早期生长反应因子-1在小鼠近视眼形成中的研究

发布时间：2018-05-18 06:05

本文选题：早期生长反应因子-1 + 近视眼　；参考：《中南大学》2010年博士论文

【摘要】： 第一章含小鼠Egr-1基因的发夹结构RNA干扰质粒的构建及干扰效率鉴定目的构建及筛选高效率针对小鼠早期生长反应因子-1(Egr-1)发夹结构RNA干扰(shRNA)的质粒。方法根据小鼠Egr-1基因mRNA序列(NM007913),设计有发夹结构的3条寡核苷酸序列,合成靶序列的Oligo DNA,退火形成双链DNA,经AgeⅠ和EcoRⅠ双酶切后的pGCSIL-GFP载体连接产生含shRNA的重组质粒1#、2#及3#,PCR筛选阳性克隆,测序测定。设计构建不针对任何特异基因的NC质粒作为阴性对照。将3个shRNA表达质粒及NC质粒转染HEK293细胞,设不做任何处理的细胞为空白对照。通过对绿色荧光蛋白(GFP)表达量的观察,实时荧光定量聚合酶链反应(RQ-PCR)及免疫印迹法(western blotting)检测Egr-1基因mRNA及蛋白的表达,鉴定shRNA表达质粒对Egr-1基因的抑制效率。组间比较采用单因素方差分析,实验组3个序列两两比较采用q检验。结果针对小鼠Egr-1基因进行RNA干扰的1#、2#及3#序列中,1#序列的质粒明显抑制了细胞内Egr-1的mRNA及蛋白表达(FmRNA=118.819, P=0.000; Fprotein=71.605, P=0.000) 结论成功构建了针对小鼠Egr-1基因的高效shRNA表达的质粒。第二章含小鼠Egr-1 shRNA慢病毒载体构建及转染小鼠视网膜的研究目的构建含绿色荧光蛋白(GFP)和小鼠早期生长反应因子-1(Egr-1)发夹结构RNA干扰(shRNA)共表达的慢病毒载体,并将其转染至小鼠视网膜组织,探索合适的给药方式。方法将前期实验已经筛选确定的小鼠Egr-1基因shRNA有效靶序列的质粒,命名为LV-shRNA(Egr-1)。将LV-shRNA(Egr-1)重组质粒及pHelper 1.0、pHelper2.0慢病毒包装用的辅助质粒共转染293T细胞,包装产生慢病毒载体,经梯度稀释后,根据荧光显微镜下绿色荧光蛋白(GFP)表达阳性细胞来计算病毒滴度。将包装好的慢病毒载体分别通过玻璃体腔和视网膜下注射的途径转染至C57BL/6小鼠视网膜,于实验后2周,取小鼠眼球制作冰冻切片,荧光显微镜观察视网膜GFP表达情况。结果①成功构建含GFP并携带靶向小鼠Egr-1基因的shRNA慢病毒载体,经孔稀释法测定病毒滴度为4×108TU/ml;②利用该慢病毒载体系统转染小鼠视网膜组织,发现经玻璃体腔注射途径转染后的小鼠,GFP广泛分布于视网膜全层包括视网膜色素上皮层(RPE)；而经视网膜下注射途径转染后的小鼠,GFP局限分布于视网膜外层。结论成功构建含GFP并携带靶向小鼠Egr-1基因的shRNA慢病毒载体,通过玻璃体腔注射较视网膜下注射转染效率高,分布范围广,为后期的体内实验提供了实验基础。第三章Egr-1基因对小鼠近视眼的调控作用目的将针对小鼠Egr-1基因的shRNA慢病毒载体行玻璃体腔注射后,观察小鼠屈光度及眼轴长度的变化,阐明Egr-1基因在小鼠近视眼形成中的调控作用。方法15日龄C57BL/6小鼠,共180只,等量随机分为3组：实验组、阴性对照组及空白对照组。其中实验组小鼠右眼玻璃体腔注入前期研究中构造的LV-shRNA(Egr-1)'慢病毒载体；阴性对照组小鼠右眼玻璃体腔注入阴性对照LV-NC慢病毒载体；空白对照组小鼠不做任何处理。分别于实验后1周、2周、3周测量各组小鼠右眼屈光度后将其麻醉处死,分别测量各组小鼠右眼眼轴长度；制作冰冻切片,荧光显微镜下观察视网膜GFP表达,判断转染情况；荧光定量聚合酶链反应(RQ-PCR)、免疫印迹法(western blotting)及免疫荧光检测小鼠视网膜Egr-1基因的表达；切片HE染色后,显微镜下观察视网膜形态有无变化。组间比较采用单因素方差分析,两两比较采用q检验。结果①RQ-PCR、western blotting、免疫荧光检测发现实验组注射眼内Egr-1的表达明显下调,其中第1周Egr-1下调最为显著(FmRNA=184.383,P=0.000; Fprotein=170.470, P=0.000);②实验组和阴性对照组的慢病毒载体注射后1周后即可在荧光显微镜下观察到视网膜全层GFP分布明显,第2周后荧光强度开始衰减,第3周后GFP表达微弱；③在实验后第1周(F屈光度=157.793,P=0.000；F眼轴长度=10.005,P=0.000)及第2周(F屈光度=182.603,P=0.000；F眼轴长度=5.273,P=0.007),实验组小鼠注射眼出现了明显的近视化发展,且伴有明显的眼轴延长；但在实验后第3周(F屈光度=1.259,P=0.290；F眼轴长度=1.004,P=0.371)实验组小鼠注射眼与阴性对照组注射眼及空白对照眼的屈光度及眼轴长度相比较,无统计学差异；阴性对照组的注射眼在实验各个阶段与空白对照眼的屈光度及眼轴长度相比较,均无统计学差异(p0.05)④在实验后1周,小鼠近视化发展趋势最明显,取这周小鼠实验组及阴性对照组的注射眼、空白对照眼切片HE染色后观察,发现各组视网膜形态无明显变化。结论通过针对小鼠Egr-1基因的shRNA慢病毒载体转染小鼠视网膜后,出现了小鼠视网膜Egr-1基因的下调,小鼠屈光度及眼轴长度向近视趋势发展,证实了Egr-1基因在小鼠近视眼形成中所起的重要作用,同时发现慢病毒载体转染小鼠视网膜安全有效。实验结果为近视眼未来的基因治疗提供了思考的方向。
[Abstract]:Chapter 1 construction of RNA interference plasmid containing mouse Egr-1 gene hairpin structure and identification of interference efficiency
Objective to construct and screen plasmids with high efficiency against RNA interference (shRNA) of mouse early growth response factor -1 (Egr-1) hairpin structure.
Methods according to the mRNA sequence of mouse Egr-1 gene (NM007913), 3 oligonucleotide sequences with hairpin structure were designed, Oligo DNA of the target sequence was synthesized, and the double stranded DNA was formed by annealing. The recombinant plasmid containing shRNA was linked by pGCSIL-GFP carrier after Age I and EcoR I to produce a recombinant plasmid containing shRNA 1#, 2# and 3 of the positive clones were screened and sequenced. Design construction was not targeted. NC plasmid of any specific gene was used as negative control. 3 shRNA expression plasmids and NC plasmids were transfected into HEK293 cells. The cells without any treatment were blank control. By observing the expression of green fluorescent protein (GFP), real-time fluorescent quantitative polymerase chain reaction (RQ-PCR) and immunoblotting (Western blotting) were used to detect the Egr-1 gene mRN. The expression of A and protein was expressed and the inhibition efficiency of the shRNA expression plasmid on Egr-1 gene was identified. The single factor variance analysis was used among the groups, and the 3 sequence 22 of the experimental group was compared with the Q test.
Results in 1#, 2# and 3# sequences of RNA interference in mouse Egr-1 gene, the plasmid of 1# sequence obviously inhibited the mRNA and protein expression of Egr-1 in the cell (FmRNA=118.819, P=0.000; Fprotein=71.605, P=0.000).
Conclusion a highly efficient shRNA expression plasmid targeting mouse Egr-1 gene was successfully constructed.
The second chapter is the construction of murine Egr-1 shRNA lentiviral vector and its transfection into mouse retina.
Objective to construct a lentivirus vector containing green fluorescent protein (GFP) and mouse early growth response factor -1 (Egr-1) hairpin structure RNA interference (shRNA), and transfect it into the mouse retina tissue and explore the appropriate way of administration.
Methods the plasmids of the effective target sequence of the mouse Egr-1 gene shRNA were selected and named as LV-shRNA (Egr-1). The recombinant plasmid of LV-shRNA (Egr-1) and pHelper 1 and the auxiliary plasmid of pHelper2.0 lentivirus package were co transfected to 293T cells, and the slow virus vector was packaged, and after the gradient dilution, it was green under the fluorescence microscope. Fluorescent protein (GFP) expression positive cells were used to calculate the virus titer. The packaged lentivirus vectors were transfected into the retina of C57BL/6 mice by intravitreal and subretinal injections. 2 weeks after the experiment, the frozen section of the mice's eyeballs was made and the GFP expression in the retina was observed by fluorescence microscope.
Results (1) the shRNA lentivirus carrier containing GFP and the target mouse Egr-1 gene was successfully constructed. The virus titer was 4 x 108TU/ml by the pore dilution method. 2. The mice were transfected with the lentivirus vector system and the mice were transfected through the intravitreal injection pathway, and GFP was widely distributed in the retina whole layer including retinal pigment. The epithelial layer (RPE), whereas the mice transfected by subretinal injection route, GFP was localized in the outer layer of the retina.
Conclusion the shRNA lentivirus carrier containing GFP and target mouse Egr-1 gene was successfully constructed. The transfection efficiency of the intravitreal injection by intravitreal injection is high and the distribution range is wide, which provides the experimental basis for the later experiment in vivo.
The third chapter is about the regulation of Egr-1 gene on myopia in mice.
Objective To observe the changes of diopter and axial length of mice after intravitreal injection of shRNA lentivirus vector of Egr-1 gene in mice, and to elucidate the regulation of Egr-1 gene in the formation of myopia in mice.
Methods a total of 180 C57BL/6 mice of 15 days of age were randomly divided into 3 groups: experimental group, negative control group and blank control group. The experimental group was injected with LV-shRNA (Egr-1) 'lentivirus vector in the early study of right eye, and negative control group was injected with negative control LV-NC lentivirus vector in the right eye of the negative control group; blank control group. The mice were treated without any treatment. After 1 weeks, 2 weeks, and 3 weeks after the experiment, they were killed in each group of mice. The length of the eye axis of the right eye was measured in each group. The frozen section was made and the expression of GFP in the retina was observed under the fluorescence microscope to determine the transfection condition; the fluorescence quantitative polymerase chain reaction (RQ-PCR) and Western blot were used. The expression of Egr-1 gene in the retina of mice was detected by Western blotting and immunofluorescence, and the morphological changes of the retina were observed under microscope after HE staining. The single factor analysis of variance was used among the groups, and 22 was compared with Q test.
Results (1) RQ-PCR, Western blotting, immunofluorescence detection showed that the expression of Egr-1 in the experimental group was obviously downregulated in the experimental group, and the most significant down regulation of Egr-1 was first weeks (FmRNA=184.383, P=0.000; Fprotein=170.470, P=0.000). 2. The experimental group and the negative control group could be observed under the fluorescence microscope after 1 weeks after the injection of the lentivirus. The total GFP distribution of the membrane was obvious. After second weeks, the fluorescence intensity began to attenuate, and the expression of GFP was weak after third weeks. (3) first weeks after the experiment (F =157.793, P=0.000; F eye axis =10.005, P=0.000) and the first 2 weeks (F flexion =182.603, P=0.000, F eye axis length), the mice in the experimental group had obvious myopia development, and the experimental mice had obvious myopia development, and the experimental mice had obvious myopia development, and the experimental mice injection eyes showed obvious myopia development, and the experimental group had obvious myopia development, and the mice injection eyes showed obvious myopia development, and There was an obvious lengthening of the ocular axis, but there was no significant difference in the diopter of the injection eyes and the blank control eye in the third weeks after the experiment (F =1.259, P=0.290; F eye axis length =1.004, P=0.371), and there was no statistical difference between the injection eyes and the blank control eye. There was no significant difference in the diopter of the eye and the axial length (P0.05) (4). In the 1 weeks after the experiment, the development trend of myopia in mice was the most obvious. Taking the injection eyes of the mice and negative control groups this week, the blank control eye slices were observed after HE staining, and there was no obvious change in the form of retina.
Conclusion after transfecting mouse retina with shRNA lentivirus vector of Egr-1 gene in mice, the Egr-1 gene of mouse retina was downregulated. The diopter and axial length of mice developed to myopia, which confirmed the important role of Egr-1 gene in the formation of myopia in mice, and found that the lentivirus vector transfected into the retina of mice. Safe and effective. The experimental results provide a direction for future gene therapy in myopia.
【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】：R778.11

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