AAV載體介導(dǎo)的BDNF表達(dá)對(duì)大鼠糖尿病模型中的RGC的保護(hù)作用
發(fā)布時(shí)間：2018-07-15 13:07
【摘要】：目的：構(gòu)建pAAV-EGFP及pAAV-BDNF重組腺相關(guān)病毒,測(cè)定其感染滴度。觀察AAV-BDNF對(duì)鏈脲佐菌素(STZ)造模大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞數(shù)量影響,通過檢測(cè)大鼠存活的熒光金(Fluorogold,FG)染料逆標(biāo)的RGCs進(jìn)行數(shù)量統(tǒng)計(jì)以及檢測(cè)視覺誘發(fā)電位及視網(wǎng)膜電圖來評(píng)價(jià)表達(dá)BDNF的重組AAV載體介導(dǎo)的基因治療的神經(jīng)保護(hù)效果。 方法： 1.pAAV-EGFP構(gòu)建：從pCAGGS中克隆CBA promoter,并與pSEWB同時(shí)用Apa和EcoRI進(jìn)行酶切消化,隨后進(jìn)行膠回收純化以及連接轉(zhuǎn)化,獲得pCBA-EGFP-WPRE。使用限制性內(nèi)切酶酶切和PCR方法檢測(cè)構(gòu)建質(zhì)粒的準(zhǔn)確性。使用磷酸鈣轉(zhuǎn)染方法檢測(cè)質(zhì)粒的基因表達(dá)活性。 2. pAAV-BDNF構(gòu)建：從大鼠腦中抽提總RNA,使用RT-PCR方法克隆融合有H i s-tag的大鼠BDNF基因,并將pCBA-EGFP-WPRE同時(shí)用HindIII和EcoRI進(jìn)行酶切消化,隨后膠回收純化及連接轉(zhuǎn)化,進(jìn)而獲得pCBA-BDNF-WPRE。使用限制性內(nèi)切酶酶切和PCR方法檢測(cè)構(gòu)建質(zhì)粒的準(zhǔn)確性。使用磷酸鈣轉(zhuǎn)染方法檢測(cè)質(zhì)粒的基因表達(dá)活性。Western-blotting分析視網(wǎng)膜中轉(zhuǎn)染病毒獲取的蛋白含量。 3.建立糖尿病大鼠模型,玻璃體腔注射EGFP或BDNF/His-tag融合蛋白的重組AAV病毒載體,不同時(shí)間點(diǎn)觀察存活的熒光金(Fluorogold,FG)染料逆標(biāo)的RGCs數(shù)量,并進(jìn)行視網(wǎng)膜電圖及視覺誘發(fā)電位檢測(cè)。 4. BDNF受體TrkB阻斷實(shí)驗(yàn)檢測(cè)未被轉(zhuǎn)染AAV-BDNF而存活的RGC數(shù)量,并進(jìn)行視網(wǎng)膜電圖及視覺誘發(fā)電位檢測(cè)。 結(jié)果： 1.成功構(gòu)建了pAAV-EGFP-WPRE及pAAV-BDNF-WPRE重組質(zhì)粒。包裝制備了腺相關(guān)病毒(rAAV-EGFP及rAAV-BDNF),經(jīng)純化、濃縮后病毒滴度為3.0×109／ml。rAAV-EGFP主射大鼠玻璃體腔3周后,視網(wǎng)膜冰凍切片,熒光顯微鏡下可觀察到熒光表達(dá),Western-blotting分析顯示視網(wǎng)膜組織高表達(dá)EGFP及BDNF蛋白。 2.應(yīng)用表達(dá)BDNF的重組AAV病毒載體進(jìn)行基因治療3個(gè)月、6個(gè)月、9個(gè)月之后,分別進(jìn)行了總視網(wǎng)膜、中心視網(wǎng)膜、周邊視網(wǎng)膜RGC存活數(shù)量的統(tǒng)計(jì)。治療組和對(duì)照組FG逆標(biāo)的RGC數(shù)量都明顯少于正常視網(wǎng)膜(P0.05),但在各時(shí)間點(diǎn),AAV-BDNF臺(tái)療組存活的RGC數(shù)量都明顯多于AAV-EGFP對(duì)照組(P0.05),同時(shí)AAV-BDNF治療組存活的RGC數(shù)量在6個(gè)月后就不再發(fā)生明顯減少(P0.05)：基因治療3個(gè)月后發(fā)現(xiàn),AAV-BDNF治療組中被重組AAV病毒載體轉(zhuǎn)染的表達(dá)BDNF的RGC數(shù)量明顯多于AAV-EGFP對(duì)照組中表達(dá)EGFP的細(xì)胞數(shù)量,說明了良好的治療效果。同時(shí),治療組中沒有被轉(zhuǎn)染的EGC數(shù)量也明顯多于對(duì)照組。BDNF受體TrkB阻斷實(shí)驗(yàn)表明,AAV-BDNF治療組中未被轉(zhuǎn)染的神經(jīng)節(jié)細(xì)胞的數(shù)量發(fā)生明顯下降,說明了活的細(xì)胞可能是通過吸收周圍被轉(zhuǎn)染的細(xì)胞所分泌的BDNF而獲得保護(hù)。 3.視覺誘發(fā)電位檢測(cè)分析,AAV-BDNF臺(tái)療組和AAV-EGFP對(duì)照組P波振幅值明顯小于正常視網(wǎng)膜(P0.05),同時(shí)從V-BDNF治療組P波振幅值明顯大于對(duì)照組(P0.05),說明基因治療不僅使更多的RGC被保護(hù)而存活,而且存活的RGC能夠發(fā)揮其正常生理功能。視網(wǎng)膜電圖檢測(cè)分析：1個(gè)月正常對(duì)照組OPs振幅與AAV-EGFP對(duì)照組比較就有顯著性差異(P0.05),3個(gè)月正常對(duì)照組OPs振幅與AAV-EGFP對(duì)照組比較有非常顯著性差異(P0.01)。且AAV-BDNF治療組與AAV-EGFP對(duì)照組相比,OPs波振幅值明顯大于AAV-EGFP對(duì)照組(P0.05)。AAV-EGFP對(duì)照組的視網(wǎng)膜電圖b波波幅在糖尿病6個(gè)月時(shí)與正常對(duì)照組比較有顯著性差異(P0.05),9個(gè)月正常對(duì)照組b波振幅與AAV-EGFP對(duì)照組比較有非常顯著性差異(P0.01),然而AAV-BDNF治療組在治療6個(gè)月及9個(gè)月時(shí)b波幅值明顯大于AAV-EGFP對(duì)照組。說明基因治療對(duì)糖尿病模型大鼠視網(wǎng)膜功能恢復(fù)有一定幫助。 結(jié)論： 1.構(gòu)建的rAAV-BDNF和rAAV-EGFP能高效轉(zhuǎn)染視網(wǎng)膜組織,并在視網(wǎng)膜上成功表達(dá)BDNF蛋白和綠色熒光蛋白。 2.治療組中被重組AAV病毒載體轉(zhuǎn)染的表達(dá)BDNF的RGC數(shù)量明顯多于對(duì)照組中表達(dá)EGFP的細(xì)胞數(shù)量,說明了良好的治療效果。同時(shí)也可以保護(hù)未被AAV-BDNF轉(zhuǎn)染的一部分視網(wǎng)膜神經(jīng)節(jié)細(xì)胞。 3.糖尿病大鼠模型中給予重組AAV病毒載體介導(dǎo)的BDNF基因治療,對(duì)于視網(wǎng)膜神經(jīng)節(jié)細(xì)胞數(shù)量以及功都能產(chǎn)生良好的治療效果。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R774.1
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3 馮軍基;童川;趙留濤;劉欣辛;肖傳斌;劉忠虎;;中華大蟾蜍BDNF基因的克隆與原核表達(dá)載體的構(gòu)建[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)動(dòng)物解剖學(xué)及組織胚胎學(xué)分會(huì)第十六次學(xué)術(shù)研討會(huì)論文集[C];2010年
4 江佩芳;袁天明;顧偉忠;詹燦陽(yáng);孫逸;俞惠民;;宮內(nèi)炎癥新生鼠腦白質(zhì)損傷后的神經(jīng)再生及BDNF作用機(jī)制研究[A];2011年浙江省醫(yī)學(xué)會(huì)兒科學(xué)分會(huì)學(xué)術(shù)年會(huì)暨兒內(nèi)科疾病診治新進(jìn)展國(guó)家級(jí)學(xué)習(xí)班論文匯編[C];2011年
5 曾文;朱楚洪;;BDNF通過促進(jìn)干細(xì)胞歸巢從而提高小直徑工程血管的通暢率[A];中國(guó)解剖學(xué)會(huì)2011年年會(huì)論文文摘匯編[C];2011年
6 周利君;鐘yN;任文杰;劉先國(guó);;BDNF對(duì)脊髓背角C-纖維誘發(fā)電位的影響及其機(jī)制的探討[A];中國(guó)生理學(xué)會(huì)張錫鈞基金會(huì)第十屆全國(guó)青年優(yōu)秀生理學(xué)學(xué)術(shù)論文綜合摘要[C];2009年
7 呂慎金;楊燕;;梅花鹿BDNF基因與行為性狀的相關(guān)分析[A];第十二次全國(guó)畜禽遺傳標(biāo)記研討會(huì)論文集[C];2010年
8 王迎新;張向榮;張志s，
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