慢病毒介导PTL基因干预的星形胶质细胞在帕金森病模型中的保护作用研究

发布时间：2018-05-03 20:43

本文选题：胰甘油三酯脂酶 + 慢病毒载体　；参考：《苏州大学》2014年博士论文

【摘要】：第一部分大鼠PTL基因RNA干扰和过表达重组慢病毒载体的构建目的：构建胰甘油三酯脂酶(pancreatic triglyceride lipase, PTL)基因RNA干扰慢病毒载体和胰甘油三酯脂酶基因慢病毒过表达载体及鉴定，并进行包装和生产高滴度高纯度的慢病毒颗粒。方法：针对PTL基因序列，合成4对靶向大鼠PTL基因特异性干扰序列，其两端含酶切位点粘端，直接连入酶切后的RNA干扰慢病毒载体pLenO-THM上。将连接好的产物转入制备好的细菌感受态细胞，对长出的克隆进行PCR鉴定和测序比对后，鉴定阳性的克隆即为构建成功的重组慢病毒干扰质粒。以钙转法将重组质粒与慢病毒包装质粒共转染293T细胞，包装生产慢病毒颗粒，并依据绿色荧光蛋白(GFP)表达水平检测病毒滴度；从含有PTL基因的质粒克隆模板PCR扩增后，将PTL基因与pLenO-DCE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PTL慢病毒过表达载体，将其转染293T细胞，培养48h后，荧光显微镜观察融合蛋白GFP/PTL的表达。同时收集细胞培养上清液，将其浓缩后在293T细胞中测定病毒滴度。结果：成功构建PTL基因RNA干扰慢病毒载体和PTL基因慢病毒过表达载体，PCR和DNA测序证实阳性克隆序列正确，转染293T细胞后镜下可见荧光强度强烈，并且能被高效转染，这样能确保病毒的稳定表达，表明病毒包装成功。病毒的滴度大约在2×108TU/ml。结论：成功制备PTL的RNA干扰和过表达重组慢病毒载体，为后续转染两型星形胶质细胞以获得高效稳定的基因干预效果提供可靠技术，为更深入的研究PTL基因干预后对星形胶质细胞及神经元细胞生物学特性的影响奠定坚实基础。第二部分PTL基因干预的星形胶质细胞生物学特性研究目的：将已成功构建的PTL基因RNA干扰慢病毒和过表达PTL基因慢病毒颗粒分别转染1型星形胶质细胞(type1astrocytes, T1As)和2型星形胶质细胞(type2astrocytes,, T2As)，观察其转染能力和报告基因的表达水平，分析这种干预对星形胶质细胞生物学特性改变的影响，将PTL基因干预的星形胶质细胞与原代神经元共培养，观察这种干预对神经元细胞生物学特性的影响。方法：将已成功构建的PTL基因RNA干扰慢病毒和过表达PTL基因慢病毒颗粒分别转染星形胶质细胞，镜下观察上述感染后的星形胶质细胞GFP的表达、转导效率，荧光定量PCR和Western blot分别检测PTL mRNA、蛋白的表达情况，MTT法检测基因干预后星形胶质细胞的存活率，将PTL基因干预的两型星形胶质细胞分别与原代大脑皮层神经元共培养，观察共培养后神经元的形态学变化，免疫荧光检测各组神经元轴突生长相关蛋白[神经丝蛋白(NF-200)、Ⅲ型β微管蛋白(β-Ⅲtubulin)和生长相关蛋白-43(GAP43)]的表达，Western blot检测星形胶质细胞及神经元中胶质源性神经营养因子[胶质细胞源性神经营养因子(GDNF)、中脑星形胶质细胞源性神经营养因子(MANF)]的表达水平。结果：(1)转染PTL RNA干扰病毒的两型星形胶质细胞中PTL表达明显降低，干扰效率达90%以上；而转染PTL过表达病毒的T1As中PTL表达水平显著升高。PTL过表达的星形胶质细胞(尤其是T1As)增殖能力显著提高。(2)与PTL过表达的T1As共培养的原代神经元生长迁移能力明显增强，形成较规则的细胞索带，免疫荧光检测显示形成的细胞索带中神经元标记物(NF-200, β-Ⅲtubulin和GAP-43)阳性率高，并在该组胶质细胞和处理的神经元中均发现较高的MANF表达。结论：星形胶质细胞和维持中枢神经脂质代谢稳定密切相关，PTL过表达的T1As可能通过分泌某些神经营养因子促进神经元存活和增殖迁移。第三部分星形胶质细胞PTL基因过表达对PD细胞模型的保护作用研究目的：探讨慢病毒介导PTL基因过表达的星形胶质细胞对6-羟基多巴胺(6-OHDA)诱导的PC12细胞帕金森病模型的作用及其分子机制。方法：采用PC12细胞，以6-OHDA制作帕金森病细胞模型。分6组：细胞模型组、正常对照组、LV-PTL-T1As组、LV-GFP-T1As组、LV-PTL-T2As组、LV-GFP-T2As组。细胞模型组采用6-OHDA处理PC12细胞24h，正常对照组给予PC12细胞DMEM+10%胎牛血清培养，余下四组先将转染PTL基因过表达(LV-pLenO-DCE-PTL)和LV-pLenO-DCE-GFP (空质粒载体)的两型星形胶质细胞(LV-PTL-T1As, LV-GFP-T1As, LV-PTL-T2As, LV-GFP-T2As)分别与PC12细胞共培养24h，然后再换最佳作用浓度(100μM)6-OHDA处理24h。上述各组细胞处理后，采用MTT法检测细胞存活率，光镜观察各组PC12细胞生长形态学变化，Western blot检测细胞凋亡(Caspase-3、Bax、Bcl-2)、自噬(LC3Ⅱ)、内质网应激关键分子GRP78及促炎症因子NF-κΒ p65表达，比色法进行谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)的活性测定，分析PTL基因过表达的星形胶质细胞对帕金森病细胞模型的影响；最后，通过Western blot检测上述6组PC12细胞中Akt、p-Akt、GSK-3β、p-GSK-3β的蛋白表达；在细胞模型组、LV-PTL-T1As组及LV-PTL-T2As组，先用Akt信号通路抑制剂LY294002处理1h，再用6-OHDA处理24h，以探讨Akt/GSK-3β信号通路在PTL基因过表达的星形胶质细胞对6-OHDA致PC12细胞损伤保护中的作用。结果:(1)成功构建6-OHDA诱导的PC12细胞PD模型，应用MTT法检测发现，在一定浓度范围内(25μM-200μM)，随6-OHDA浓度增高细胞活性逐步下降；在同一浓度，作用时间越长，细胞活性下降越明显，存在明显时间和剂量依赖性，其中100μM6-OHDA是建立PD细胞模型的最佳作用浓度，最适作用时间是24h (细胞存活率为58.7±10.3%)。(2)100μM6-OHDA处理24h后可以诱导PC12细胞出现明显凋亡的形态学变化，而PTL基因过表达的星形胶质细胞可明显延缓PC12细胞损伤，其中LV-PTL-T1As组细胞损伤程度明显减轻，可见PC12细胞PD模型有比较完整的突起结构。(3)6-OHDA使PC12细胞PD模型的Bax/Bcl-2比率、Caspase-3和LC3Ⅱ的表达增加；PTL过表达的星形胶质细胞可使6-OHDA处理的PC12细胞存活率增加，其中LV-PTL-T1As组PC12细胞存活率较其余处理组高；PTL基因过表达的星形胶质细胞(尤其是LV-PTL-T1As组)使6-OHDA处理的PC12细胞凋亡和自噬相关指标均明显下调。(4)通过与转染PTL基因过表达的星形胶质细胞(尤其是LV-PTL-T1As组)共培养，6-OHDA处理引起的PC12细胞中上调的GRP78和NF-κΒ p65水平明显下降。(5)细胞模型组中SOD、GSH-Px水平较正常对照组有显著下降。PTL基因过表达的星形胶质细胞使6-OHDA处理的PC12细胞SOD、GSH-Px水平显著升高。(6) PTL基因过表达的星形胶质细胞(尤其是LV-PTL-T1As组)使PC12细胞PD模型p-Akt和p-GSK-3β表达增加；LY294002拮抗PTL基因过表达的星形胶质细胞对6-OHDA引起的PC12细胞损伤的保护作用。结论：过表达PTL基因的星形胶质细胞对6-OHDA作用的PC12细胞具有保护作用，，这些保护作用可能是通过Akt/GSK-3β信号通路实现的，这为PD发病机制的研究提供了新的重要线索，可能成为PD治疗的新靶向。
[Abstract]:Part one: Construction of RNA interference and over expression recombinant lentiviral vector of rat PTL gene
Objective: to construct the pancreatic triglyceride lipase (PTL) gene RNA to interfere with lentivirus vector and to identify the lentivirus overexpression vector of the lentivirus gene and the triglyceride lipase gene, and to package and produce lentivirus particles with high titer and high purity.
Methods: according to the PTL gene sequence, the PTL gene specific interference sequence of 4 pairs of target rats was synthesized. The two ends of the target rat were adhered to the enzyme cut site and directly connected to the RNA interfering lentivirus vector pLenO-THM. The linked products were transferred into the prepared bacterial receptive cells, and the PCR identification and sequence alignment of the long clones were compared, Jian Dingyang Sex cloning was a successful recombinant lentivirus interference plasmid. The recombinant plasmid and lentivirus package plasmid were co transfected to 293T cells by calcium transfer method and packaged to produce lentivirus particles, and the virus titer was detected according to the expression level of green fluorescent protein (GFP). The PTL gene and pLenO-D were amplified from the PTL gene of the plasmid cloned template containing PTL gene. The CE carrier was double enzyme cut. After purification of the enzyme cut product, the products were connected or reorganized, and the product transformed the bacterial receptive cells. PCR identification was carried out for the long clones. The PTL lentivirus overexpression vector was successfully constructed and transfected to 293T cells. After 48h, the fusion protein GFP/PTL was observed by the fluorescence microscope. Meanwhile, the supernatant of cell culture was collected and concentrated. The titer of virus was detected in 293T cells.
Results: the PTL gene RNA interfered with the lentivirus vector and the PTL gene lentivirus overexpression vector. PCR and DNA sequencing confirmed that the positive clones were correct. The fluorescence intensity of the transfected 293T cells was strong and the transfection could be transfected efficiently. This could ensure the stability of the virus, which showed that the virus package was successful. The virus titer was about 2 X 108TU/ml.
Conclusion: the successful preparation of PTL RNA interference and overexpression of recombinant lentivirus vector provides a reliable technique for the subsequent transfection of type two astrocytes in order to achieve efficient and stable gene intervention. It lays a solid foundation for the further study of the effect of PTL gene on the biological properties of astrocytes and neuron cells.
The second part is about the biological characteristics of astrocytes intervened by PTL gene.
Objective: the successfully constructed PTL gene RNA interferes with lentivirus and overexpressed PTL gene lentivirus particles in type 1 astrocytes (type1astrocytes, T1As) and type 2 astrocytes (type2astrocytes, T2As), to observe the transfection ability and the expression level of the reporter gene, and analyze the intervention to astrocyte organisms. The effects of the changes in the characteristics of the PTL gene were co cultured with the astrocytes and the primary neurons, and the effects of this intervention on the biological characteristics of neurons were observed.
Methods: the successfully constructed PTL gene RNA interfered with lentivirus and the overexpressed PTL gene lentivirus particles were transfected into astrocytes respectively. The expression of GFP in the astrocytes after the infection was observed under the microscope, the transduction efficiency, the fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression of PTL, and the MTT method was used to determine the prognosis of the gene. The survival rate of astrocytes was co cultured with the primary cerebral cortex neurons of type two astrocytes intervened by PTL gene respectively. The morphological changes of neurons were observed after co culture. Immunofluorescent detection of neurofilament protein (NF-200), type III beta microtubule (beta III tubulin) and growth phase of neuron axon growth in each group The expression of -43 (GAP43)] and the expression level of glial derived neurotrophic factor [glial cell derived neurotrophic factor (GDNF) and astroglial derived neurotrophic factor (MANF)] in astrocytes and neurons were detected by Western blot.
Results: (1) the expression of PTL in type two astrocytes transfected with PTL RNA interfering virus was significantly reduced, and the interference efficiency was more than 90%, while the PTL expression level in T1As transfected with PTL overexpressed virus significantly increased the proliferation ability of astrocytes (especially T1As) with.PTL overexpression. (2) co culture of T1As with PTL overexpressed T1As The ability of neuronal growth and migration was significantly enhanced, and a more regular cell cord was formed. Immunofluorescence detection showed that the positive rate of neuronal markers (NF-200, beta - III tubulin and GAP-43) in the cell cord of the cells was high, and a higher expression of MANF was found in the glial cells and the treated neurons.
Conclusion: astrocytes are closely related to the maintenance of lipid metabolism in the central nervous system. The overexpressed T1As in PTL may promote the survival and proliferation of neurons by secreting some neurotrophic factors.
The third part is the protective effect of PTL gene overexpression on PD cell model.
Objective: To investigate the role and molecular mechanism of the PTL gene overexpressed by the lentivirus mediated astrocytes in the 6- hydroxydopamine (6-OHDA) induced PC12 cell Parkinson's disease model.
Methods: using PC12 cells, the Parkinson disease cell model was made by 6-OHDA. The cell model group, the normal control group, the normal control group, the LV-PTL-T1As group, the LV-GFP-T1As group, the LV-PTL-T2As group, the LV-GFP-T2As group. The cell model group was treated with 6-OHDA to treat PC12 cells 24h, the normal control group was given PC12 cell DMEM+10% fetal bovine serum culture, the remaining four groups first transfected PT. L gene overexpression (LV-pLenO-DCE-PTL) and LV-pLenO-DCE-GFP (empty plasmid vector) type two astrocytes (LV-PTL-T1As, LV-GFP-T1As, LV-PTL-T2As, LV-GFP-T2As) co culture 24h with PC12 cells respectively, and then change the optimal concentration (100 mu M) 6-OHDA at the 6-OHDA 24h. cells. The morphological changes of PC12 cells were observed by light microscopy. Western blot was used to detect apoptosis (Caspase-3, Bax, Bcl-2), autophagy (LC3 II), the key molecule GRP78 of endoplasmic reticulum stress and the expression of NF- kappa p65, and the activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) was measured by colorimetric method. The effects of astrocytes on the Parkinson's disease cell model were obtained. Finally, the protein expression of Akt, p-Akt, GSK-3 beta and p-GSK-3 beta in the 6 groups of PC12 cells was detected by Western blot. In the cell model group, LV-PTL-T1As group and LV-PTL-T2As group, 1H was first treated with Akt signaling inhibitor LY294002. The role of 3 beta signaling pathway in the protection of PTL gene overexpression astrocytes against 6-OHDA induced PC12 cell injury.
Results: (1) the PD model of PC12 cells induced by 6-OHDA was successfully constructed. The MTT method was used to detect the cell activity in a certain concentration range (25 mu M-200 mu M), and the cell activity decreased gradually with the increase of 6-OHDA concentration; the longer the time the action time was, the more obvious the cell activity decreased, and there was a significant time and dose dependence, of which 100 mu M6-OHDA was the establishment of PD. The optimal action concentration of the cell model was 24h (58.7 + 10.3%). (2) 100 mu M6-OHDA could induce morphological changes of apoptosis in PC12 cells, while the PTL gene overexpressed astrocytes could significantly delay the PC12 cell damage, of which the degree of cell injury in the LV-PTL-T1As group was significantly reduced. The PD model of PC12 cells showed a relatively complete protuberance structure. (3) 6-OHDA increased the Bax/Bcl-2 ratio of PD model in PC12 cells, and the expression of Caspase-3 and LC3 II increased; PTL overexpressed astrocytes could increase the survival rate of PC12 cells treated by 6-OHDA, and the survival rate of LV-PTL-T1As group cells was higher than that of the rest treatment group. The expression of astrocytes (especially in LV-PTL-T1As group) reduced the apoptosis and autophagy related indexes of 6-OHDA treated PC12 cells. (4) the level of GRP78 and NF- kappa p65 in PC12 cells induced by 6-OHDA treatment was significantly decreased by co culture with astrocytes transfected with PTL gene over expression of astrocytes (especially LV-PTL-T1As). (5) The level of SOD and GSH-Px in the cell model group was significantly lower than that in the normal control group. The.PTL gene overexpressed astrocytes made the 6-OHDA treated PC12 cells SOD, the GSH-Px level increased significantly. (6) the PTL gene overexpressed astrocytes (especially the LV-PTL-T1As group) increased the PC12 PD model p-Akt and the expression of the beta. The protective effect of PTL gene overexpression astrocytes on 6-OHDA induced PC12 cell injury.
Conclusion: the astrocytes overexpressing the PTL gene have protective effects on the PC12 cells acting on 6-OHDA. These protective effects may be achieved through the Akt/GSK-3 beta signaling pathway. This provides a new important clue for the study of the pathogenesis of PD and may be a new target for the treatment of PD.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.5

【参考文献】