AAV载体介导的BDNF表达对大鼠糖尿病模型中的RGC的保护作用
发布时间：2018-07-15 13:07
【摘要】：目的：构建pAAV-EGFP及pAAV-BDNF重组腺相关病毒,测定其感染滴度。观察AAV-BDNF对链脲佐菌素(STZ)造模大鼠视网膜神经节细胞数量影响,通过检测大鼠存活的荧光金(Fluorogold,FG)染料逆标的RGCs进行数量统计以及检测视觉诱发电位及视网膜电图来评价表达BDNF的重组AAV载体介导的基因治疗的神经保护效果。 方法： 1.pAAV-EGFP构建：从pCAGGS中克隆CBA promoter,并与pSEWB同时用Apa和EcoRI进行酶切消化,随后进行胶回收纯化以及连接转化,获得pCBA-EGFP-WPRE。使用限制性内切酶酶切和PCR方法检测构建质粒的准确性。使用磷酸钙转染方法检测质粒的基因表达活性。 2. pAAV-BDNF构建：从大鼠脑中抽提总RNA,使用RT-PCR方法克隆融合有H i s-tag的大鼠BDNF基因,并将pCBA-EGFP-WPRE同时用HindIII和EcoRI进行酶切消化,随后胶回收纯化及连接转化,进而获得pCBA-BDNF-WPRE。使用限制性内切酶酶切和PCR方法检测构建质粒的准确性。使用磷酸钙转染方法检测质粒的基因表达活性。Western-blotting分析视网膜中转染病毒获取的蛋白含量。 3.建立糖尿病大鼠模型,玻璃体腔注射EGFP或BDNF/His-tag融合蛋白的重组AAV病毒载体,不同时间点观察存活的荧光金(Fluorogold,FG)染料逆标的RGCs数量,并进行视网膜电图及视觉诱发电位检测。 4. BDNF受体TrkB阻断实验检测未被转染AAV-BDNF而存活的RGC数量,并进行视网膜电图及视觉诱发电位检测。 结果： 1.成功构建了pAAV-EGFP-WPRE及pAAV-BDNF-WPRE重组质粒。包装制备了腺相关病毒(rAAV-EGFP及rAAV-BDNF),经纯化、浓缩后病毒滴度为3.0×109／ml。rAAV-EGFP主射大鼠玻璃体腔3周后,视网膜冰冻切片,荧光显微镜下可观察到荧光表达,Western-blotting分析显示视网膜组织高表达EGFP及BDNF蛋白。 2.应用表达BDNF的重组AAV病毒载体进行基因治疗3个月、6个月、9个月之后,分别进行了总视网膜、中心视网膜、周边视网膜RGC存活数量的统计。治疗组和对照组FG逆标的RGC数量都明显少于正常视网膜(P0.05),但在各时间点,AAV-BDNF台疗组存活的RGC数量都明显多于AAV-EGFP对照组(P0.05),同时AAV-BDNF治疗组存活的RGC数量在6个月后就不再发生明显减少(P0.05)：基因治疗3个月后发现,AAV-BDNF治疗组中被重组AAV病毒载体转染的表达BDNF的RGC数量明显多于AAV-EGFP对照组中表达EGFP的细胞数量,说明了良好的治疗效果。同时,治疗组中没有被转染的EGC数量也明显多于对照组。BDNF受体TrkB阻断实验表明,AAV-BDNF治疗组中未被转染的神经节细胞的数量发生明显下降,说明了活的细胞可能是通过吸收周围被转染的细胞所分泌的BDNF而获得保护。 3.视觉诱发电位检测分析,AAV-BDNF台疗组和AAV-EGFP对照组P波振幅值明显小于正常视网膜(P0.05),同时从V-BDNF治疗组P波振幅值明显大于对照组(P0.05),说明基因治疗不仅使更多的RGC被保护而存活,而且存活的RGC能够发挥其正常生理功能。视网膜电图检测分析：1个月正常对照组OPs振幅与AAV-EGFP对照组比较就有显著性差异(P0.05),3个月正常对照组OPs振幅与AAV-EGFP对照组比较有非常显著性差异(P0.01)。且AAV-BDNF治疗组与AAV-EGFP对照组相比,OPs波振幅值明显大于AAV-EGFP对照组(P0.05)。AAV-EGFP对照组的视网膜电图b波波幅在糖尿病6个月时与正常对照组比较有显著性差异(P0.05),9个月正常对照组b波振幅与AAV-EGFP对照组比较有非常显著性差异(P0.01),然而AAV-BDNF治疗组在治疗6个月及9个月时b波幅值明显大于AAV-EGFP对照组。说明基因治疗对糖尿病模型大鼠视网膜功能恢复有一定帮助。 结论： 1.构建的rAAV-BDNF和rAAV-EGFP能高效转染视网膜组织,并在视网膜上成功表达BDNF蛋白和绿色荧光蛋白。 2.治疗组中被重组AAV病毒载体转染的表达BDNF的RGC数量明显多于对照组中表达EGFP的细胞数量,说明了良好的治疗效果。同时也可以保护未被AAV-BDNF转染的一部分视网膜神经节细胞。 3.糖尿病大鼠模型中给予重组AAV病毒载体介导的BDNF基因治疗,对于视网膜神经节细胞数量以及功都能产生良好的治疗效果。
[Abstract]:Objective: to construct pAAV-EGFP and pAAV-BDNF recombinant adeno-related virus and determine its infection titer. The effect of AAV-BDNF on the number of retinal ganglion cells in rat model of streptozotocin (STZ) was observed. The quantitative statistics of RGCs and the detection of visual evoked potential and retina electricity were detected by detecting the RGCs of the surviving Fluorogold, FG dyestuff RGCs in rats. To evaluate the neuroprotective effect of recombinant AAV vector mediated gene therapy for BDNF expression.
Method:
1.pAAV-EGFP Construction: CBA promoter was cloned from pCAGGS, and pSEWB was digested with Apa and EcoRI at the same time with pSEWB, then recycled and purified by glue and connection and transformation. The accuracy of pCBA-EGFP-WPRE. using restriction endonuclease digestion and PCR method to detect the construction of plasmid was obtained. The gene expression of plasmid was detected by calcium phosphate transfection method. Sex.
2. pAAV-BDNF Construction: extracting total RNA from rat brain, using RT-PCR method to clone the BDNF gene of rat with H I s-tag, and digesting pCBA-EGFP-WPRE at the same time as HindIII and EcoRI, then recycled and purified and connected transformation, and then obtain pCBA-BDNF-WPRE. using restriction endonuclease digestion and PCR method to detect construction plasmids. The accuracy of the transfection method was used to detect the gene expression activity of the plasmid..Western-blotting was used to analyze the protein content of the transfected virus in the retina.
3. the diabetic rat model was established. The recombinant AAV virus vector was injected with EGFP or BDNF/His-tag fusion protein in the vitreous cavity. The number of RGCs of the surviving fluorescent (Fluorogold, FG) dyestuff was observed at different time points, and the electroretinogram and visual evoked potential were detected.
4. the BDNF receptor TrkB blocking assay was used to detect the number of RGC surviving without transfection of AAV-BDNF, and electroretinogram and visual evoked potential were detected.
Result:
1. the recombinant plasmid of pAAV-EGFP-WPRE and pAAV-BDNF-WPRE was successfully constructed. The adeno-related virus (rAAV-EGFP and rAAV-BDNF) was packed and prepared. After purification, the virus titer was 3 * 109 / ml.rAAV-EGFP in the vitreous cavity for 3 weeks, the frozen section of the retina, fluorescence microscopy could be observed under the fluorescence microscope, and Western-blotting analysis showed that EGFP and BDNF protein are highly expressed in the retina tissue.
2. the recombinant AAV virus vector expressing BDNF was used for gene therapy for 3 months, 6 months and 9 months later, the total retina, central retina, and peripheral retina RGC survived, respectively. The number of RGC in the treatment group and the control group was significantly less than that of the normal retina (P0.05), but at every time point, the survival R of the AAV-BDNF therapy group. The number of GC was significantly more than that of the AAV-EGFP control group (P0.05), and the number of surviving RGC in the AAV-BDNF group no longer decreased significantly after 6 months (P0.05). After 3 months of gene therapy, the number of RGC expressing BDNF by the recombinant AAV virus vector in the AAV-BDNF treatment group was significantly more than the number of EGFP cells expressed in the AAV-EGFP control group. At the same time, the number of non transfected EGC in the treatment group was also significantly more than the control group.BDNF receptor TrkB blocking experiment, which showed that the number of ganglion cells that were not transfected in the AAV-BDNF treatment group decreased obviously, indicating that the living cells may be secreted by the BDNF secreted by the transfected cells. And get protection.
3. visual evoked potential analysis showed that the amplitude of P wave in AAV-BDNF group and AAV-EGFP control group was significantly lower than that of normal retina (P0.05), and the amplitude of P wave in V-BDNF treatment group was significantly greater than that of control group (P0.05), indicating that gene therapy not only made more RGC be protected and survived, but the surviving RGC could play its normal physiological function. Omentogram analysis: the amplitude of OPs in the 1 months normal control group was significantly different from that of the AAV-EGFP control group (P0.05). The amplitude of OPs in the normal control group was significantly different from that of the AAV-EGFP control group (P0.01). The amplitude of OPs wave in the AAV-BDNF treatment group was significantly higher than that of the AAV-EGFP control group, and the amplitude of the OPs wave was significantly greater than that of the AAV-EGFP control group (P0.05) The b wave amplitude of the electroretinogram in the.AAV-EGFP control group was significantly different from that of the normal control group at 6 months of diabetes (P0.05). The amplitude of the b wave amplitude in the normal control group was significantly different from that of the AAV-EGFP control group (P0.01) in the normal control group (P0.01), but the b wave amplitude of the AAV-BDNF treatment group was significantly greater than the AAV-EGFP control at 6 months and 9 months. Conclusion: gene therapy is helpful to the recovery of retinal function in diabetic rats.
Conclusion:
1. the constructed rAAV-BDNF and rAAV-EGFP can efficiently transfect retinal tissue and express BDNF and GFP successfully on the retina.
2. the number of RGC expressing BDNF transfected by recombinant AAV virus vector in the treatment group was significantly more than the number of cells expressing EGFP in the control group, indicating a good therapeutic effect. Meanwhile, a part of the retinal ganglion cells that were not transfected by AAV-BDNF could also be protected.
3. the BDNF gene therapy mediated by recombinant AAV virus vector in the diabetic rat model can produce good therapeutic effect on the number and work of retinal ganglion cells.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
[Abstract]:Objective: to construct pAAV-EGFP and pAAV-BDNF recombinant adeno-related virus and determine its infection titer. The effect of AAV-BDNF on the number of retinal ganglion cells in rat model of streptozotocin (STZ) was observed. The quantitative statistics of RGCs and the detection of visual evoked potential and retina electricity were detected by detecting the RGCs of the surviving Fluorogold, FG dyestuff RGCs in rats. To evaluate the neuroprotective effect of recombinant AAV vector mediated gene therapy for BDNF expression.
Method:
1.pAAV-EGFP Construction: CBA promoter was cloned from pCAGGS, and pSEWB was digested with Apa and EcoRI at the same time with pSEWB, then recycled and purified by glue and connection and transformation. The accuracy of pCBA-EGFP-WPRE. using restriction endonuclease digestion and PCR method to detect the construction of plasmid was obtained. The gene expression of plasmid was detected by calcium phosphate transfection method. Sex.
2. pAAV-BDNF Construction: extracting total RNA from rat brain, using RT-PCR method to clone the BDNF gene of rat with H I s-tag, and digesting pCBA-EGFP-WPRE at the same time as HindIII and EcoRI, then recycled and purified and connected transformation, and then obtain pCBA-BDNF-WPRE. using restriction endonuclease digestion and PCR method to detect construction plasmids. The accuracy of the transfection method was used to detect the gene expression activity of the plasmid..Western-blotting was used to analyze the protein content of the transfected virus in the retina.
3. the diabetic rat model was established. The recombinant AAV virus vector was injected with EGFP or BDNF/His-tag fusion protein in the vitreous cavity. The number of RGCs of the surviving fluorescent (Fluorogold, FG) dyestuff was observed at different time points, and the electroretinogram and visual evoked potential were detected.
4. the BDNF receptor TrkB blocking assay was used to detect the number of RGC surviving without transfection of AAV-BDNF, and electroretinogram and visual evoked potential were detected.
Result:
1. the recombinant plasmid of pAAV-EGFP-WPRE and pAAV-BDNF-WPRE was successfully constructed. The adeno-related virus (rAAV-EGFP and rAAV-BDNF) was packed and prepared. After purification, the virus titer was 3 * 109 / ml.rAAV-EGFP in the vitreous cavity for 3 weeks, the frozen section of the retina, fluorescence microscopy could be observed under the fluorescence microscope, and Western-blotting analysis showed that EGFP and BDNF protein are highly expressed in the retina tissue.
2. the recombinant AAV virus vector expressing BDNF was used for gene therapy for 3 months, 6 months and 9 months later, the total retina, central retina, and peripheral retina RGC survived, respectively. The number of RGC in the treatment group and the control group was significantly less than that of the normal retina (P0.05), but at every time point, the survival R of the AAV-BDNF therapy group. The number of GC was significantly more than that of the AAV-EGFP control group (P0.05), and the number of surviving RGC in the AAV-BDNF group no longer decreased significantly after 6 months (P0.05). After 3 months of gene therapy, the number of RGC expressing BDNF by the recombinant AAV virus vector in the AAV-BDNF treatment group was significantly more than the number of EGFP cells expressed in the AAV-EGFP control group. At the same time, the number of non transfected EGC in the treatment group was also significantly more than the control group.BDNF receptor TrkB blocking experiment, which showed that the number of ganglion cells that were not transfected in the AAV-BDNF treatment group decreased obviously, indicating that the living cells may be secreted by the BDNF secreted by the transfected cells. And get protection.
3. visual evoked potential analysis showed that the amplitude of P wave in AAV-BDNF group and AAV-EGFP control group was significantly lower than that of normal retina (P0.05), and the amplitude of P wave in V-BDNF treatment group was significantly greater than that of control group (P0.05), indicating that gene therapy not only made more RGC be protected and survived, but the surviving RGC could play its normal physiological function. Omentogram analysis: the amplitude of OPs in the 1 months normal control group was significantly different from that of the AAV-EGFP control group (P0.05). The amplitude of OPs in the normal control group was significantly different from that of the AAV-EGFP control group (P0.01). The amplitude of OPs wave in the AAV-BDNF treatment group was significantly higher than that of the AAV-EGFP control group, and the amplitude of the OPs wave was significantly greater than that of the AAV-EGFP control group (P0.05) The b wave amplitude of the electroretinogram in the.AAV-EGFP control group was significantly different from that of the normal control group at 6 months of diabetes (P0.05). The amplitude of the b wave amplitude in the normal control group was significantly different from that of the AAV-EGFP control group (P0.01) in the normal control group (P0.01), but the b wave amplitude of the AAV-BDNF treatment group was significantly greater than the AAV-EGFP control at 6 months and 9 months. Conclusion: gene therapy is helpful to the recovery of retinal function in diabetic rats.
Conclusion:
1. the constructed rAAV-BDNF and rAAV-EGFP can efficiently transfect retinal tissue and express BDNF and GFP successfully on the retina.
2. the number of RGC expressing BDNF transfected by recombinant AAV virus vector in the treatment group was significantly more than the number of cells expressing EGFP in the control group, indicating a good therapeutic effect. Meanwhile, a part of the retinal ganglion cells that were not transfected by AAV-BDNF could also be protected.
3. the BDNF gene therapy mediated by recombinant AAV virus vector in the diabetic rat model can produce good therapeutic effect on the number and work of retinal ganglion cells.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
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