本文選題：嗅鞘細(xì)胞　切入點(diǎn)：絲素蛋白　出處：《蘇州大學(xué)》2011年碩士論文

【摘要】：目的探討不同直徑絲素蛋白納米纖維(silk fibroin nanofibers,SFS)對(duì)嗅鞘細(xì)胞(olfactory ensheathing cells,OECs)生長(zhǎng)及遷移的影響,為脊髓損傷(spinal cord injury,SCI)修復(fù)材料的選擇提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 方法(1)SFS的制備:運(yùn)用靜電紡絲技術(shù)制備SFS,并通過掃描電鏡(scanning electron microscope,SEM)觀察。(2)OECs的純化培養(yǎng):取SD大鼠嗅球新鮮分離的OECs,采用改良差速貼壁法純化培養(yǎng)。(3) OECs與SFS的復(fù)合培養(yǎng):將純化后的OECs分別接種至以左旋多聚賴氨酸(poly-l-lysine,PLL)(對(duì)照組)和SFS(400nm,1200nm)(實(shí)驗(yàn)組)包被蓋玻片的培養(yǎng)皿中進(jìn)行復(fù)合培養(yǎng)。(4)檢測(cè)指標(biāo):復(fù)合培養(yǎng)不同時(shí)間點(diǎn)行倒置相差顯微鏡和掃描電鏡觀察細(xì)胞形態(tài)、生長(zhǎng)、分布及與材料黏附情況。神經(jīng)生長(zhǎng)因子受體p75(nerve growth factor receptor p75,NGFR p75)及膠質(zhì)原纖維酸性蛋白(glial fibrillary acidic protein,GFAP)免疫熒光染色測(cè)定細(xì)胞表現(xiàn)型,噻唑蘭(Thiazoyl Blue Tetrazolium Bromide,MTT)法測(cè)定支架材料上OECs的增殖情況,死活細(xì)胞染色試劑及流式細(xì)胞術(shù)檢測(cè)細(xì)胞生存率,細(xì)胞的遷移通過Leica AF6000活細(xì)胞工作站拍攝得到圖像,活細(xì)胞工作站動(dòng)態(tài)跟蹤觀察OECs生長(zhǎng)遷移并計(jì)算細(xì)胞遷移軌跡、遷移行為、遷移效率、遷移速率。 結(jié)果(1)倒置相差顯微鏡示SFS上培養(yǎng)的OECs與對(duì)照組相比,細(xì)胞沿絲素纖維遷移,細(xì)胞突起方向走向較一致,形態(tài)特征無顯著差異。(2)掃描電鏡觀察顯示SFS平均直徑約為(395±3)nm、(1210±7)nm,纖維呈三維立體網(wǎng)狀結(jié)構(gòu),分布均勻,OECs與材料結(jié)合緊密,且細(xì)胞突起方向與纖維走向較一致,形態(tài)與對(duì)照組無顯著性差異。(2)免疫熒光染色:培養(yǎng)4d時(shí),免疫熒光染色顯示實(shí)驗(yàn)組NGFRp75/GFAP陽性,OECs在SFS材料上仍保留其特有的表現(xiàn)型。(3)MTT檢測(cè)顯示:培養(yǎng)第4d時(shí),對(duì)照組OECs的吸光度值較1200nm直徑的材料OECs的吸光度值有顯著差異(p0.05),培養(yǎng)第7d時(shí),對(duì)照組OECs的吸光度值較實(shí)驗(yàn)組吸光度值有顯著差異(p0.05),而且OECs在400nmSFS材料上的吸光度值比在1200nmSFS材料上的吸光度值高,有統(tǒng)計(jì)學(xué)意義(p0.05)。400nmSFS相比1200nmSFS更能促進(jìn)OECs的增殖。(4)死活細(xì)胞染色試劑檢測(cè)顯示:在培養(yǎng)第4、7天時(shí),實(shí)驗(yàn)組細(xì)胞形態(tài)較對(duì)照組正常,成活率較高,兩組死亡細(xì)胞數(shù)比較無顯著性差異。(5)流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示:在培養(yǎng)第4天時(shí),實(shí)驗(yàn)組OECs較對(duì)照組OECs的凋亡率無顯著差異(p0.05)。(6)活細(xì)胞工作站觀察顯示:細(xì)胞的遷移速率,遷移效率和對(duì)照組相比均無顯著地統(tǒng)計(jì)學(xué)差異(p0.05),兩種直徑的SFS對(duì)OECs在纖維上的遷移速率、遷移效率沒有顯著影響。 結(jié)論不同直徑絲素蛋白納米纖維對(duì)嗅鞘細(xì)胞的生長(zhǎng)與遷移均具有支持和引導(dǎo)作用,SFS有望成為一種新型的組織工程支架材料結(jié)合OECs移植修復(fù)脊髓損傷。
[Abstract]:Objective to investigate the effects of fibroin nanofibers (SFSs) of different diameters on the growth and migration of olfactory ensheathing cells (OECs) in olfactory ensheathing cells (OECs), and to provide experimental and theoretical basis for the selection of repair materials for spinal cord injury-induced spinal cord injury (sci).Methods preparation of SFS: SFSs were prepared by electrospinning technique, and the purification and culture of OECs from olfactory bulb of SD rats were observed by scanning electron microscopy (SEM). The cells were freshly isolated from olfactory bulb of SD rats and cultured by modified differential adherent method. The compound culture of OECs and SFS was carried out.Culture: the purified OECs was inoculated into a culture plate coated with L-poly-l-lysineine (control group) and SFS400nmnmnmnmnmnmnmnmnmnmHN (experimental group) respectively. The detection index: inverted phase contrast microscopy was performed at different time points in the compound culture.The morphology of cells was observed by microscope and scanning electron microscope.Growth, distribution and adhesion to materials.Neuronal growth factor receptor p75(nerve growth factor receptor p75 (NGFR-p75) and glial fibrillary acidic protein (GFAP) were used to detect the phenotype of the cells. The proliferation of OECs on the scaffold was measured by Thiazoyl Blue Tetrazolium BromideMTTassay.The survival rate of cells was detected by cell staining reagent and flow cytometry. The cell migration was captured by Leica AF6000 living cell workstation. The living cell workstation dynamically tracked the growth and migration of OECs and calculated the migration path and migration behavior.Migration efficiency, migration rate.Results (1) the inverted phase contrast microscope showed that the OECs cultured on SFS moved along the fibroin fibers and in the same direction as the cells in the control group.The average diameter of SFS was about 1210 鹵7 nm, the fiber was three-dimensional reticular structure, the distribution of OECs was close to the material, and the direction of cell process was consistent with the direction of fiber, and the average diameter of SFS was about 1210 鹵7 nm by scanning electron microscope (SEM), which showed that the fiber had a three-dimensional reticular structure, and the direction of cell process was the same as that of the fiber.There was no significant difference in morphology between the control group and the control group. Immunofluorescence staining showed that the NGFRp75/GFAP positive cells of the experimental group still retained their specific phenotype on the SFS material at the 4th day of culture.The absorbance value of OECs in control group was significantly different from that of OECs in diameter of 1200nm.The absorbance value of OECs in the control group was significantly higher than that in the experimental group, and the absorbance value of OECs on 400nmSFS was higher than that on 1200nmSFS.Compared with 1200nmSFS, P0.05N 路400nmSFS could promote the proliferation of OECs. The staining reagent showed that the cell morphology of the experimental group was normal and the survival rate was higher than that of the control group at the 7th day of culture.There was no significant difference in the number of dead cells between the two groups. The results of flow cytometry showed that the apoptosis rate of OECs in experimental group was not significantly different from that of OECs in control group on the 4th day of culture. The observation of living cell workstation showed that the migration rate of cells was higher than that of control group.There was no significant difference in migration efficiency between the two groups. There was no significant difference between the two diameters of SFS on the migration rate and efficiency of OECs on fiber.Conclusion fibroin nanofibers of different diameters can support and guide the growth and migration of olfactory ensheathing cells. SFS is expected to be a new tissue engineering scaffold combined with OECs transplantation to repair spinal cord injury.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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