本文關(guān)鍵詞： 慢病毒 Nurr1 神經(jīng)干細(xì)胞 移植 帕金森病　出處：《昆明醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：[目的]在體外研究Nurr1基因能夠促進(jìn)神經(jīng)干細(xì)胞向多巴胺能神經(jīng)元分化的基礎(chǔ)上,采用慢病毒介導(dǎo),建立過(guò)表達(dá)Nurr1的神經(jīng)干細(xì)胞,進(jìn)一步研究過(guò)表達(dá)Nurr1的神經(jīng)干細(xì)胞移植治療帕金森病大鼠模型的療效,初步探討其作用機(jī)制,為神經(jīng)干細(xì)胞移植治療帕金森病探索新途徑。[方法]1、使用PCR技術(shù)擴(kuò)增目的基因Nurr1后,將其與pLenO-DCE慢病毒載體分別進(jìn)行雙酶切。純化酶切產(chǎn)物后進(jìn)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(duì)長(zhǎng)出的克隆進(jìn)行PCR鑒定、測(cè)序和分析比對(duì),得到構(gòu)建成功的Nurr1慢病毒過(guò)表達(dá)載體,并轉(zhuǎn)染293T細(xì)胞,收集細(xì)胞培養(yǎng)上清液,將其濃縮后測(cè)定病毒滴度。2、采用無(wú)血清培養(yǎng)技術(shù)從孕14天的胎鼠中腦組織分離培養(yǎng)神經(jīng)干細(xì)胞,每天觀察細(xì)胞生長(zhǎng)情況,5-7d后傳代培養(yǎng)。用Nestin免疫熒光檢測(cè)驗(yàn)證其干細(xì)胞特性,并用特異性標(biāo)記物β-Ⅲ-Tubulin、TH、GFAP鑒定其多分化潛能。3、通過(guò)慢病毒感染NSC細(xì)胞預(yù)實(shí)驗(yàn),確定感染復(fù)數(shù)M0I,攜帶Nurr1的慢病毒以MOI=20感染NSC后,通過(guò)熒光觀察、PCR及和Wtetern blot檢測(cè)分析感染后Nurr1的表達(dá)情況。4、SD大鼠經(jīng)6-OHDA定點(diǎn)毀損DA能神經(jīng)元纖維投射的前腦內(nèi)側(cè)束、腹側(cè)被蓋區(qū)和尾狀核后,阿樸嗎啡誘導(dǎo)旋轉(zhuǎn)試驗(yàn)計(jì)數(shù)30min內(nèi)的平均旋轉(zhuǎn)速度,檢測(cè)PD大鼠模型制作情況。5、將成功PD大鼠模型隨機(jī)分為3組進(jìn)行細(xì)胞移植:假手術(shù)對(duì)照組移植等體積PBS替代NSC;NSC組移植轉(zhuǎn)染慢病毒空載體的NSC;Nurr1組移植轉(zhuǎn)染Nurr1慢病毒載體的NSC。分別在移植治療后2周、5周、8周用阿樸嗎啡腹腔注射誘發(fā)旋轉(zhuǎn)實(shí)驗(yàn)觀察移植后大鼠行為的改善情況。并分別于上述時(shí)間點(diǎn)以Western blot技術(shù)檢測(cè)腦內(nèi)TH、DAT蛋白的表達(dá)變化;于8W灌注取腦,以免疫組織熒光技術(shù)觀察腦組織形態(tài)及TH表達(dá)情況;分析比較治療效果及其作用機(jī)理。[結(jié)果]1、本實(shí)驗(yàn)成功構(gòu)建了過(guò)表達(dá)Nurr1基因的慢病毒載體pLenO-DCE-Nurr1。2、胎鼠中腦細(xì)胞懸液原代培養(yǎng)7d左右可形成數(shù)百個(gè)懸浮生長(zhǎng)直徑約200 μm的神經(jīng)球,經(jīng)Nestin免疫熒光檢測(cè)呈陽(yáng)性。經(jīng)誘導(dǎo)分化培養(yǎng)后檢測(cè)到β-Ⅲ-Tubulin、TH、GFAP陽(yáng)性細(xì)胞,表明神經(jīng)干細(xì)胞有向多巴胺能神經(jīng)元、星型膠質(zhì)細(xì)胞等多向分化能力:3、將過(guò)表達(dá)Nurr1的慢病毒顆粒感染原代培養(yǎng)神經(jīng)干細(xì)胞后48h后可見(jiàn)綠色熒光蛋白表達(dá),感染5d后熒光表達(dá)最強(qiáng)。其中陰性對(duì)照組、Nurr1組可見(jiàn)綠色熒光,且每一視野下熒光細(xì)胞數(shù)相似,說(shuō)明導(dǎo)入的目的基因不影響慢病毒重組質(zhì)粒的感染效率;用RT-PCR和Western blot的方法能檢測(cè)出Nurr1在神經(jīng)干細(xì)胞中轉(zhuǎn)錄和表達(dá)。4、SD大鼠立體定向注射6-OHDA術(shù)后,阿樸嗎啡誘導(dǎo)試驗(yàn)檢測(cè)發(fā)現(xiàn)大鼠出現(xiàn)無(wú)法控制的異常向毀損對(duì)側(cè)連續(xù)旋轉(zhuǎn)行為。發(fā)現(xiàn)第2周始誘導(dǎo)出現(xiàn)旋轉(zhuǎn)的大鼠數(shù)量明顯增加,旋轉(zhuǎn)速度較前增快。4周后增加趨勢(shì)減慢;至術(shù)后6周,誘導(dǎo)出現(xiàn)旋轉(zhuǎn)的大鼠數(shù)量及旋轉(zhuǎn)速度相對(duì)穩(wěn)定。最終有43只大鼠旋轉(zhuǎn)行為穩(wěn)定并7rpm/min,可認(rèn)定為成功的PD模型,成模率58.1%。5、細(xì)胞移植術(shù)后各實(shí)驗(yàn)組大鼠經(jīng)阿樸嗎啡誘導(dǎo)的旋轉(zhuǎn)行為學(xué)觀察表明:注射PBS的對(duì)照組移植后PD大鼠的旋轉(zhuǎn)情況沒(méi)有明顯改善;與對(duì)照組比較,單純NSC移植組大鼠異常旋轉(zhuǎn)圈數(shù)從第2周開(kāi)始下降,異常旋轉(zhuǎn)行為在一定程度上有所改善;移植2周后,Nurr1移植組大鼠的旋轉(zhuǎn)行為較NSC移植組明顯改善,隨時(shí)間進(jìn)行性好轉(zhuǎn),但至8周仍不能達(dá)到正常水平。6、各實(shí)驗(yàn)組移植后移植區(qū)TH免疫熒光檢測(cè)表明:過(guò)表達(dá)Nurr1的神經(jīng)干細(xì)胞移植組較單純NSC移植組有更多的移植細(xì)胞存活,且移植細(xì)胞分化率更高,在體內(nèi)GFP熒光表達(dá)時(shí)間不低于8周;神經(jīng)干細(xì)胞移植可促進(jìn)內(nèi)源性TH陽(yáng)性細(xì)胞表達(dá)。7、各實(shí)驗(yàn)組移植后Western blot檢測(cè)表明:Nurr1移植組動(dòng)物腦內(nèi)TH、DAT蛋白表達(dá)量顯著性高于NSC移植治療組,隨時(shí)間推移進(jìn)行性增多。此兩種蛋白的表達(dá)水平同動(dòng)物的行為學(xué)改善情況呈平行關(guān)系。[結(jié)論]本研究成功構(gòu)建了攜帶Nurr1基因的慢病毒載體,過(guò)表達(dá)Nurr1的NSC腦內(nèi)移植治療PD模型大鼠后,在一定時(shí)間內(nèi)能顯著提高NSC移植治療的效果。Nurr1可作為細(xì)胞移植治療PD的重要基因靶點(diǎn),為今后臨床治療提供了實(shí)驗(yàn)依據(jù)。
[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.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R742.5;R-332

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