本文選題：神經(jīng)干細胞 + 雪旺細胞共培養(yǎng)　； 參考：《上海交通大學》2015年博士論文

【摘要】：第一部分大鼠雪旺細胞與神經(jīng)干細胞共培養(yǎng)的體外實驗研究目的:觀察大鼠雪旺細胞(Schwann cells,SCs)與神經(jīng)干細胞(Neural Stem Cells,NSCs)體外共培養(yǎng)時對神經(jīng)干細胞存活、分化方向以及BDNF、GDNF分泌量的影響。材料和方法:取p2-p4代雪旺細胞與p2代神經(jīng)干細胞在誘導培養(yǎng)基中共培養(yǎng)。共分為三組:雪旺細胞組、神經(jīng)干細胞組、雪旺細胞與神經(jīng)干細胞共培養(yǎng)組。倒置相差顯微鏡下,每天觀察三組細胞形態(tài)并計數(shù);細胞免疫熒光化學染色:SCs用S100標記,NSCs用Nestin巢蛋白標記,分化的神經(jīng)元細胞用Map2、Neu N標記,星形膠質(zhì)細胞用GFAP標記;MTT(四甲基偶氮唑藍)比色法檢測各細胞組存活率;酶聯(lián)免疫吸附法(ELISA)檢測各組細胞懸液1,3,5,7天BDNF、GDNF分泌情況。結(jié)果:雪旺細胞與神經(jīng)干細胞共培養(yǎng)組細胞狀態(tài)更好、細胞突起細長、交織成網(wǎng)狀,神經(jīng)干細胞大多分化為神經(jīng)元細胞;共培養(yǎng)組細胞存活率及BDNF、GDNF也高于其他兩組(p0.05)。結(jié)論:雪旺細胞與神經(jīng)干細胞共培養(yǎng)可以促進神經(jīng)干細胞向神經(jīng)元分化,兩者共培養(yǎng)可以通過分泌更多神經(jīng)營養(yǎng)因子改善神經(jīng)再生微環(huán)境。第二部分組織工程化人工神經(jīng)的體外構(gòu)建目的:通過比較雪旺細胞和神經(jīng)干細胞共同培養(yǎng)與單獨一種細胞(雪旺細胞或神經(jīng)干細胞)種植于三種材料上,細胞形態(tài)學變化及細胞增殖能力的變化,以探索進行構(gòu)建組織工程化人工神經(jīng)體外實驗的可行性。材料和方法:雪旺細胞、神經(jīng)干細胞以及雪旺細胞-神經(jīng)干細胞(1:1)分別與PLGA、c PLGA、lc PLGA三種材料共同于分化培養(yǎng)基下培養(yǎng),構(gòu)建層粘連蛋白(Laminin)及殼聚糖(Chitosan)涂層的內(nèi)置納米纖維細絲的新型PLGA神經(jīng)導管。MTT法檢測雪旺細胞和神經(jīng)干細胞共培養(yǎng)以及單獨一種細胞的生物相容性和細胞毒性。結(jié)果:細胞與生物材料共培養(yǎng)七天后,掃描電子顯微鏡下觀察發(fā)現(xiàn),所有細胞組在lc PLGA材料上相較其他兩種材料上具有更好的形態(tài)學表現(xiàn),細胞沿管腔內(nèi)縱行排列的納米纖維細絲定向生長。而倒置相差顯微鏡下觀察到,不論在哪一種材料上生長,雪旺細胞與神經(jīng)干細胞共培養(yǎng)組細胞密度更大,細胞的突起更為細長且交織成網(wǎng)狀。這項研究表明lc PLGA具有良好的生物相容性和較小的細胞毒性,而管腔內(nèi)的納米纖維細絲具有引導細胞定向生長的作用。結(jié)論:雪旺細胞和神經(jīng)干細胞共同培養(yǎng)較單一細胞種植于神經(jīng)導管生物材料上更為有利,這些發(fā)現(xiàn)為人工神經(jīng)導管移植修復周圍神經(jīng)損傷提供了一個生物學基礎(chǔ)。第三部分以PLGA為支架的神經(jīng)干細胞-雪旺細胞組織工程人工神經(jīng)橋接技術(shù)修復大鼠喉返神經(jīng)損傷的研究目的:采用形態(tài)學、分子生物學和電生理學等方法,檢測PLGA-神經(jīng)干細胞-雪旺細胞組織工程人工3D神經(jīng)橋接修復大鼠喉返神經(jīng)缺損的功能效果。材料和方法:將72只150 g健康SD大鼠隨機均分為6組。制作大鼠10 mm的喉返神經(jīng)缺損模型;A組為神經(jīng)干細胞+雪旺細胞+PLGA導管組,B組為神經(jīng)干細胞+PLGA導管組,C組為雪旺細胞+PLGA導管組,D組為PLGA空導管組,E組為自體神經(jīng)移植組。F組為假手術(shù)組。術(shù)后8周及12周,切取再生神經(jīng)中間片段切片,用于免疫熒光染色。并在透射電鏡下觀察導管周圍的神經(jīng)細胞的形態(tài)學改變。術(shù)后8周及12周檢測再生神經(jīng)干動作電位,并計算其潛伏期和振幅。結(jié)果:免疫熒光實驗結(jié)果顯示,在共培養(yǎng)組中,有大量神經(jīng)微絲免疫反應性的纖維和S100免疫反應性纖維,并圍繞在神經(jīng)纖維周圍,提示髓鞘化的神經(jīng)纖維已成功再生。透射電鏡下可見不同階段細胞類型和有髓鞘結(jié)構(gòu)的顯著差異,手術(shù)8周和12周后,CO組顯示有大量再生神經(jīng)纖維,并且其髓鞘較厚,發(fā)育成熟,纖維束之間結(jié)締組織較少,再生軸突發(fā)育好且排列有序。術(shù)后8周的動作電位顯示,潛伏期:共培養(yǎng)組(CO)小于神經(jīng)干細胞(NSC),雪旺細胞(SC)、空導管(null),和SC組有顯著差異(p0.0001),但和自體神經(jīng)移植組(atograft)無差異;波幅:各組之間無明顯差異性。術(shù)后12周的動作電位顯示,潛伏期:共培養(yǎng)組(CO)小于神經(jīng)干細胞(NSC),雪旺細胞(SC)、空導管(null)以及自體神經(jīng)移植組(atograft);.波幅:自體神經(jīng)移植組優(yōu)于共培養(yǎng)、雪旺細胞、神經(jīng)干細胞以及空導管組,其它幾組之間無明顯差異性。結(jié)論:PLGA-神經(jīng)干細胞-雪旺細胞組織工程化人工3D神經(jīng)可增強大鼠喉返神經(jīng)的再生
[Abstract]:Part 1: in the first part of the co culture of rat Schwann cells and neural stem cells in vitro, the effects of Schwann cells (SCs) and neural stem cells (Neural Stem Cells, NSCs) on the survival of neural stem cells, the direction of differentiation, and the effect of BDNF and GDNF secretion were observed. Materials and methods: take the P2-P4 generation of Schwann cells. P2 generation neural stem cells were cultured in the inducible medium. They were divided into three groups: Schwann cell group, neural stem cell group, Schwann cell and neural stem cell co culture group. Under the inverted phase contrast microscope, three groups of cell morphology were observed and counted. Cell immunofluorescence staining: SCs was marked with S100, and NSCs was marked with Nestin nestin. Neuron cells were labeled with Map2, Neu N, astrocytes were labeled with GFAP, MTT (four methylazazolium blue) was used to detect the survival rate of each cell group; enzyme linked immunosorbent assay (ELISA) was used to detect the 1,3,5,7 day BDNF and GDNF secretion in each group of cells. Results: the cell state of the co culture group of Schwann cells and neural stem cells was better, and the cell protruding was fine. Long, interwoven into reticulate, neural stem cells mostly differentiate into neuron cells; the survival rate of cell and BDNF and GDNF in co culture group are also higher than that of other two groups (P0.05). Conclusion: co culture of Schwann cells and neural stem cells can promote neural stem cells to differentiate into neurons, and the co culture can improve the nerve by secreting more neurotrophic factors. Second parts of tissue engineered artificial nerve in vitro construction objective: To explore the construction of tissue engineering human by comparing Schwann cells and neural stem cells together with a single cell (Schwann cells or neural stem cells) planted on three materials, cell morphological changes and cell proliferation energy changes. Materials and methods: the materials and methods: Schwann cells, neural stem cells and Schwann cells - neural stem cells (1:1), together with PLGA, C PLGA, LC PLGA, respectively, were cultured under the differentiation medium, and constructed a new PLGA neuro conductance of nanofiber filaments of laminin (Laminin) and chitosan (Chitosan) coating. .MTT method was used to test the co culture of Schwann cells and neural stem cells and the biocompatibility and cytotoxicity of a single cell. Results: seven days after co culture of cells and biomaterials, it was observed under scanning electron microscope that all the cell groups had better morphological features on the LC PLGA material than the other two materials. Nanofiber filaments aligned vertically in the cavity were directed to grow. Under the inverted phase contrast microscope, the cell density was larger and the cell protruding was more elongated and reticulate, no matter which material was grown on one of the material, and the LC PLGA had good biocompatibility and smaller size. Conclusion: the co culture of Schwann cells and neural stem cells is more favorable than the single cell cultivation on the nerve conduit biomaterials. These findings provide a biological basis for the repair of peripheral nerve injury by artificial nerve conduit transplantation. The three part of the study on the repair of recurrent laryngeal nerve injury in rats with PLGA supported neural stem cells - Schwann cell tissue engineering artificial nerve bridging technique: morphological, molecular and electrophysiologic methods were used to detect the repair of recurrent laryngeal nerve defect in rats by PLGA- neural stem cells - Schwann cell tissue engineering human 3D nerve bridging Functional effects. Materials and methods: 72 150 g healthy SD rats were randomly divided into 6 groups. The rat model of recurrent laryngeal nerve defect was made by 10 mm. Group A was neural stem cells + Schwann cell +PLGA catheter group, B group was neural stem cell +PLGA catheter group, C group was a +PLGA catheter group of Schwann cells, D group was PLGA air catheter group, E group was autotransplantation group of autologous nerve group. In the sham operation group, 8 and 12 weeks after the operation, the intermediate segments of the regenerated nerve were cut and used for immunofluorescence staining. The morphological changes of the nerve cells around the catheter were observed under the transmission electron microscope. The regenerative nerve action potential was detected at 8 and 12 weeks after the operation, and the incubation period and amplitude were calculated. Results: the results of immunofluorescence test showed that in common. In the culture group, there are a large number of neurofilament immunoreactive fibers and S100 immunoreactive fibers and around the nerve fibers, suggesting that myelinated nerve fibers have been successfully regenerated. Under transmission electron microscopy, there are significant differences in cell types and myelin structures in different stages. 8 weeks and 12 weeks after hand surgery, group CO shows a large number of regenerative nerves. Fiber, and its myelin sheath is thicker, mature and less connective tissue between the fiber bundles, and the regeneration axons are well developed and ordered. The action potential 8 weeks after the operation shows that the incubation period is less than the neural stem cells (NSC), SC, null, and SC group (P0.0001), but with the autologous nerve graft group (atograf). T) no difference. There was no significant difference between each group. The action potential of 12 weeks after operation showed that the incubation period: the co culture group (CO) was less than the neural stem cells (NSC), the Schwann cells (SC), the empty catheter (null) and the autologous nerve graft group (atograft); the amplitude: the self body nerve graft group was superior to co culture, Schwann cells, neural stem cells, and empty catheterization groups. Conclusion: PLGA- neural stem cell Schwann cell tissue engineered artificial 3D nerve can enhance the regeneration of the recurrent laryngeal nerve in rats.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R651.3

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相關(guān)期刊論文 前1條

1 孫曉宇;;雪旺細胞對于周圍神經(jīng)再生的功能與作用[J];內(nèi)蒙古民族大學學報(自然科學版);2010年01期

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本文編號：1900323