本文選題：視網(wǎng)膜色素變性　切入點(diǎn)：RNA干擾　出處：《第二軍醫(yī)大學(xué)》2010年博士論文

【摘要】： 目的 1.本實驗通過基因打靶技術(shù)構(gòu)建攜帶人突變RHO基因的視網(wǎng)膜變性小鼠。對構(gòu)建成功的不同鼠齡小鼠視網(wǎng)膜進(jìn)行組織病理變化研究,明確其病理改變特點(diǎn)。 2.制備靶向RHO基因的LV-GFP-shRNA。通過顯微注射器將慢病毒載體注射入小鼠視網(wǎng)膜下腔內(nèi)。研究注射后不同時間hRHO的mRNA和蛋白表達(dá)變化；不同時間視網(wǎng)膜轉(zhuǎn)染率和視網(wǎng)膜形態(tài)學(xué)變化。從而評估RNA干擾技術(shù)治療效果,為臨床應(yīng)用提供實驗依據(jù)。 方法 1.構(gòu)建mRHOp-hRHO-SV40A-PBS質(zhì)粒,進(jìn)行小鼠原核受精卵注射,繁育出轉(zhuǎn)基因小鼠。RT-PCR及western-blot方法鑒定轉(zhuǎn)基因小鼠。 2.18只RP小鼠隨機(jī)分成4組。在小鼠出生后15天、30天、60天各處死6只。5只健康小鼠進(jìn)行對照。過量戊巴比妥鈉腹腔注射處死小鼠后迅速完整摘取小鼠眼球,沿視神經(jīng)做3mm厚度的切片。分別行HE染色、原位細(xì)胞凋亡檢測和電鏡超微檢測。比較不同時間視網(wǎng)膜外核層厚度的變化、感光細(xì)胞凋亡陽性率及光感受器細(xì)胞超微結(jié)構(gòu)的改變。結(jié)果采用組間T檢驗,spss13.0統(tǒng)計軟件進(jìn)行統(tǒng)計學(xué)分析。 3.利用前期實驗證實體外轉(zhuǎn)染基因沉默效率為79.14%的靶向hRHO的siRNA序列,構(gòu)建pGCL-GFP-shRNA慢病毒穿梭質(zhì)粒,并制備LV-GFP-shRNA。 4.80只15天鼠齡RP小鼠,左眼作為對照組,不做任何處理；右眼視網(wǎng)膜下腔注射LV-GFP-shRNA。分別在注射后15天、30天、45天、60天隨機(jī)各處死20只RP小鼠。應(yīng)用半定量RT-PCR及western-blot方法檢測治療后不同時間RP小鼠視網(wǎng)膜hRHO mRNA及蛋白表達(dá)變化。視網(wǎng)膜鋪片觀察治療后不同時間視網(wǎng)膜轉(zhuǎn)染率,視網(wǎng)膜色素細(xì)胞熒光染色的范圍用0到3級評估。(0：沒有發(fā)現(xiàn)熒光細(xì)胞；1：散在的陽性細(xì)胞；2：部分陽性細(xì)胞連在一起；3：連續(xù)的陽性細(xì)胞)。行免疫熒光染色觀察視網(wǎng)膜RHO表達(dá),行HE染色觀察視網(wǎng)膜外核層厚度變化。 結(jié)果 1. mRHOp-hRHO-SV40-PBS質(zhì)粒經(jīng)各種酶切鑒定位置正確后鑒定測序。質(zhì)粒線性化位點(diǎn)為NotI和Pvul,雙酶切后可切成3037bp(回收)和1679bp、1043bp,回收3037bp用于顯微注射。小鼠受精卵注射后成功繁育出表達(dá)hRHO突變基因的RP小鼠。 2.RP小鼠出生后15天,視網(wǎng)膜外核層及內(nèi)核層已經(jīng)可以分出層次。正常對照組視網(wǎng)膜外核層在各個時間點(diǎn)沒有明顯的變化(9.30±0.37),而RP小鼠出生15天外核層開始變薄(7.05±0.25),30天明顯變薄(5.29±0.24),60天外核層減至單層(2.04±0.28)。TUNEL染色正常對照組為陰性,RP小鼠15天出現(xiàn)散在陽性顆粒,60天出現(xiàn)大片陽性顆粒。提示光感受器細(xì)胞出現(xiàn)大量壞死及凋亡。透射電鏡顯示：早期視網(wǎng)膜視桿細(xì)胞外節(jié)正常、外界膜連續(xù),30天開始視桿細(xì)胞外節(jié)縮短,外界膜開始變細(xì),不連續(xù)。盤膜出現(xiàn)空泡狀改變,內(nèi)節(jié)線粒體、內(nèi)質(zhì)網(wǎng)均有不同程度變性,溶解；細(xì)胞核出現(xiàn)核濃縮,發(fā)生凋亡。 3.成功構(gòu)建靶向hRHO的pGCL-GFP-shRNA'慢病毒穿梭質(zhì)粒,并制備出滴度為1.0×1010ifu/ml的LV-GFP-shRNA。 4.RP小鼠在進(jìn)行右眼LV-GFP-shRNA注射后30天時RHO mRNA表達(dá)開始下降,到60天時明顯下降。組內(nèi)比較：在45天、60天mRNA表達(dá)水平LV-GFP-shRNA組較陰性對照組有明顯下降(P0.05)。在蛋白表達(dá)水平上,LV-GFP-shRNA組在30天時較對照組表達(dá)開始下降,45天時進(jìn)一步下降,60天時又趨于平穩(wěn)。組內(nèi)比較：在45天、60天蛋白表達(dá)水平LV-GFP-shRNA組較陰性對照組有明顯下降(P0.05)。視網(wǎng)膜ONL層數(shù)改變LV-GFP-shRNA組較陰性對照組在15天、30天、45天均沒有明顯差異,到60天時出現(xiàn)LV-GFP-shRNA組較陰性對照組ONL層數(shù)增厚(P0.05)。 結(jié)論 1.利用轉(zhuǎn)基因技術(shù)成功構(gòu)建出表達(dá)hRHO突變基因的視網(wǎng)膜變性小鼠。為后續(xù)研究治療提供可靠的動物模型。 2.本實驗構(gòu)建出的RP小鼠模型中視網(wǎng)膜組織病理學(xué)改變與人類RHO基因突變引起的視網(wǎng)膜色素變性的病理改變類似,為下一步行基因治療療效判定提供形態(tài)學(xué)依據(jù)。 3.成功構(gòu)建靶向hRHO的pGCL-GFP-shRNA慢病毒穿梭質(zhì)粒,并制備出滴度為1.0×1010ifu/ml的LV-GFP-shRNA。 4. LV-GFP-shRNA視網(wǎng)膜下腔注射后短期可以明顯下調(diào)RHO蛋白表達(dá)。為RNA干擾技術(shù)治療人類視網(wǎng)膜色素變性提供可靠的動物實驗依據(jù)。
[Abstract]:Purpose



1 . In this experiment , retinal degeneration mice bearing human mutation RHO gene were constructed by gene targeting technique . The pathological changes of retinal tissues of different mouse - age mice were studied .



2 . preparing the LV - GFP - shRNA targeting the RHO gene , injecting the slow viral vector into the subretinal cavity of the mouse through a micro injector , and researching the mRNA and protein expression changes of hRHO after injection ;
The effect of RNA interference was evaluated and the experimental basis was provided for clinical application .



method



1 . Construction of mRHO - SV40A - PBS plasmid was carried out in mice , and the transgenic mice were bred by RT - PCR and western - blot .



2 . 18 RP mice were randomly divided into 4 groups . 6 . 5 healthy mice were sacrificed 15 days , 30 days and 60 days after birth .



3 . The siRNA sequence targeting hRHO with the silencing efficiency of 79.14 % in vitro was confirmed by previous experiments , and the pGCL - GFP - shRNA lentivirus shuttle plasmid was constructed and LV - GFP - shRNA was prepared .



4.80 days old RP mice , left eye as control group , do not do any treatment ;
Left - eye subretinal cavity injected with LV - GFP - shRNA . 20 RP mice were sacrificed on 15 days , 30 days , 45 days and 60 days after injection . The changes of hRHO mRNA and protein expression in the retina of RP mice after treatment were detected by semi - quantitative RT - PCR and western - blot .
1 : scattered positive cells ;
2 : part of the positive cells were linked together ;
3 : continuous positive cells ) . The retinal RHO expression was observed by immunofluorescence staining , and the thickness of the outer nucleus of the retina was observed by HE staining .



Results



1. mRHOp-hRHO-SV40-PBS璐ㄧ矑緇忓悇縐嶉叾鍒囬壌瀹氫綅緗紜悗閴村畾嫻嬪簭.璐ㄧ矑綰挎，

本文編號：1715295