed. The expression of dopamine transporter mRNA was detected by RT-PCR, and the expression of dopamine transporter and basic fibroblast growth factor protein was detected by Western blot method and the content of dopamine was detected by high performance liquid chromatography electrochemistry method by Western blot method, and the dopamine transport was observed by TUNEL staining. Protein antisense oligonucleotides have no toxic effects on the retina.
Results: the average diopter A group was +0.917 + 0.303 D, B group was -3.542 + 0.579 D, C group was -0.125 + 0.628 D, D group was -3.583 0.665 D. 05) the axial length of the experimental eye in the.A group was 6.625 + 0.108mm, the group B was 8.052 + 0.317 mm, the group C was 6.702 + 0.140 mm, the D group was 7.755 + 0.120 mm, the E group was 7.893 + 0.249 mm, the C group, the group and the group had statistical significance. .05). There was no significant difference in corneal curvature between each group (F=0.305, P0.05). Dopamine transporter antisense oligonucleotides could reduce the expression of dopamine transporter mRNA and protein (P0.05) in the pigmented epithelium of the guinea pig retina (P0.05), and increase the expression of basic fibroblast growth factor (P0.05) with normal eye (P0.05). The expression level of basic fibroblast growth factor was negatively correlated (r=-0.973, P0.05).
Conclusion: 1. dopamine transporter antisense oligonucleotides can inhibit the myopia caused by form deprivation and the corresponding ocular axis extension, and can induce down regulation of dopamine transporter expression in the retinal pigment epithelium and the regulation of.2. dopamine transporter in the expression of basic fibroblast growth factor may be regulated by the regulation of dopamine transporter. The expression of basic fibroblast growth factor in retinal pigment epithelium affects the formation of form deprivation myopia in guinea pigs.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R778
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