【摘要】： 目的：建立豚鼠形覺(jué)剝奪性近視眼動(dòng)物模型,觀察多巴胺轉(zhuǎn)運(yùn)蛋白、多巴胺及堿性成纖維細(xì)胞生長(zhǎng)因子在視網(wǎng)膜色素上皮中的表達(dá)。 方法：隨機(jī)選取三周齡豚鼠30只,分為3組, A組：未行干預(yù)作為正常對(duì)照組；B組：右眼采用半透明自制眼罩遮蓋2周；C組：右眼采用半透明自制眼罩遮蓋1周,去遮蓋1周。對(duì)各組進(jìn)行視網(wǎng)膜檢影測(cè)屈光度、A超測(cè)量眼軸及角膜曲率計(jì)測(cè)量角膜曲率。采用免疫組織化學(xué)法檢測(cè)多巴胺轉(zhuǎn)運(yùn)蛋白在視網(wǎng)膜的表達(dá)定位,RT-PCR檢測(cè)多巴胺轉(zhuǎn)運(yùn)蛋白mRNA的表達(dá),Western blot法檢測(cè)多巴胺轉(zhuǎn)運(yùn)蛋白及堿性成纖維細(xì)胞生長(zhǎng)因子蛋白的表達(dá)及高效液相色譜-電化學(xué)法檢測(cè)多巴胺的含量。 結(jié)果：平均屈光度A組為+0.917±0.303 D,B組為-3.541±0.579 D,C組為+0.750±0.223 D；B組與A組差異有統(tǒng)計(jì)學(xué)意義(P0.05),其余各組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。眼軸長(zhǎng)度A組為6.663±0.176mm,B組為8.045±0.318 mm,C組為6.767±0.118 mm;B組與A組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),其余各組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。各組實(shí)驗(yàn)眼角膜曲率差異無(wú)統(tǒng)計(jì)學(xué)意義(F=0.583,P0.05)。多巴胺轉(zhuǎn)運(yùn)蛋白主要表達(dá)于視網(wǎng)膜色素上皮層,脈絡(luò)膜中無(wú)表達(dá);在B組呈強(qiáng)陽(yáng)性表達(dá),在其余各組呈陽(yáng)性表達(dá)；B組多巴胺轉(zhuǎn)運(yùn)蛋白免疫反應(yīng)增強(qiáng)主要表現(xiàn)在視網(wǎng)膜色素上皮。豚鼠形覺(jué)剝奪性近視眼視網(wǎng)膜色素上皮多巴胺轉(zhuǎn)運(yùn)蛋白及多巴胺較正常眼表達(dá)上調(diào)(P0.05),堿性成纖維細(xì)胞生長(zhǎng)因子較正常眼表達(dá)下調(diào)(P0.05)。多巴胺轉(zhuǎn)運(yùn)蛋白的表達(dá)水平與免疫組織化學(xué)的反應(yīng)強(qiáng)度呈密切正相關(guān)(r=0.893,P0.05)；多巴胺轉(zhuǎn)運(yùn)蛋白的表達(dá)水平與多巴胺含量呈密切正相關(guān)(r=0.902,P0.05)；多巴胺轉(zhuǎn)運(yùn)蛋白與堿性成纖維細(xì)胞生長(zhǎng)因子的表達(dá)水平呈密切負(fù)相關(guān)(r=-0.976,P0.05)。 結(jié)論：1.多巴胺轉(zhuǎn)運(yùn)蛋白、多巴胺及堿性成纖維細(xì)胞生長(zhǎng)因子在豚鼠視網(wǎng)膜色素上皮均有表達(dá)；多巴胺轉(zhuǎn)運(yùn)蛋白在脈絡(luò)膜中無(wú)表達(dá)。2.豚鼠形覺(jué)剝奪性近視眼視網(wǎng)膜色素上皮多巴胺轉(zhuǎn)運(yùn)蛋白表達(dá)上調(diào),堿性成纖維細(xì)胞生長(zhǎng)因子表達(dá)下調(diào)；多巴胺轉(zhuǎn)運(yùn)蛋白與堿性成纖維細(xì)胞生長(zhǎng)因子的表達(dá)水平呈密切負(fù)相關(guān)。 目的：觀察外源性多巴胺轉(zhuǎn)運(yùn)蛋白反義寡核苷酸作用下,豚鼠形覺(jué)剝奪性近視眼視網(wǎng)膜色素上皮多巴胺轉(zhuǎn)運(yùn)蛋白及堿性成纖維細(xì)胞生長(zhǎng)因子的表達(dá)改變,探討多巴胺轉(zhuǎn)運(yùn)蛋白對(duì)豚鼠形覺(jué)剝奪性近視眼視網(wǎng)膜色素上皮堿性成纖維細(xì)胞生長(zhǎng)因子表達(dá)的調(diào)控。 方法：隨機(jī)選取三周齡豚鼠50只,分為5組, A組：未行干預(yù)作為正常對(duì)照組；B組：右眼采用半透明自制眼罩遮蓋2周；C組：右眼采用半透明自制眼罩遮蓋2周,同時(shí)予玻璃體腔內(nèi)注射多巴胺轉(zhuǎn)運(yùn)蛋白反義寡核苷酸,濃度為0.02nmol／ul;D組：右眼采用半透明自制眼罩遮蓋2周,同時(shí)予玻璃體腔內(nèi)注射多巴胺轉(zhuǎn)運(yùn)蛋白核苷酸,濃度為0.02nmol／ul;E組：右眼采用半透明自制眼罩遮蓋2周,同時(shí)予玻璃體腔內(nèi)注射等量生理鹽水作為陰性對(duì)照。對(duì)各組進(jìn)行視網(wǎng)膜檢影測(cè)屈光度、A超測(cè)量眼軸及角膜曲率計(jì)測(cè)量角膜曲率。采用RT-PCR檢測(cè)多巴胺轉(zhuǎn)運(yùn)蛋白mRNA的表達(dá)；Western blot法檢測(cè)多巴胺轉(zhuǎn)運(yùn)蛋白及堿性成纖維細(xì)胞生長(zhǎng)因子蛋白的表達(dá)及高效液相色譜-電化學(xué)法檢測(cè)多巴胺的含量；并采用TUNEL染色,觀察多巴胺轉(zhuǎn)運(yùn)蛋白反義寡核苷酸對(duì)視網(wǎng)膜有無(wú)毒性作用。 結(jié)果：平均屈光度A組為+0.917±0.303 D,B組為-3.542±0.579 D,C組為-0.125±0.628 D,D組為-3.583±0.665 D,E組為-3.792±1.111D；B組、C組、D組及E組與A組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),C組與B組差異有統(tǒng)計(jì)學(xué)意義(P0.05),D組、E組與B組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。A組實(shí)驗(yàn)眼眼軸長(zhǎng)度為6.625±0.108mm,B組為8.052±0.317 mm,C組為6.702±0.140 mm,D組為7.755±0.120 mm,E組為7.893±0.249 mm；B組、C組、D組及E組與A組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),C組與B組差異有統(tǒng)計(jì)學(xué)意義(P0.05),D組、E組與B組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。各組實(shí)驗(yàn)眼角膜曲率差異無(wú)統(tǒng)計(jì)學(xué)意義(F=0.305,P0.05)。多巴胺轉(zhuǎn)運(yùn)蛋白反義寡核苷酸可使豚鼠視網(wǎng)膜色素上皮多巴胺轉(zhuǎn)運(yùn)蛋白mRNA及蛋白表達(dá)下調(diào)(P0.05),同時(shí)可使堿性成纖維細(xì)胞生長(zhǎng)因子較正常眼表達(dá)上調(diào)(P0.05)。多巴胺轉(zhuǎn)運(yùn)蛋白與堿性成纖維細(xì)胞生長(zhǎng)因子蛋白的表達(dá)水平呈密切負(fù)相關(guān)(r=-0.973,P0.05)。 結(jié)論：1.多巴胺轉(zhuǎn)運(yùn)蛋白反義寡核苷酸可抑制豚鼠由于形覺(jué)剝奪所引起的近視眼以及相應(yīng)的眼軸延長(zhǎng),同時(shí)可引起視網(wǎng)膜色素上皮中的多巴胺轉(zhuǎn)運(yùn)蛋白表達(dá)下調(diào)及堿性成纖維細(xì)胞生長(zhǎng)因子表達(dá)上調(diào)。2.多巴胺轉(zhuǎn)運(yùn)蛋白可能通過(guò)調(diào)控視網(wǎng)膜色素上皮堿性成纖維細(xì)胞生長(zhǎng)因子的表達(dá),從而影響豚鼠形覺(jué)剝奪性近視眼的形成。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R778

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