本文選題：神經(jīng)干細(xì)胞 + 激活態(tài)雪旺細(xì)胞��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：【目的】本課題擬通過(guò)檢測(cè)激活態(tài)雪旺細(xì)胞(activated Schwann cells,ASCs)和非激活態(tài)雪旺細(xì)胞(Schwann cells,SCs)之間營(yíng)養(yǎng)因子的表達(dá)差異,探究體外培養(yǎng)對(duì)ASCs的功能狀態(tài)影響,及ASCs、SCs誘導(dǎo)神經(jīng)干細(xì)胞(neural stem cells,NSCs)向神經(jīng)元分化的作用;通過(guò)培養(yǎng)ASCs、SCs于靜電紡絲聚己內(nèi)酯(PCL)纖維支架上,對(duì)比ASCs、SCs與PCL纖維支架的生物相容性;通過(guò)培養(yǎng)NSCs或聯(lián)合ASCs于靜電紡絲PCL纖維支架上,觀察細(xì)胞分布、增殖、分化,探究ASCs和NSCs共培養(yǎng)體系與靜電紡絲PCL纖維支架的生物相容性。【方法】通過(guò)結(jié)扎Wistar大鼠坐骨神經(jīng)7天,模擬坐骨神經(jīng)挫傷模型,提取ASCs;由正常坐骨神經(jīng)提取SCs,分別培養(yǎng)至第3代,提取總蛋白,通過(guò)Western Blot對(duì)比分析ASCs和SCs的營(yíng)養(yǎng)因子在蛋白水平的表達(dá)差異;提取Wister孕鼠E14.5天胚胎海馬區(qū)NSCs,通過(guò)免疫熒光染色進(jìn)行細(xì)胞鑒定及觀察NSCs的多向分化能力;通過(guò)ASCs和SCs的條件培養(yǎng)基半量換液誘導(dǎo)NSCs分化,觀察神經(jīng)元分化比例及軸突長(zhǎng)度;3D培養(yǎng)ASCs和SCs在靜電紡絲PCL纖維支架上,通過(guò)CCK-8實(shí)驗(yàn)檢測(cè)細(xì)胞活性與增殖,結(jié)晶紫染色觀察細(xì)胞分布及尋找合適細(xì)胞密度,激光共聚焦顯微鏡觀察細(xì)胞粘附和形態(tài);將NSCs單純或聯(lián)合ASCs培養(yǎng)于靜電紡絲PCL纖維支架上,通過(guò)CCK-8實(shí)驗(yàn)檢測(cè)NSCs增殖,通過(guò)激光掃描電鏡觀察細(xì)胞形態(tài)和分布,通過(guò)激光共聚焦顯微鏡觀察NSCs神經(jīng)元的分化和ASCs MBP的表達(dá)。【結(jié)果】1.傳至第3代的ASCs較SCs高表達(dá)BDNF、NGF(P0.05),而NT-3、MBP的表達(dá)則低于SCs(P0.05);半量換液7天后,ASCM組,神經(jīng)元分化比例為36.06±7.04%;SCM組分化比例為17.22±3.78%;M組分化比例為5.78±3.03%。ASCM組神經(jīng)元分化比例高于其他兩組(P0.05)。ASCM組,神經(jīng)元軸突長(zhǎng)度為161.33±67.44μm;SCM組神經(jīng)元軸突長(zhǎng)度為110.33±60.34μm;M組,神經(jīng)元軸突長(zhǎng)度為97.55±54.09μm。ASCM組神經(jīng)元軸突長(zhǎng)于其余兩組(P0.05)。2.靜電紡絲PCL纖維支架的纖維直徑集中在7.93±1.41μm,纖維隨機(jī)分布能形成3D培養(yǎng)結(jié)構(gòu);結(jié)晶紫染色觀察:一定數(shù)量的雪旺細(xì)胞緊貼纖維縱向分布,且細(xì)胞種植密度為2×10^4個(gè)/cm2時(shí)細(xì)胞增殖良好;通過(guò)S-100染色,激光共聚焦顯微鏡觀察:細(xì)胞呈典型的梭型,并且細(xì)胞緊貼纖維分布,與結(jié)晶紫染色觀察結(jié)果相一致;CCK-8實(shí)驗(yàn)檢測(cè):ASCs在PCL支架上比SCs的增殖更快,第7天OD值高于SCs(P0.05)。3.神經(jīng)干細(xì)胞在靜電紡絲PCL纖維支架上培養(yǎng),1-7天OD值逐漸增大,在第6-7天時(shí),趨于平緩,在第7天時(shí)細(xì)胞增殖達(dá)到最高峰;通過(guò)βⅢ-Tubulin、GFAP、O4免疫熒光染色,利用激光共聚焦顯微鏡進(jìn)行觀察:神經(jīng)干細(xì)胞分化7天后,能夠分化為星形膠質(zhì)細(xì)胞,神經(jīng)元和少突膠質(zhì)細(xì)胞;NSCs與ASCs共培養(yǎng)于靜電紡絲PCL支架上后,分化7天進(jìn)行疫熒光染色共聚焦顯微鏡觀察:一定數(shù)量的ASCs能夠表達(dá)MBP,神經(jīng)干細(xì)胞能夠分化為神經(jīng)元,并且神經(jīng)元多分布于表達(dá)MBP的ASCs周?chē)！窘Y(jié)論】1.體外培養(yǎng)條件下傳代至第3代的ASCs仍能保持一定的激活表型,且能促進(jìn)NSCs向神經(jīng)元方向分化;2.靜電紡絲PCL支架具有3D培養(yǎng)結(jié)構(gòu),與ASCs具有良好的生物相容性,且纖維對(duì)細(xì)胞分布具有一定導(dǎo)向性作用;3.NSCs、ASCs共培養(yǎng)體系在靜電紡絲PCL支架上能夠形成3D培養(yǎng),可以進(jìn)行體內(nèi)移植探究。
[Abstract]:[Objective] to investigate the difference in the expression of nutritional factors between activated Schwann cells (ASCs) and non active Schwann cells (Schwann cells, SCs) in the active state, and to explore the effect of in vitro culture on the functional state of ASCs, and the effect of ASCs and SCs induced neural stem cells (neural stem) to neuron differentiation. The biocompatibility of ASCs, SCs and PCL fiber scaffolds was compared by ASCs and SCs on the electrostatic spinning polyhexyl (PCL) fiber scaffold. The biocompatibility of the cell distribution, proliferation and differentiation between the NSCs or the combined ASCs on the electrostatic spinning PCL fiber scaffold was observed and the biocompatibility between the ASCs and NSCs co culture system and the electrostatic spinning PCL fiber scaffold was investigated. Methods by ligating the sciatic nerve of Wistar rats for 7 days, the sciatic nerve contusion model was simulated and ASCs was extracted. SCs was extracted from the normal sciatic nerve and cultured to third generations. The total protein was extracted respectively. The expression difference between ASCs and SCs in the protein level was analyzed by Western Blot, and NSCs in the hippocampus of E14.5 Day Embryo of Wister pregnant mice was extracted. Immunofluorescence staining was used to identify and observe the multidirectional differentiation of NSCs; NSCs differentiation was induced by semi mass transfer of ASCs and SCs conditioned medium, and the proportion of neuron differentiation and the length of axon were observed. 3D culture ASCs and SCs were used to detect cell activity and proliferation by CCK-8 test and crystal violet staining. The cells were distributed and looked for the appropriate cell density. The adhesion and morphology of the cells were observed by laser confocal microscopy. The NSCs pure or combined ASCs was cultured on the electrospun PCL fiber scaffold. The proliferation of NSCs was detected by the CCK-8 experiment. The morphology and distribution of the cells were observed by laser scanning electron microscopy. The points of NSCs neurons were observed by laser confocal microscopy. Expression of ASCs and MBP. [results] 1. to third generation of ASCs higher than SCs expressed BDNF, NGF (P0.05), and NT-3, MBP was lower than SCs (P0.05), 7 days after the half volume of liquid, ASCM group, the proportion of neuron differentiation was 36.06 + 7.04%, the proportion of differentiation was 17.22 + 3.78%, the proportion of differentiation group of 5.78 + neurons differentiation was higher than the other In the two group (P0.05) group.ASCM, the axon length of the neuron was 161.33 + 67.44 m, and the axon length of the SCM group was 110.33 + 60.34 mu m; the axon of the neuron axon length was 97.55 + 54.09 mu m.ASCM, and the axon of the neuron was longer than the other two groups (P0.05). The fiber diameter of the.2. electrostatic spinning PCL fiber stent was 7.93 + 1.41 mu m, and the random distribution of fiber could form 3D. Culture structure; crystal violet staining observation: a certain number of Schwann cells were closely attached to the fiber longitudinal distribution, and the cell proliferation was good when the cell planting density was 2 x 10^4 /cm2; the cells were observed by S-100 staining and laser confocal microscope: the cells showed a typical spindle type, and the cells were closely attached to the fiber distribution, and the results were in accordance with the crystal violet staining results; CCK-8 Experimental detection: the proliferation of ASCs on the PCL scaffold is faster than that of SCs. The seventh day OD value is higher than that of SCs (P0.05).3. neural stem cells on the electrospun PCL fiber scaffold. On the 1-7 day, the OD value gradually increases. At the 6-7 day, it tends to be gentle and the cell proliferation reaches the peak at seventh days; through the beta III -Tubulin, GFAP, O4 immunofluorescence staining, and laser copolymerization. The focal microscope was observed: 7 days after differentiation, the neural stem cells differentiated into astrocytes, neurons and oligodendrocytes. NSCs and ASCs were co cultured on the electrospun PCL scaffold and were differentiated for 7 days by immunofluorescence staining confocal microscopy: a certain number of ASCs could express MBP, and neural stem cells could differentiate into nerves. And the neurons were distributed around the ASCs of the expression of MBP. [Conclusion] 1. in vitro culture, the third generation of ASCs still can maintain a certain activation phenotype, and can promote the differentiation of NSCs to the neuron; 2. electrospun PCL scaffold has a 3D culture structure, and has good biocompatibility with ASCs, and the fiber is distributed to the cells. 3.NSCs ASCs co culture system can form 3D culture on electrospinning PCL scaffolds and can be transplanted in vivo.
【學(xué)位授予單位】：天津醫(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)】：R651.2;R318.08

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 Ellen Merete Hagen;;Acute complications of spinal cord injuries[J];World Journal of Orthopedics;2015年01期

2 Nebahat Sezer;Selami Akku?;Fatma Gül?in U?urlu;;Chronic complications of spinal cord injury[J];World Journal of Orthopedics;2015年01期

3 Tevfik Y?lmaz;Erkan Kaptano?lu;;Current and future medical therapeutic strategies for the functional repair of spinal cord injury[J];World Journal of Orthopedics;2015年01期

4 KAM-SZE TSANG;;Induction of Functional Recovery by Co-transplantation of Neural Stem Cells and Schwann Cells in a Rat Spinal Cord Contusion Injury Model[J];Biomedical and Environmental Sciences;2007年03期

5 ;Effect of Rat Schwann Cell Secretion on Proliferation and Differentiation of Human Neural Stem Cells[J];Biomedical and Environmental Sciences;2003年01期

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本文編號(hào)：1994986