视黄醇脱氢酶和视网膜G蛋白偶联受体在形觉剥夺性近视眼小鼠中表达特征的实验研究

doi:10.3877/cma.j.issn.2095-2007.2022.01.002

目的

探讨视黄醇脱氢酶(RDH)和视网膜G蛋白偶联受体(RGR)在形觉剥夺性近视眼小鼠中的表达特征。

方法

采用数字表法将4周龄C57BL6/J小鼠100只(200只眼)随机分为对照组和近视组，每组各50只(100只眼)。采用自制的遮光眼罩形觉剥夺小鼠的单眼视力，构建近视眼动物模型。右眼形觉剥夺4周后，采用红外偏心摄影验光仪测量小鼠的屈光度。采用手持数字显微镜测量小鼠的眼轴长度。采用实时逆转录聚合酶链反应测量小鼠全视网膜组织中Rdh5、Rdh10及Rgr基因信使核糖核酸(mRNA)的相对表达量。采用蛋白质免疫印迹法测量小鼠全视网膜组织中RDH5、RDH10及RGR蛋白的表达水平。两组小鼠屈光度、眼轴长度、基因的mRNA相对表达量及蛋白质相对灰度值采用±s表示。两组小鼠屈光度的比较采用配对t检验；眼轴长度、基因mRNA相对表达量及蛋白质相对灰度值的总体差异采用单因素方差分析，当差异有统计学意义时，再进一步采用LSD检验两两比较。

结果

近视组小鼠遮盖眼和对侧眼的屈光度分别为(-5.631±4.052)D和(4.231±2.828)D。近视组小鼠遮盖眼呈现出明显的近视漂移，两者比较的差异有统计学意义(t＝-10.91，P<0.05)。对照组右眼和左眼的屈光度分别为(3.774±4.079)D和(4.171±4.425)D，两者比较的差异无统计学意义(t＝-1.344，P>0.05)。近视组小鼠遮盖眼、对侧眼及对照组眼轴分别为(3305.0±86.4)μm、(3221.0±90.0)μm及(3232.0±68.6)μm。遮盖眼眼轴最长，三者比较的差异均有统计学意义(F＝6.248，P<0.05)。近视组小鼠遮盖眼Rdh5、Rdh10及Rgr基因mRNA的相对表达量分别为(2.032±0.162)、(2.611±0.258)及(3.876±0.576)，均略低于对侧眼。近视组小鼠遮盖眼Rdh5、Rdh10及Rgr基因的mRNA相对表达量高于对照组。经单因素方差分析，两组比较的差异均有统计学意义(F＝38.671，88.510，78.557；P<0.05)。近视组和对照组小鼠RDH5、RDH10及RGR蛋白质相对灰度值的趋势仅与基因的mRNA相对表达量趋势部分一致。近视组小鼠遮盖眼RDH5、RDH10及RGR蛋白质的相对灰度值低于对侧眼和对照组。经单因素方差分析，两组比较的差异均有统计学意义(F＝12.189，56.956，6.620；P<0.05)。

结论

单眼形觉剥夺刺激能升高Rdh5、Rdh10及Rgr基因的mRNA相对表达量，但其蛋白质相对灰度值改变不一致，近视眼小鼠视网膜的RDH5和RDH10蛋白质相对灰度值高，而RGR低。异常的视觉刺激可能抑制RDH的生成，影响视觉周期反应，促进近视眼的发展。

关键词: 形觉剥夺近视, 视黄醇脱氢酶5, 视黄醇脱氢酶10, 视网膜G蛋白偶联受体, 视网膜

Objective

The aim of this study was to examine the expression pattern differences of retinol dehydrogenase (RDH) and rpe-retinal G protein-coupled receptor (RGR) in deprivation myopic mice.

Methods

100 four-week-old C57BL6/J mice (200 eyes) were randomly distributed into myopia group and control group, 50 for each group (100 eyes). Monocular deprivation of was induced by covering the right eyes with handmade, translucent occluders, while the left eyes served as contralateral eyes. Mean-while, the control group received no treatment. After deprivation for four weeks, refraction errors were measured via an eccentric infrared photorefractor, and axial lengths were estimated using a hand-held digital microscope. The messenger ribonucleic acid (mRNA) transcript levels of Rdh5, Rdh10 and Rgr genes were detected using real-time reverse transcription polymerase chain reaction. The corresponding protein concentrations of these genes were detected by Western blot analysis. Refractive errors, axial length, mRNA levels and the relative gray value of protein were described as ±s. The refraction interocular difference was used to compare by paired t-test. One-way ANOVA was used to determine statistic differences in axial length, mRNA levels and relative gray value of protein among multiple samples, and the LSD test was used for further pairwise comparisons.

Results

The refractive error of the covered myopic eyes and contralateral eyes in myopia group were (-5.631±4.052) D, (4.231±2.828) D, resepctively. There was a significant difference between them (t=-10.914, P<0.05). The control group was found to have refractive error for right and left eyes, which were (3.774±4.079) D and (4.171±4.425) D, respectively. There was not a significant difference between them (t=-1.344, P>0.05). The axial length of covered myopic eyes, contralateral eyes and control group were (3305.0±86.4) μm, (3221.0±90.0) μm and (3232.0±68.6) μm, respectively. After one-way ANOVA, there was a statistically significant difference among them (F=6.248, P<0.05). In the retinas of covered myopic eyes, the relative mRNA levels of Rdh5, Rdh10 and Rgr genes was (2.032±0.162), (2.611±0.258) and (3.876±0.576), respectively, which were all lower than that in the contralateral retinas. Compared with control group, the mRNA levels of gene increased significantly in myopia group. After one-way ANOVA, there was a significant difference between them (F=38.671, 88.510, 78.557; P<0.05). The results of Western blot analysis showed that relative gray value of protein expression pattern of these genes in myopic group were in accordance with the mRNA expression trend. The relative gray value of proteins of RDH5, RDH10 and RGR in covered myopic eyes were lower than that in the contralateral uncovered eyes. After one-way ANOVA, there was a significant difference between them (F=12.189, 56.956, 6.620; P<0.05).

Conclusions

Early monocular deprivation induces a robust increase in mRNA levels of Rdh5, Rdh10 and Rgr genes, while the relative gray value of protein is inconsistent with that. The relative gray value of protein of RDH5 and RDH10 are high, and RGR low in covered myopic eyes. Abnormal visual stimulus is helpful to impede the expression of retinol dehydrogenases and promote the myopia progression through the potential effects of visual cycle.

Key words: Form deprivation myopia, Retinol dehydrogenase 5, Retinol dehydrogenase 10, Rpe-retinal G protein-coupled receptor, Retina

表1 实时逆转录聚合酶链反应引物序列

引物名称	引物序列(5′-3′)
GAPDH-F	GGTTGTCTCCTGCGACTTCA
GAPDH-R	TGGTCCAGGGTTTCTTACTCC
RGR-F	GTTCATGGTTTCCAGGGATTTG
RGR-R	CAGATGACATCCACACAAACAG
RDH5-F	GGAGAAACTGGACTTTTTGGTC
RDH5-R	TGTGTTCACACTCAGTACTCTC
RDH10-F	TGGGGAAGAGAGAGAATGTCTA
RDH10-R	AAACCACACCAGCATTATTGAC

表1 实时逆转录聚合酶链反应引物序列

引物名称	引物序列(5′-3′)
GAPDH-F	GGTTGTCTCCTGCGACTTCA
GAPDH-R	TGGTCCAGGGTTTCTTACTCC
RGR-F	GTTCATGGTTTCCAGGGATTTG
RGR-R	CAGATGACATCCACACAAACAG
RDH5-F	GGAGAAACTGGACTTTTTGGTC
RDH5-R	TGTGTTCACACTCAGTACTCTC
RDH10-F	TGGGGAAGAGAGAGAATGTCTA
RDH10-R	AAACCACACCAGCATTATTGAC

表2 两组小鼠形觉剥夺4周后屈光度的比较(±s，D)

组别	只数	右眼	左眼	眼间差值
近视组	50	-5.631±4.052	4.231±2.828	-9.863±4.781
对照组	50	3.774±4.079	4.171±4.425	-0.398±2.001
t值		-9.644	0.064
P值		<0.05	>0.05

表2 两组小鼠形觉剥夺4周后屈光度的比较(±s，D)

组别	只数	右眼	左眼	眼间差值
近视组	50	-5.631±4.052	4.231±2.828	-9.863±4.781
对照组	50	3.774±4.079	4.171±4.425	-0.398±2.001
t值		-9.644	0.064
P值		<0.05	>0.05

表3 两组小鼠形觉剥夺4周后眼轴长度的比较(±s，μm)

组别	只数	右眼	左眼
近视组	50	3305.0±86.4	3221.0±90.0
对照组	50		3232.0±68.6
F值		6.248
P值		<0.05

表3 两组小鼠形觉剥夺4周后眼轴长度的比较(±s，μm)

组别	只数	右眼	左眼
近视组	50	3305.0±86.4	3221.0±90.0
对照组	50		3232.0±68.6
F值		6.248
P值		<0.05

图2 两组小鼠形觉剥夺4周后全视网膜组织中RDH5、RDH10及RGR蛋白质相对表达量的比较　图2A示蛋白质印迹灰度图；图2B示蛋白条带灰度值的比值　注：RDH5，视黄醇脱氢酶5；RDH10，视黄醇脱氢酶10；RGR，视网膜G蛋白偶联受体；mRNA，信使核糖核酸；^*，差异有统计学意义

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Retinol dehydrogenase and rpe-retinal G protein-coupled receptor expression patterns in mice after monocular deprivation

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Retinol dehydrogenase and rpe-retinal G protein-coupled receptor expression patterns in mice after monocular deprivation