本文選題：早期生長反應(yīng)因子-1 + 近視眼��； 參考：《中南大學(xué)》2010年博士論文

【摘要】： 第一章含小鼠Egr-1基因的發(fā)夾結(jié)構(gòu)RNA干擾質(zhì)粒的構(gòu)建及干擾效率鑒定 目的構(gòu)建及篩選高效率針對小鼠早期生長反應(yīng)因子-1(Egr-1)發(fā)夾結(jié)構(gòu)RNA干擾(shRNA)的質(zhì)粒。 方法根據(jù)小鼠Egr-1基因mRNA序列(NM007913),設(shè)計有發(fā)夾結(jié)構(gòu)的3條寡核苷酸序列,合成靶序列的Oligo DNA,退火形成雙鏈DNA,經(jīng)AgeⅠ和EcoRⅠ雙酶切后的pGCSIL-GFP載體連接產(chǎn)生含shRNA的重組質(zhì)粒1#、2#及3#,PCR篩選陽性克隆,測序測定。設(shè)計構(gòu)建不針對任何特異基因的NC質(zhì)粒作為陰性對照。將3個shRNA表達(dá)質(zhì)粒及NC質(zhì)粒轉(zhuǎn)染HEK293細(xì)胞,設(shè)不做任何處理的細(xì)胞為空白對照。通過對綠色熒光蛋白(GFP)表達(dá)量的觀察,實時熒光定量聚合酶鏈反應(yīng)(RQ-PCR)及免疫印跡法(western blotting)檢測Egr-1基因mRNA及蛋白的表達(dá),鑒定shRNA表達(dá)質(zhì)粒對Egr-1基因的抑制效率。組間比較采用單因素方差分析,實驗組3個序列兩兩比較采用q檢驗。 結(jié)果針對小鼠Egr-1基因進(jìn)行RNA干擾的1#、2#及3#序列中,1#序列的質(zhì)粒明顯抑制了細(xì)胞內(nèi)Egr-1的mRNA及蛋白表達(dá)(FmRNA=118.819, P=0.000; Fprotein=71.605, P=0.000) 結(jié)論成功構(gòu)建了針對小鼠Egr-1基因的高效shRNA表達(dá)的質(zhì)粒。 第二章含小鼠Egr-1 shRNA慢病毒載體構(gòu)建及轉(zhuǎn)染小鼠視網(wǎng)膜的研究 目的構(gòu)建含綠色熒光蛋白(GFP)和小鼠早期生長反應(yīng)因子-1(Egr-1)發(fā)夾結(jié)構(gòu)RNA干擾(shRNA)共表達(dá)的慢病毒載體,并將其轉(zhuǎn)染至小鼠視網(wǎng)膜組織,探索合適的給藥方式。 方法將前期實驗已經(jīng)篩選確定的小鼠Egr-1基因shRNA有效靶序列的質(zhì)粒,命名為LV-shRNA(Egr-1)。將LV-shRNA(Egr-1)重組質(zhì)粒及pHelper 1.0、pHelper2.0慢病毒包裝用的輔助質(zhì)粒共轉(zhuǎn)染293T細(xì)胞,包裝產(chǎn)生慢病毒載體,經(jīng)梯度稀釋后,根據(jù)熒光顯微鏡下綠色熒光蛋白(GFP)表達(dá)陽性細(xì)胞來計算病毒滴度。將包裝好的慢病毒載體分別通過玻璃體腔和視網(wǎng)膜下注射的途徑轉(zhuǎn)染至C57BL/6小鼠視網(wǎng)膜,于實驗后2周,取小鼠眼球制作冰凍切片,熒光顯微鏡觀察視網(wǎng)膜GFP表達(dá)情況。 結(jié)果①成功構(gòu)建含GFP并攜帶靶向小鼠Egr-1基因的shRNA慢病毒載體,經(jīng)孔稀釋法測定病毒滴度為4×108TU/ml;②利用該慢病毒載體系統(tǒng)轉(zhuǎn)染小鼠視網(wǎng)膜組織,發(fā)現(xiàn)經(jīng)玻璃體腔注射途徑轉(zhuǎn)染后的小鼠,GFP廣泛分布于視網(wǎng)膜全層包括視網(wǎng)膜色素上皮層(RPE)；而經(jīng)視網(wǎng)膜下注射途徑轉(zhuǎn)染后的小鼠,GFP局限分布于視網(wǎng)膜外層。 結(jié)論成功構(gòu)建含GFP并攜帶靶向小鼠Egr-1基因的shRNA慢病毒載體,通過玻璃體腔注射較視網(wǎng)膜下注射轉(zhuǎn)染效率高,分布范圍廣,為后期的體內(nèi)實驗提供了實驗基礎(chǔ)。 第三章Egr-1基因?qū)π∈蠼曆鄣恼{(diào)控作用 目的將針對小鼠Egr-1基因的shRNA慢病毒載體行玻璃體腔注射后,觀察小鼠屈光度及眼軸長度的變化,闡明Egr-1基因在小鼠近視眼形成中的調(diào)控作用。 方法15日齡C57BL/6小鼠,共180只,等量隨機(jī)分為3組：實驗組、陰性對照組及空白對照組。其中實驗組小鼠右眼玻璃體腔注入前期研究中構(gòu)造的LV-shRNA(Egr-1)'慢病毒載體；陰性對照組小鼠右眼玻璃體腔注入陰性對照LV-NC慢病毒載體；空白對照組小鼠不做任何處理。分別于實驗后1周、2周、3周測量各組小鼠右眼屈光度后將其麻醉處死,分別測量各組小鼠右眼眼軸長度；制作冰凍切片,熒光顯微鏡下觀察視網(wǎng)膜GFP表達(dá),判斷轉(zhuǎn)染情況；熒光定量聚合酶鏈反應(yīng)(RQ-PCR)、免疫印跡法(western blotting)及免疫熒光檢測小鼠視網(wǎng)膜Egr-1基因的表達(dá)；切片HE染色后,顯微鏡下觀察視網(wǎng)膜形態(tài)有無變化。組間比較采用單因素方差分析,兩兩比較采用q檢驗。 結(jié)果①RQ-PCR、western blotting、免疫熒光檢測發(fā)現(xiàn)實驗組注射眼內(nèi)Egr-1的表達(dá)明顯下調(diào),其中第1周Egr-1下調(diào)最為顯著(FmRNA=184.383,P=0.000; Fprotein=170.470, P=0.000);②實驗組和陰性對照組的慢病毒載體注射后1周后即可在熒光顯微鏡下觀察到視網(wǎng)膜全層GFP分布明顯,第2周后熒光強(qiáng)度開始衰減,第3周后GFP表達(dá)微弱；③在實驗后第1周(F屈光度=157.793,P=0.000；F眼軸長度=10.005,P=0.000)及第2周(F屈光度=182.603,P=0.000；F眼軸長度=5.273,P=0.007),實驗組小鼠注射眼出現(xiàn)了明顯的近視化發(fā)展,且伴有明顯的眼軸延長；但在實驗后第3周(F屈光度=1.259,P=0.290；F眼軸長度=1.004,P=0.371)實驗組小鼠注射眼與陰性對照組注射眼及空白對照眼的屈光度及眼軸長度相比較,無統(tǒng)計學(xué)差異；陰性對照組的注射眼在實驗各個階段與空白對照眼的屈光度及眼軸長度相比較,均無統(tǒng)計學(xué)差異(p0.05)④在實驗后1周,小鼠近視化發(fā)展趨勢最明顯,取這周小鼠實驗組及陰性對照組的注射眼、空白對照眼切片HE染色后觀察,發(fā)現(xiàn)各組視網(wǎng)膜形態(tài)無明顯變化。 結(jié)論通過針對小鼠Egr-1基因的shRNA慢病毒載體轉(zhuǎn)染小鼠視網(wǎng)膜后,出現(xiàn)了小鼠視網(wǎng)膜Egr-1基因的下調(diào),小鼠屈光度及眼軸長度向近視趨勢發(fā)展,證實了Egr-1基因在小鼠近視眼形成中所起的重要作用,同時發(fā)現(xiàn)慢病毒載體轉(zhuǎn)染小鼠視網(wǎng)膜安全有效。實驗結(jié)果為近視眼未來的基因治療提供了思考的方向。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R778.11

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