RNA干扰治疗视网膜色素变性的动物实验研究
摘要
目的
1.本实验通过基因打靶技术构建携带人突变RHO基因的视网膜变性小鼠。对构建成功的不同鼠龄小鼠视网膜进行组织病理变化研究,明确其病理改变特点。
2.制备靶向RHO基因的LV-GFP-shRNA。通过显微注射器将慢病毒载体注射入小鼠视网膜下腔内。研究注射后不同时间hRHO的mRNA和蛋白表达变化；不同时间视网膜转染率和视网膜形态学变化。从而评估RNA干扰技术治疗效果,为临床应用提供实验依据。
方法
1.构建mRHOp-hRHO-SV40A-PBS质粒,进行小鼠原核受精卵注射,繁育出转基因小鼠。RT-PCR及western-blot方法鉴定转基因小鼠。
2.18只RP小鼠随机分成4组。在小鼠出生后15天、30天、60天各处死6只。5只健康小鼠进行对照。过量戊巴比妥钠腹腔注射处死小鼠后迅速完整摘取小鼠眼球,沿视神经做3mm厚度的切片。分别行HE染色、原位细胞凋亡检测和电镜超微检测。比较不同时间视网膜外核层厚度的变化、感光细胞凋亡阳性率及光感受器细胞超微结构的改变。结果采用组间T检验,spss13.0统计软件进行统计学分析。
3.利用前期实验证实体外转染基因沉默效率为79.14%的靶向hRHO的siRNA序列,构建pGCL-GFP-shRNA慢病毒穿梭质粒,并制备LV-GFP-shRNA。
4.80只15天鼠龄RP小鼠,左眼作为对照组,不做任何处理；右眼视网膜下腔注射LV-GFP-shRNA。分别在注射后15天、30天、45天、60天随机各处死20只RP小鼠。应用半定量RT-PCR及western-blot方法检测治疗后不同时间RP小鼠视网膜hRHO mRNA及蛋白表达变化。视网膜铺片观察治疗后不同时间视网膜转染率,视网膜色素细胞荧光染色的范围用0到3级评估。(0：没有发现荧光细胞；1：散在的阳性细胞；2：部分阳性细胞连在一起；3：连续的阳性细胞)。行免疫荧光染色观察视网膜RHO表达,行HE染色观察视网膜外核层厚度变化。
结果
1. mRHOp-hRHO-SV40-PBS质粒经各种酶切鉴定位置正确后鉴定测序。质粒线性化位点为NotI和Pvul,双酶切后可切成3037bp(回收)和1679bp、1043bp,回收3037bp用于显微注射。小鼠受精卵注射后成功繁育出表达hRHO突变基因的RP小鼠。
2.RP小鼠出生后15天,视网膜外核层及内核层已经可以分出层次。正常对照组视网膜外核层在各个时间点没有明显的变化(9.30±0.37),而RP小鼠出生15天外核层开始变薄(7.05±0.25),30天明显变薄(5.29±0.24),60天外核层减至单层(2.04±0.28)。TUNEL染色正常对照组为阴性,RP小鼠15天出现散在阳性颗粒,60天出现大片阳性颗粒。提示光感受器细胞出现大量坏死及凋亡。透射电镜显示：早期视网膜视杆细胞外节正常、外界膜连续,30天开始视杆细胞外节缩短,外界膜开始变细,不连续。盘膜出现空泡状改变,内节线粒体、内质网均有不同程度变性,溶解；细胞核出现核浓缩,发生凋亡。
3.成功构建靶向hRHO的pGCL-GFP-shRNA'慢病毒穿梭质粒,并制备出滴度为1.0×1010ifu/ml的LV-GFP-shRNA。
4.RP小鼠在进行右眼LV-GFP-shRNA注射后30天时RHO mRNA表达开始下降,到60天时明显下降。组内比较：在45天、60天mRNA表达水平LV-GFP-shRNA组较阴性对照组有明显下降(P<0.05)。在蛋白表达水平上,LV-GFP-shRNA组在30天时较对照组表达开始下降,45天时进一步下降,60天时又趋于平稳。组内比较：在45天、60天蛋白表达水平LV-GFP-shRNA组较阴性对照组有明显下降(P<0.05)。视网膜ONL层数改变LV-GFP-shRNA组较阴性对照组在15天、30天、45天均没有明显差异,到60天时出现LV-GFP-shRNA组较阴性对照组ONL层数增厚(P<0.05)。
结论
1.利用转基因技术成功构建出表达hRHO突变基因的视网膜变性小鼠。为后续研究治疗提供可靠的动物模型。
2.本实验构建出的RP小鼠模型中视网膜组织病理学改变与人类RHO基因突变引起的视网膜色素变性的病理改变类似,为下一步行基因治疗疗效判定提供形态学依据。
3.成功构建靶向hRHO的pGCL-GFP-shRNA慢病毒穿梭质粒,并制备出滴度为1.0×1010ifu/ml的LV-GFP-shRNA。
4. LV-GFP-shRNA视网膜下腔注射后短期可以明显下调RHO蛋白表达。为RNA干扰技术治疗人类视网膜色素变性提供可靠的动物实验依据。
Objective
1. To design a mouse model of autosomal dominant retinitis pigmentosa who carry by human mutant RHO through gene targeting. To identify pathological character in different age.
2. To construct the LV-GFP virus which could express the specific siRNA targeted to hRHO and evaluate its inhibitory effect in vivo. Mice treat with LV-GFP-shRNA were injected subretinally. To investigate the hRHO degeneration effect by using hRHO RNAi.
Methods
1. To construct mRHOp-hRHO-SV40A-PBS, targeting inject in ertilized egg. To identify RP mice by using RT-PCR and western-blot.
2. Eighteen RP mice were sacrificed at different time namely postnatal days 15 and 30,60. The eyeball were immediately enuleated and fixed in 10% formaldehyde or 2.5% glutaral solution.Routine pathologic procedure were performed and sections were stained by hematoxylin and eosin(HE) and terminal deoxytransferase-mediated dUTP nicked labeling(TUNEL).The eyeball fixed with glutaral solution were examined by electron-microscopy.
3. In our prophase study, we had successfully inhibited the expression of mutant hRHO genes effectively in cultured rat rod cells by applying chemical synthetic siRNA, and sieved out one high-performance RNA interference chisequence which gene silencing efficiency is 79.14%. To construct the LV-GFP virus which could express the specific siRNA targeted to mutant hRHO.
4. Eighty RP mice were used in this research (postnatal day 15). LV-GFP-shRNA was injected on in the right eyes, and left eyes were neither injected with saline or with control virus as blank control. Mice were euthanized by pentobarbital sodium after injection day15,30,45,60,and then eyes were removed. To investigate the hRHO degeneration effect by using hRHO RNAi. To confirm the transfection efficiency after injected subretinally in different time. To evaluate the range of transfection efficiency through 3 point (0:none; 1: sporadic masccline; 2:part of masccline connected; 3:consecutive masccline).To observe the layer number of outer nuclear layer by immunofluorescence.
Results
1. We had constructed the mRHOp-hRHO-SV40-PBS and made the amplification and depuration of it. mRHOp-hRHO-SV40-PBS were microinjected in amphicytula, and breed RP mice who carry by human mutant RHO successfully.
2. The layer number of retinal outer nuclear layer cells was gradually decreased from day 15 (7.05±0.25) to day 60 (2.04±0.28). At the day 30, (5.29±0.24) layer were observed. Compared with RP mice, the layer number of retinal outer nuclear layer cells in wild type mice were observed (9.30±0.37) stable in all the time. The apoptosis of outer layer cells were seen on day 15 and increased deeply in the day 60. External limiting membrane were discontinuation on day 30, vacuole occurred in the outer segmen disk. Degeneration in bioblast and endocytoplasmic reticulum were altered and the apoptosis of outer layer cells were seen by the electro-microscopy in RP mice.
3. We had constructed the LV-GFP for expressing siRNA and made the amplification and depuration of it. The tite of LV-GFP-shRNA was 1.0×1010 particles per ml.
4. Our in vivo test showed 15 and 60 days after LV-GFP-shRNA injected subretinally, the GFP could be detected in retina sections,which meant that LV-GFP-shRNA had successfully transduced into rod cells and the shRNA being expressed. RT-PCR test showed the recombinant virus vector could efficiently inhibited the RHO mRNA in rod cells after injected 45 days (P<0.05).Western blot test also showed the recombinant virus vector could efficiently inhibited the RHO protein in rod cells after injected 45 days (P< 0.05) compared with control.
Conclutions
1.We created a mouse model of autosomal dominant retinitis pigmentosa who carry by human mutant RHO through gene targeting successfully and provide RP animal model for the further research.
2. We found the process of histopathology in the RP model were similar with human who suffered from RP.
3. We had constructed the LV-GFP for expressing si RNA and made the amplification and depuration of it. The tite of LV-GFP-shRNA was 1.0×1010 particles per ml.
4. After injected subretinally, LV-GFP-shRNA could depress of the expression of RHO either mRNA or protein in short period.
1.本实验通过基因打靶技术构建携带人突变RHO基因的视网膜变性小鼠。对构建成功的不同鼠龄小鼠视网膜进行组织病理变化研究,明确其病理改变特点。
2.制备靶向RHO基因的LV-GFP-shRNA。通过显微注射器将慢病毒载体注射入小鼠视网膜下腔内。研究注射后不同时间hRHO的mRNA和蛋白表达变化；不同时间视网膜转染率和视网膜形态学变化。从而评估RNA干扰技术治疗效果,为临床应用提供实验依据。
方法
1.构建mRHOp-hRHO-SV40A-PBS质粒,进行小鼠原核受精卵注射,繁育出转基因小鼠。RT-PCR及western-blot方法鉴定转基因小鼠。
2.18只RP小鼠随机分成4组。在小鼠出生后15天、30天、60天各处死6只。5只健康小鼠进行对照。过量戊巴比妥钠腹腔注射处死小鼠后迅速完整摘取小鼠眼球,沿视神经做3mm厚度的切片。分别行HE染色、原位细胞凋亡检测和电镜超微检测。比较不同时间视网膜外核层厚度的变化、感光细胞凋亡阳性率及光感受器细胞超微结构的改变。结果采用组间T检验,spss13.0统计软件进行统计学分析。
3.利用前期实验证实体外转染基因沉默效率为79.14%的靶向hRHO的siRNA序列,构建pGCL-GFP-shRNA慢病毒穿梭质粒,并制备LV-GFP-shRNA。
4.80只15天鼠龄RP小鼠,左眼作为对照组,不做任何处理；右眼视网膜下腔注射LV-GFP-shRNA。分别在注射后15天、30天、45天、60天随机各处死20只RP小鼠。应用半定量RT-PCR及western-blot方法检测治疗后不同时间RP小鼠视网膜hRHO mRNA及蛋白表达变化。视网膜铺片观察治疗后不同时间视网膜转染率,视网膜色素细胞荧光染色的范围用0到3级评估。(0：没有发现荧光细胞；1：散在的阳性细胞；2：部分阳性细胞连在一起；3：连续的阳性细胞)。行免疫荧光染色观察视网膜RHO表达,行HE染色观察视网膜外核层厚度变化。
结果
1. mRHOp-hRHO-SV40-PBS质粒经各种酶切鉴定位置正确后鉴定测序。质粒线性化位点为NotI和Pvul,双酶切后可切成3037bp(回收)和1679bp、1043bp,回收3037bp用于显微注射。小鼠受精卵注射后成功繁育出表达hRHO突变基因的RP小鼠。
2.RP小鼠出生后15天,视网膜外核层及内核层已经可以分出层次。正常对照组视网膜外核层在各个时间点没有明显的变化(9.30±0.37),而RP小鼠出生15天外核层开始变薄(7.05±0.25),30天明显变薄(5.29±0.24),60天外核层减至单层(2.04±0.28)。TUNEL染色正常对照组为阴性,RP小鼠15天出现散在阳性颗粒,60天出现大片阳性颗粒。提示光感受器细胞出现大量坏死及凋亡。透射电镜显示：早期视网膜视杆细胞外节正常、外界膜连续,30天开始视杆细胞外节缩短,外界膜开始变细,不连续。盘膜出现空泡状改变,内节线粒体、内质网均有不同程度变性,溶解；细胞核出现核浓缩,发生凋亡。
3.成功构建靶向hRHO的pGCL-GFP-shRNA'慢病毒穿梭质粒,并制备出滴度为1.0×1010ifu/ml的LV-GFP-shRNA。
4.RP小鼠在进行右眼LV-GFP-shRNA注射后30天时RHO mRNA表达开始下降,到60天时明显下降。组内比较：在45天、60天mRNA表达水平LV-GFP-shRNA组较阴性对照组有明显下降(P<0.05)。在蛋白表达水平上,LV-GFP-shRNA组在30天时较对照组表达开始下降,45天时进一步下降,60天时又趋于平稳。组内比较：在45天、60天蛋白表达水平LV-GFP-shRNA组较阴性对照组有明显下降(P<0.05)。视网膜ONL层数改变LV-GFP-shRNA组较阴性对照组在15天、30天、45天均没有明显差异,到60天时出现LV-GFP-shRNA组较阴性对照组ONL层数增厚(P<0.05)。
结论
1.利用转基因技术成功构建出表达hRHO突变基因的视网膜变性小鼠。为后续研究治疗提供可靠的动物模型。
2.本实验构建出的RP小鼠模型中视网膜组织病理学改变与人类RHO基因突变引起的视网膜色素变性的病理改变类似,为下一步行基因治疗疗效判定提供形态学依据。
3.成功构建靶向hRHO的pGCL-GFP-shRNA慢病毒穿梭质粒,并制备出滴度为1.0×1010ifu/ml的LV-GFP-shRNA。
4. LV-GFP-shRNA视网膜下腔注射后短期可以明显下调RHO蛋白表达。为RNA干扰技术治疗人类视网膜色素变性提供可靠的动物实验依据。
Objective
1. To design a mouse model of autosomal dominant retinitis pigmentosa who carry by human mutant RHO through gene targeting. To identify pathological character in different age.
2. To construct the LV-GFP virus which could express the specific siRNA targeted to hRHO and evaluate its inhibitory effect in vivo. Mice treat with LV-GFP-shRNA were injected subretinally. To investigate the hRHO degeneration effect by using hRHO RNAi.
Methods
1. To construct mRHOp-hRHO-SV40A-PBS, targeting inject in ertilized egg. To identify RP mice by using RT-PCR and western-blot.
2. Eighteen RP mice were sacrificed at different time namely postnatal days 15 and 30,60. The eyeball were immediately enuleated and fixed in 10% formaldehyde or 2.5% glutaral solution.Routine pathologic procedure were performed and sections were stained by hematoxylin and eosin(HE) and terminal deoxytransferase-mediated dUTP nicked labeling(TUNEL).The eyeball fixed with glutaral solution were examined by electron-microscopy.
3. In our prophase study, we had successfully inhibited the expression of mutant hRHO genes effectively in cultured rat rod cells by applying chemical synthetic siRNA, and sieved out one high-performance RNA interference chisequence which gene silencing efficiency is 79.14%. To construct the LV-GFP virus which could express the specific siRNA targeted to mutant hRHO.
4. Eighty RP mice were used in this research (postnatal day 15). LV-GFP-shRNA was injected on in the right eyes, and left eyes were neither injected with saline or with control virus as blank control. Mice were euthanized by pentobarbital sodium after injection day15,30,45,60,and then eyes were removed. To investigate the hRHO degeneration effect by using hRHO RNAi. To confirm the transfection efficiency after injected subretinally in different time. To evaluate the range of transfection efficiency through 3 point (0:none; 1: sporadic masccline; 2:part of masccline connected; 3:consecutive masccline).To observe the layer number of outer nuclear layer by immunofluorescence.
Results
1. We had constructed the mRHOp-hRHO-SV40-PBS and made the amplification and depuration of it. mRHOp-hRHO-SV40-PBS were microinjected in amphicytula, and breed RP mice who carry by human mutant RHO successfully.
2. The layer number of retinal outer nuclear layer cells was gradually decreased from day 15 (7.05±0.25) to day 60 (2.04±0.28). At the day 30, (5.29±0.24) layer were observed. Compared with RP mice, the layer number of retinal outer nuclear layer cells in wild type mice were observed (9.30±0.37) stable in all the time. The apoptosis of outer layer cells were seen on day 15 and increased deeply in the day 60. External limiting membrane were discontinuation on day 30, vacuole occurred in the outer segmen disk. Degeneration in bioblast and endocytoplasmic reticulum were altered and the apoptosis of outer layer cells were seen by the electro-microscopy in RP mice.
3. We had constructed the LV-GFP for expressing siRNA and made the amplification and depuration of it. The tite of LV-GFP-shRNA was 1.0×1010 particles per ml.
4. Our in vivo test showed 15 and 60 days after LV-GFP-shRNA injected subretinally, the GFP could be detected in retina sections,which meant that LV-GFP-shRNA had successfully transduced into rod cells and the shRNA being expressed. RT-PCR test showed the recombinant virus vector could efficiently inhibited the RHO mRNA in rod cells after injected 45 days (P<0.05).Western blot test also showed the recombinant virus vector could efficiently inhibited the RHO protein in rod cells after injected 45 days (P< 0.05) compared with control.
Conclutions
1.We created a mouse model of autosomal dominant retinitis pigmentosa who carry by human mutant RHO through gene targeting successfully and provide RP animal model for the further research.
2. We found the process of histopathology in the RP model were similar with human who suffered from RP.
3. We had constructed the LV-GFP for expressing si RNA and made the amplification and depuration of it. The tite of LV-GFP-shRNA was 1.0×1010 particles per ml.
4. After injected subretinally, LV-GFP-shRNA could depress of the expression of RHO either mRNA or protein in short period.
引文
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