【作者】 蒋晶晶；

【导师】 刘双珍；

【作者基本信息】 中南大学 ， 眼科学， 2010， 博士

【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(p>0.05)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。更多还原

【Abstract】 Chapter one:Construction and identification of plasmid mediated short hairpin RNA interference targeting the Egr-1 gene of miceObjective To construct and screen high-efficiency plasmid mediated short hairpin RNA interference (shRNA) targeting the early growth response factor-1 (Egr-1) of mice.Methods Based on the Egr-1 gene mRNA sequence of mouse (NM007913), we designed 3 hairpin oligonucleotide sequences, synthesized Oligo DNA of target sequences, annealed to form double-stranded DNA. By Age I and EcoR I digested the pGCSIL-GFP vector to generate recombinant plasmids containing shRNA 1#,2# and 3#, PCR screened clones and sequenced. Designed and constructed not for any specific gene plasmid as a negative control, named NC. The three shRNA expression plasmids and NC plasmid were co-transfected to HEK293 cells. Cells without any treatment have as blank control. Through observed the green fluorescent protein (GFP) expression and detected Egr-1 gene expression of mRNA and protein in cells by real-time fluorescence quantitative polymerase chain reaction (RQ-PCR) and western blotting, we identified the inhibitory efficiency of shRNA expression plasmid tageting Egr-1 gene. Groups were analyzed using ANOVA, between 3 sequences of experimental group were compared with q-test. Results The RNA interference sequences for Egr-1 gene of mice:1 #,2# and 3#, the plasmid of 1# sequence significantly inhibited the mRNA and protein expression of Egr-1 in cells (F mRNA=118.819, P=0.000; Fprotein=71.605, P=0.000).Conclusion We successfully constructed the efficient shRNA expression plasmid targeting murine Egr-1 gene.Chapter two:Construction the lentivirus vector mediated short hairpin RNA interference targeting the Egr-1 gene of mice and transfection to the retina of miceObjective To construct the lentivirus vector containing green fluorescent protein (GFP) and short hairpin RNA interference (shRNA) targeting the early growth response factor-1 (Egr-1) of mice and transfect to murine retina, exploring the appropriate method for drug delivery.Methods shRNA plasmid which is most efficient in inhibiting Egr-1 expression was named LV-shRNA (Egr-1). The LV-shRNA (Egr-1) recombinant plasmid and pHelper1.0, pHelper2.0 what the auxiliary plasmids of packaging of lentivirus were co-transfected to 293T cells, packaging and produce vector, diluted by the gradient, according to green fluorescent protein (GFP) positive cells under fluorescence microscope to calculate the virus titer. The packaged lentivirus vector was separately transfected to retina of C57BL/6 mice through intravitreal injection and subretina injection. Eyes of mice were enucleated for histological analysis GFP expression in retina was observed under fluorescence microscopy.Results①We successfully constructed the lentivirus vector containing GFP and shRNA targeting the Egr-1 gene of mice, method of dilution holes for the virus titer was 4×108 TU/ml;②Using the lentivirus vector system transfect retina of mice, we found GFP widely distributed in all retina layers including the retina pigment epithelium (RPE) cell layer after intravitreal injection; GFP restricted expressed in the outer retina after subretina injection.Conclusion We successfully constructed the lentivirus vector containing GFP and shRNA targeting the Egr-1 gene of mice, compared with the two methods of drug delivery, intravitreal injection was more efficient in transfection and wide distribution, provided a basis for experiments in vivo.Chapter three:The regulation effect of Egr-1 gene in mice myopiaObjective Have the intravitreal injection of lentivirus vector containing shRNA targeting the Egr-1 gene, observed the changes of refraction and axial length of mice, clarify the regulation of Egr-1 gene in the development of myopia in mice. Methods 180 15-days-old C57BL/6 mice were randomly divided into 3 groups:experimental group, negative control group and blank control group. The experimental group of mice right eyes had injected the lentivirus vector of LV-shRNA (Egr-1) which had preliminary study; negative control group of mice right eyes have injected the negative control lentivirus vector of LV-NC; without any treatment mice as blank control group. After 1,2 and 3 weeks of experiment, we measured the diopter of right eye of mice in each group, then killed mice after anesthesia, measured axial length of right eye of mice in each group; producted frozen sections, observe the GFP expression in the retina under the microscope to determine transfection efficiency; through using real-time fluorescence quantitative polymerase chain reaction (RQ-PCR), western blotting and immunofluorescence, we detect the expression of Egr-1 gene in murine retina; we observed retina changes under the microscope. Groups were analyzed using ANOVA, between 3 groups were compared with q-test..Results①Though RQ-PCR, western blotting and immunofluorescence, we found that the expression of Egr-1 was significantly reduced, in which experimental group injected eyes, especially the expression of Egr-1 have down-regulation the most significant after 1 week of experiment (F mRNA= 184.383, P= 0.000; F protein= 170.470, P= 0.000);②After 1 week of experiment, expression of GFP in murine retina was found in the experimental group and the negative control group after the injection of the lentivirus vector. After 2 weeks of experiment, the fluorescence intensity began to decay. After 3 weeks of experiment, expression of GFP in retina was weak;③After 1 week of experiment, (F diopter= 157.793,P=0.000; F axial length= 10.005, P=0.000) and after 2 weeks of experiment (Fdiopter= 182.603, P= 0.000; F axial length= 5.273, P= 0.007), the mice in experimental group which the injected eyes has a significant development of myopia, and with obvious extension of axial length; but After 3 weeks of experiment (F diopter= 1.259, P= 0.290; F axial length= 1.004, P= 0.371) the mice in experimental group which the injected eyes compare with the negative control group which injected eyes and control eyes, the refraction and axial length has no significant differences; negative control group which injected eyes at all stages of the experiment compare with the blank control eyes, the refraction and axial length has no significant difference (p> 0.05)④After 1 week of experiment, the most obvious trend of development of myopia in mice, take sections of this week and have HE staining, showed no significant morphological changes in the retina among 3 groups.Conclusion Through transfected the lentivirus vector containing shRNA targeting the Egr-1 gene to murine retina, the Egr-1 gene in mice retina have reduced, the refractive error and axial length tune the trend of myopia, confirmed that Egr-1 gene play an important role in the development of myopia in mice. We also found that it was safe and effective for lentivirus to transfected murine retina. The results provide the direction of thinking for gene therapy in myopia in the future.更多还原