本文選題：胰甘油三酯脂酶 + 慢病毒載體�。� 參考：《蘇州大學(xué)》2014年博士論文

【摘要】：第一部分大鼠PTL基因RNA干擾和過表達重組慢病毒載體的構(gòu)建 目的：構(gòu)建胰甘油三酯脂酶(pancreatic triglyceride lipase, PTL)基因RNA干擾慢病毒載體和胰甘油三酯脂酶基因慢病毒過表達載體及鑒定，并進行包裝和生產(chǎn)高滴度高純度的慢病毒顆粒。 方法：針對PTL基因序列，合成4對靶向大鼠PTL基因特異性干擾序列，其兩端含酶切位點粘端，直接連入酶切后的RNA干擾慢病毒載體pLenO-THM上。將連接好的產(chǎn)物轉(zhuǎn)入制備好的細(xì)菌感受態(tài)細(xì)胞，對長出的克隆進行PCR鑒定和測序比對后，鑒定陽性的克隆即為構(gòu)建成功的重組慢病毒干擾質(zhì)粒。以鈣轉(zhuǎn)法將重組質(zhì)粒與慢病毒包裝質(zhì)粒共轉(zhuǎn)染293T細(xì)胞，包裝生產(chǎn)慢病毒顆粒，并依據(jù)綠色熒光蛋白(GFP)表達水平檢測病毒滴度；從含有PTL基因的質(zhì)�？寺∧０錚CR擴增后，將PTL基因與pLenO-DCE載體分別進行雙酶切。純化酶切產(chǎn)物后進行定向連接或重組，其產(chǎn)物轉(zhuǎn)化細(xì)菌感受態(tài)細(xì)胞，對長出的克隆進行PCR鑒定、測序和分析比對，得到構(gòu)建成功的PTL慢病毒過表達載體，將其轉(zhuǎn)染293T細(xì)胞，培養(yǎng)48h后，熒光顯微鏡觀察融合蛋白GFP/PTL的表達。同時收集細(xì)胞培養(yǎng)上清液，將其濃縮后在293T細(xì)胞中測定病毒滴度。 結(jié)果：成功構(gòu)建PTL基因RNA干擾慢病毒載體和PTL基因慢病毒過表達載體，PCR和DNA測序證實陽性克隆序列正確，轉(zhuǎn)染293T細(xì)胞后鏡下可見熒光強度強烈，并且能被高效轉(zhuǎn)染，這樣能確保病毒的穩(wěn)定表達，表明病毒包裝成功。病毒的滴度大約在2×108TU/ml。 結(jié)論：成功制備PTL的RNA干擾和過表達重組慢病毒載體，為后續(xù)轉(zhuǎn)染兩型星形膠質(zhì)細(xì)胞以獲得高效穩(wěn)定的基因干預(yù)效果提供可靠技術(shù)，為更深入的研究PTL基因干預(yù)后對星形膠質(zhì)細(xì)胞及神經(jīng)元細(xì)胞生物學(xué)特性的影響奠定堅實基礎(chǔ)。 第二部分PTL基因干預(yù)的星形膠質(zhì)細(xì)胞生物學(xué)特性研究 目的：將已成功構(gòu)建的PTL基因RNA干擾慢病毒和過表達PTL基因慢病毒顆粒分別轉(zhuǎn)染1型星形膠質(zhì)細(xì)胞(type1astrocytes, T1As)和2型星形膠質(zhì)細(xì)胞(type2astrocytes,, T2As)，觀察其轉(zhuǎn)染能力和報告基因的表達水平，分析這種干預(yù)對星形膠質(zhì)細(xì)胞生物學(xué)特性改變的影響，將PTL基因干預(yù)的星形膠質(zhì)細(xì)胞與原代神經(jīng)元共培養(yǎng)，觀察這種干預(yù)對神經(jīng)元細(xì)胞生物學(xué)特性的影響。 方法：將已成功構(gòu)建的PTL基因RNA干擾慢病毒和過表達PTL基因慢病毒顆粒分別轉(zhuǎn)染星形膠質(zhì)細(xì)胞，鏡下觀察上述感染后的星形膠質(zhì)細(xì)胞GFP的表達、轉(zhuǎn)導(dǎo)效率，熒光定量PCR和Western blot分別檢測PTL mRNA、蛋白的表達情況，MTT法檢測基因干預(yù)后星形膠質(zhì)細(xì)胞的存活率，將PTL基因干預(yù)的兩型星形膠質(zhì)細(xì)胞分別與原代大腦皮層神經(jīng)元共培養(yǎng)，觀察共培養(yǎng)后神經(jīng)元的形態(tài)學(xué)變化，免疫熒光檢測各組神經(jīng)元軸突生長相關(guān)蛋白[神經(jīng)絲蛋白(NF-200)、Ⅲ型β微管蛋白(β-Ⅲtubulin)和生長相關(guān)蛋白-43(GAP43)]的表達，Western blot檢測星形膠質(zhì)細(xì)胞及神經(jīng)元中膠質(zhì)源性神經(jīng)營養(yǎng)因子[膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子(GDNF)、中腦星形膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子(MANF)]的表達水平。 結(jié)果：(1)轉(zhuǎn)染PTL RNA干擾病毒的兩型星形膠質(zhì)細(xì)胞中PTL表達明顯降低，干擾效率達90%以上；而轉(zhuǎn)染PTL過表達病毒的T1As中PTL表達水平顯著升高。PTL過表達的星形膠質(zhì)細(xì)胞(尤其是T1As)增殖能力顯著提高。(2)與PTL過表達的T1As共培養(yǎng)的原代神經(jīng)元生長遷移能力明顯增強，形成較規(guī)則的細(xì)胞索帶，免疫熒光檢測顯示形成的細(xì)胞索帶中神經(jīng)元標(biāo)記物(NF-200, β-Ⅲtubulin和GAP-43)陽性率高，并在該組膠質(zhì)細(xì)胞和處理的神經(jīng)元中均發(fā)現(xiàn)較高的MANF表達。 結(jié)論：星形膠質(zhì)細(xì)胞和維持中樞神經(jīng)脂質(zhì)代謝穩(wěn)定密切相關(guān)，PTL過表達的T1As可能通過分泌某些神經(jīng)營養(yǎng)因子促進神經(jīng)元存活和增殖遷移。 第三部分星形膠質(zhì)細(xì)胞PTL基因過表達對PD細(xì)胞模型的保護作用研究 目的：探討慢病毒介導(dǎo)PTL基因過表達的星形膠質(zhì)細(xì)胞對6-羥基多巴胺(6-OHDA)誘導(dǎo)的PC12細(xì)胞帕金森病模型的作用及其分子機制。 方法：采用PC12細(xì)胞，以6-OHDA制作帕金森病細(xì)胞模型。分6組：細(xì)胞模型組、正常對照組、LV-PTL-T1As組、LV-GFP-T1As組、LV-PTL-T2As組、LV-GFP-T2As組。細(xì)胞模型組采用6-OHDA處理PC12細(xì)胞24h，正常對照組給予PC12細(xì)胞DMEM+10%胎牛血清培養(yǎng)，余下四組先將轉(zhuǎn)染PTL基因過表達(LV-pLenO-DCE-PTL)和LV-pLenO-DCE-GFP (空質(zhì)粒載體)的兩型星形膠質(zhì)細(xì)胞(LV-PTL-T1As, LV-GFP-T1As, LV-PTL-T2As, LV-GFP-T2As)分別與PC12細(xì)胞共培養(yǎng)24h，然后再換最佳作用濃度(100μM)6-OHDA處理24h。上述各組細(xì)胞處理后，采用MTT法檢測細(xì)胞存活率，光鏡觀察各組PC12細(xì)胞生長形態(tài)學(xué)變化，Western blot檢測細(xì)胞凋亡(Caspase-3、Bax、Bcl-2)、自噬(LC3Ⅱ)、內(nèi)質(zhì)網(wǎng)應(yīng)激關(guān)鍵分子GRP78及促炎癥因子NF-κΒ p65表達，比色法進行谷胱甘肽過氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)的活性測定，分析PTL基因過表達的星形膠質(zhì)細(xì)胞對帕金森病細(xì)胞模型的影響；最后，通過Western blot檢測上述6組PC12細(xì)胞中Akt、p-Akt、GSK-3β、p-GSK-3β的蛋白表達；在細(xì)胞模型組、LV-PTL-T1As組及LV-PTL-T2As組，先用Akt信號通路抑制劑LY294002處理1h，再用6-OHDA處理24h，以探討Akt/GSK-3β信號通路在PTL基因過表達的星形膠質(zhì)細(xì)胞對6-OHDA致PC12細(xì)胞損傷保護中的作用。 結(jié)果:(1)成功構(gòu)建6-OHDA誘導(dǎo)的PC12細(xì)胞PD模型，應(yīng)用MTT法檢測發(fā)現(xiàn)，在一定濃度范圍內(nèi)(25μM-200μM)，隨6-OHDA濃度增高細(xì)胞活性逐步下降；在同一濃度，作用時間越長，細(xì)胞活性下降越明顯，存在明顯時間和劑量依賴性，其中100μM6-OHDA是建立PD細(xì)胞模型的最佳作用濃度，最適作用時間是24h (細(xì)胞存活率為58.7±10.3%)。(2)100μM6-OHDA處理24h后可以誘導(dǎo)PC12細(xì)胞出現(xiàn)明顯凋亡的形態(tài)學(xué)變化，而PTL基因過表達的星形膠質(zhì)細(xì)胞可明顯延緩PC12細(xì)胞損傷，其中LV-PTL-T1As組細(xì)胞損傷程度明顯減輕，可見PC12細(xì)胞PD模型有比較完整的突起結(jié)構(gòu)。(3)6-OHDA使PC12細(xì)胞PD模型的Bax/Bcl-2比率、Caspase-3和LC3Ⅱ的表達增加；PTL過表達的星形膠質(zhì)細(xì)胞可使6-OHDA處理的PC12細(xì)胞存活率增加，其中LV-PTL-T1As組PC12細(xì)胞存活率較其余處理組高；PTL基因過表達的星形膠質(zhì)細(xì)胞(尤其是LV-PTL-T1As組)使6-OHDA處理的PC12細(xì)胞凋亡和自噬相關(guān)指標(biāo)均明顯下調(diào)。(4)通過與轉(zhuǎn)染PTL基因過表達的星形膠質(zhì)細(xì)胞(尤其是LV-PTL-T1As組)共培養(yǎng)，6-OHDA處理引起的PC12細(xì)胞中上調(diào)的GRP78和NF-κΒ p65水平明顯下降。(5)細(xì)胞模型組中SOD、GSH-Px水平較正常對照組有顯著下降。PTL基因過表達的星形膠質(zhì)細(xì)胞使6-OHDA處理的PC12細(xì)胞SOD、GSH-Px水平顯著升高。(6) PTL基因過表達的星形膠質(zhì)細(xì)胞(尤其是LV-PTL-T1As組)使PC12細(xì)胞PD模型p-Akt和p-GSK-3β表達增加；LY294002拮抗PTL基因過表達的星形膠質(zhì)細(xì)胞對6-OHDA引起的PC12細(xì)胞損傷的保護作用。 結(jié)論：過表達PTL基因的星形膠質(zhì)細(xì)胞對6-OHDA作用的PC12細(xì)胞具有保護作用，，這些保護作用可能是通過Akt/GSK-3β信號通路實現(xiàn)的，這為PD發(fā)病機制的研究提供了新的重要線索，可能成為PD治療的新靶向。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R742.5

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