慢病毒介导过表达Nurr1基因的神经干细胞移植治疗帕金森病大鼠模型

发布时间：2018-02-27 18:41

本文关键词： 慢病毒 Nurr1 神经干细胞移植帕金森病　出处：《昆明医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]在体外研究Nurr1基因能够促进神经干细胞向多巴胺能神经元分化的基础上,采用慢病毒介导,建立过表达Nurr1的神经干细胞,进一步研究过表达Nurr1的神经干细胞移植治疗帕金森病大鼠模型的疗效,初步探讨其作用机制,为神经干细胞移植治疗帕金森病探索新途径。[方法]1、使用PCR技术扩增目的基因Nurr1后,将其与pLenO-DCE慢病毒载体分别进行双酶切。纯化酶切产物后进行定向连接或重组,其产物转化细菌感受态细胞,对长出的克隆进行PCR鉴定、测序和分析比对,得到构建成功的Nurr1慢病毒过表达载体,并转染293T细胞,收集细胞培养上清液,将其浓缩后测定病毒滴度。2、采用无血清培养技术从孕14天的胎鼠中脑组织分离培养神经干细胞,每天观察细胞生长情况,5-7d后传代培养。用Nestin免疫荧光检测验证其干细胞特性,并用特异性标记物β-Ⅲ-Tubulin、TH、GFAP鉴定其多分化潜能。3、通过慢病毒感染NSC细胞预实验,确定感染复数M0I,携带Nurr1的慢病毒以MOI=20感染NSC后,通过荧光观察、PCR及和Wtetern blot检测分析感染后Nurr1的表达情况。4、SD大鼠经6-OHDA定点毁损DA能神经元纤维投射的前脑内侧束、腹侧被盖区和尾状核后,阿朴吗啡诱导旋转试验计数30min内的平均旋转速度,检测PD大鼠模型制作情况。5、将成功PD大鼠模型随机分为3组进行细胞移植:假手术对照组移植等体积PBS替代NSC;NSC组移植转染慢病毒空载体的NSC;Nurr1组移植转染Nurr1慢病毒载体的NSC。分别在移植治疗后2周、5周、8周用阿朴吗啡腹腔注射诱发旋转实验观察移植后大鼠行为的改善情况。并分别于上述时间点以Western blot技术检测脑内TH、DAT蛋白的表达变化;于8W灌注取脑,以免疫组织荧光技术观察脑组织形态及TH表达情况;分析比较治疗效果及其作用机理。[结果]1、本实验成功构建了过表达Nurr1基因的慢病毒载体pLenO-DCE-Nurr1。2、胎鼠中脑细胞悬液原代培养7d左右可形成数百个悬浮生长直径约200 μm的神经球,经Nestin免疫荧光检测呈阳性。经诱导分化培养后检测到β-Ⅲ-Tubulin、TH、GFAP阳性细胞,表明神经干细胞有向多巴胺能神经元、星型胶质细胞等多向分化能力:3、将过表达Nurr1的慢病毒颗粒感染原代培养神经干细胞后48h后可见绿色荧光蛋白表达,感染5d后荧光表达最强。其中阴性对照组、Nurr1组可见绿色荧光,且每一视野下荧光细胞数相似,说明导入的目的基因不影响慢病毒重组质粒的感染效率;用RT-PCR和Western blot的方法能检测出Nurr1在神经干细胞中转录和表达。4、SD大鼠立体定向注射6-OHDA术后,阿朴吗啡诱导试验检测发现大鼠出现无法控制的异常向毁损对侧连续旋转行为。发现第2周始诱导出现旋转的大鼠数量明显增加,旋转速度较前增快。4周后增加趋势减慢;至术后6周,诱导出现旋转的大鼠数量及旋转速度相对稳定。最终有43只大鼠旋转行为稳定并7rpm/min,可认定为成功的PD模型,成模率58.1%。5、细胞移植术后各实验组大鼠经阿朴吗啡诱导的旋转行为学观察表明:注射PBS的对照组移植后PD大鼠的旋转情况没有明显改善;与对照组比较,单纯NSC移植组大鼠异常旋转圈数从第2周开始下降,异常旋转行为在一定程度上有所改善;移植2周后,Nurr1移植组大鼠的旋转行为较NSC移植组明显改善,随时间进行性好转,但至8周仍不能达到正常水平。6、各实验组移植后移植区TH免疫荧光检测表明:过表达Nurr1的神经干细胞移植组较单纯NSC移植组有更多的移植细胞存活,且移植细胞分化率更高,在体内GFP荧光表达时间不低于8周;神经干细胞移植可促进内源性TH阳性细胞表达。7、各实验组移植后Western blot检测表明:Nurr1移植组动物脑内TH、DAT蛋白表达量显著性高于NSC移植治疗组,随时间推移进行性增多。此两种蛋白的表达水平同动物的行为学改善情况呈平行关系。[结论]本研究成功构建了携带Nurr1基因的慢病毒载体,过表达Nurr1的NSC脑内移植治疗PD模型大鼠后,在一定时间内能显著提高NSC移植治疗的效果。Nurr1可作为细胞移植治疗PD的重要基因靶点,为今后临床治疗提供了实验依据。
[Abstract]:[Objective] to promote neural stem cells into dopaminergic neuron differentiation based on Nurr1 gene in vitro, using lentivirus mediated overexpression of Nurr1, the establishment of neural stem cells, further research on the curative effect of Nurr1 expression in rat model of neural stem cell transplantation for the treatment of Parkinson's disease and to explore its mechanism for nerve stem cell transplantation for the treatment of Parkinson's disease and explore new ways. Methods]1, Nurr1 amplification of the target gene using PCR technology, the pLenO-DCE lentiviral vectors were digested. The purified enzyme products after the connection or restructuring orientation, its products were transformed into bacterial competent cells, PCR identification of clones werescreened, sequencing analysis and comparison, obtained successfully constructed the Lentivirus Expression Vector of Nurr1 and transfected into 293T cells. The cell supernatant was collected, the concentration of the virus titer was determined by.2, serum free culture Technique of isolation and culture of neural stem cells from embryonic day 14 of fetal rat brain tissue, cell growth was observed every day, 5-7d after subculturing. Verify the characteristics of stem cells with Nestin immunofluorescence, and specific markers of beta III -Tubulin, TH, GFAP identified multipotent.3 by lentivirus infection NSC cells pre experiment, determine the multiplicity of infection M0I, lentivirus carrying Nurr1 to MOI=20 after NSC infection, were observed by fluorescence, and PCR and Wtetern blot to detect expression of.4 Nurr1 after infection, SD rats were designated 6-OHDA DA neurons damaged fiber projection of the medial forebrain bundle, ventral tegmental area and caudate nucleus after apomorphine induced rotation test average rotation speed counting 30min, detection of PD rat model made of.5, the successful model of PD rats were randomly divided into 3 groups: sham control group cell transplantation Transplantation volume PBS instead of NSC in NSC group; Transplantation of lentiviral vector NSC; Nurr1 group of transplantation of Nurr1 transfected with lentiviral vector NSC. respectively in transplantation after 2 weeks, 5 weeks, 8 weeks were observed by intraperitoneal injection of apomorphine induced rotation test to improve the behavior of rats after transplantation. And at the same time point to detect brain Western blot technology in TH. The change of the expression of DAT in 8W; brain was removed by immunohistochemistry technique to observe the morphology of brain tissue and the expression of TH; analysis and comparison of treatment effect and its mechanism of action. The results of]1, we successfully constructed lentivirus expressing vector of pLenO-DCE-Nurr1.2 Nurr1 gene, cells in the fetal rat suspension cultured about 7d the formation of hundreds of suspended growth diameter of about 200 m neurospheres by Nestin immunofluorescence was positive. The differentiation was detected after beta III -Tubulin, TH, GFAP positive cells showed that neural stem cells To dopaminergic neurons, differentiation of astrocytes: 3, the overexpression of lentivirus infected Nurr1 in primary cultured neural stem cells after 48h showed the expression of green fluorescent protein, the expression of the strongest fluorescence after 5D infection. The negative control group, Nurr1 group showed green fluorescence, and the fluorescence of each cell from the perspective of the number of similar, the infection does not affect the efficiency of the target gene into the recombinant lentiviral plasmid with RT-PCR and Western; blot can detect the expression of.4 Nurr1 in neural stem cells and transcription, SD rats with stereotactic injection of 6-OHDA after induced by apomorphine test found that rats cannot control the abnormal to on the side of damaged continuous rotation behavior. Found from the second week number of rats induced by rotation of the rotating speed is increased faster after.4 weeks after the increase trend slowed down; 6 weeks after surgery, induced by rotating the The number of rats and the rotation speed is relatively stable. Finally the rotational behavior of 43 rats and 7rpm/min, can be identified as PD model, 58.1%.5 model, cell transplantation of rats in each experimental group by rotational behavior induced by apomorphine. The results indicated: the control group was injected with PBS aftertransplantation rotation PD rats did not improve significantly; compared with the control group, simple NSC transplantation rats abnormal rotation number decreased from the beginning of the second week, the abnormal rotational behavior was improved to a certain extent; 2 weeks after transplantation, the rotational behavior of Nurr1 rats of transplantation group than NSC transplantation group were significantly improved, but improved with time. To 8 weeks still cannot reach the normal level of each experimental group.6, transplanted TH immunofluorescence assay showed that the overexpression of Nurr1 in neural stem cells transplantation group compared with NSC transplantation group had more survival of transplanted cells, and differentiation of transplanted cells was higher That time is not less than 8 weeks the expression of GFP in vivo fluorescence; neural stem cell transplantation can promote the endogenous expression of TH positive cells in the experimental group.7, Western after transplantation blot detection showed that Nurr1 transplantation group animal brain TH, DAT protein expression was significantly higher than that in the NSC transplantation group, increase of expression level over time. The two kinds of protein with the animal behavior to improve the situation is parallel. Conclusion: This study successfully constructed lentiviral vector carrying Nurr1 gene, overexpression of Nurr1 NSC in brain transplantation treatment of PD rats, in a certain period of time to significantly improve the.Nurr1 effect of NSC transplantation can be used as an important target gene cell transplantation for the treatment of PD, and provide experimental basis for future clinical treatment.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5;R-332

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