【作者】 刘全；

【导师】 余洪猛； 顾瑜蓉； 李厚勇；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2010， 硕士

【摘要】 第一部分嗅鞘细胞的体外培养、纯化及鉴定目的：纯化培养和鉴定成年大鼠嗅球嗅鞘细胞(olfactory ensheathing cells OECs),为后续实验做好准备。方法：取成年大鼠嗅球神经层,经酶消化的方法获得含有OECs的混合细胞悬液,采用多次长时差速贴壁的方法纯化OECs,用含有10%FBS的DF12培养基培养,2d后培养基中加入bFGF (20ng/ml)和forskolin(2uM)促进OECs的增殖,每隔3d半量更换培养基。倒置相差显微镜下观察OECs的生长状态,采用P75NTR和GFAP细胞免疫组化的方法鉴定OECs,荧光显微镜下观察OECs的形态并统计其纯度。结果：采用多次长时差速贴壁的方法能够去除绝大部分的污染细胞,包括成纤维细胞和星形胶质细胞,接种的OECs约24小时后贴壁,倒置相差显微镜下观察见大部分细胞胞体呈梭形、部分细胞胞体为多角形,伸出突起；细胞在接种培养的前2d生长缓慢,2d后培养基中加入bFGF (20ng/ml)和forskolin(2uM)可见细胞增殖加快,细胞突起细长,培养7d后见OECs覆盖皿底的90%,形成—细胞单层。P75NTR和GFAP染色见约92%的细胞为P75NTR(+),分布在突起、胞体,其中胞核被深染；约12%的细胞为GFAP (+)。结论：采用多次长时差速贴壁的方法能够获得实验所需纯度的OECs,培养基(DF12+10%FBS)中加入bFGF(20ng/ml)和forskolin(2uM)能够显著促进OECs的增殖。第二部分嗅鞘细胞对耳蜗螺旋神经节细胞离体培养的影响目的：探索在离体实验中OECs有无促进耳蜗听觉传入神经元—螺旋神经节细胞(spiral ganglion cells SGCs)存活的作用。方法：取成年大鼠嗅球和新生大鼠蜗轴组织块进行OECs与SGCs的培养,采用多次长时差速贴壁的方法纯化培养OECs。实验分OECs与SGCs共培养组、SGCs在OECs条件培养液中(OEC-CM)培养组和SGCs单独培养组。倒置相差显微镜下观察OECs和SGCs生长状态,P75NTR免疫组化法鉴定OECs,神经元特异性标志物βⅢ-tubulin标记SGCs。结果：OECs贴壁培养7天后形成—细胞单层,在OECs与SGCs共培养体系中,SGCs在OECs形成的细胞单层的表面生长,并伸出长突起,呈现典型的双极神经元形态；在培养的前6d,随着培养时间的延长,SGCs较接种前减少,但共培养组中SGCs存活数量明显高于SGCs单独培养组(P<0.01)；与SGCs在OEC-CM中培养比较,共培养组更能促进SGCs存活,并且单独培养组的SGCs数量在培养的第6d出现大幅度减少；在培养的第9d,单独培养组中几乎没有SGCs生长；而在共培养组,SGCs的数量未见明显变化(P>0.05)；统计发现单独培养组中存活的SGCs由第3d的14.6±1.1SGCs/视野(×200)减少到第14d的3.3±0.3SGCs/视野；OEC-CM组中的SGCs由35.3±2.1SGCs/视野下降到12.9±0.3SGCs/视野；共培养组中存活的SGCs较其它组明显增多,由第3d的66.9±1.2SGCs/视野(×200)减少到第14d的38.9×1.0 SGCs/视野。结论：OECs与SGCs共培养能够促进新生大鼠SGCs存活。第三部分嗅鞘细胞促进螺旋神经节细胞存活的分子机制目的：初步研究OECs促进SGCs体外存活的分子机制。方法：建立OECs与SGCs共培养体系,实验分三组：共培养+脑源性神经营养因子(BDNF,500 pg/ml)组；共培养+BDNF抗体(IgY型,50 ug/ml)组；对照组为OECs与SGCs共培养。共培养3d后固定,βⅢ-tubulin免疫组化染色鉴定SGCs,每个培养皿随机取4个视野,荧光显微镜下统计βⅢ-tubulin标记的SGCs,统计各培养组中的SGCs。结果：共培养+BDNF组中有大量SGCs在OECs形成的单层细胞毯表面生长,但与对照组比较,SCG的数目无统计学差异；加入BDNF抗体(IgY)的共培养组中的SGCs数量明显减少(P<0.01)。结论：OECs与SGCs共培养中加入BDNF对SGCs存活无明显影响,而加入BDNF抗体(IgY)后存活的SGCs减少。OECs分泌BDNF可能是其促进SGCs体外培养的存活,发挥对SGCs的营养保护作用的分子机制之一。更多还原

【Abstract】 Part 1 Culture, purification and identification of olfactory ensheathing cellsObjective:To culture and purify olfactory ensheathing cells(OECs). Methods:The olfactory nerve layer of adult rat olfactory bulb was dissected. The tissues were minced with a razor blade, and trypsinized using trypsin. OECs were obtained and purified based on their special rate of attachment which was different from the other harvested cell types during culture. DF12 medium supplemented with 10% fetal bovine serum and antibiotics was employed to culture the OECs. Basic fibroblast growth factor (bFGF,20ng/ml) and forskolin (2uM) were added to the medium to enhance OECs growth two days after plating. Half of the medium in each group was refreshed twice a week. OECs were immunocytochemically characterized and confirmed by expression of low-affinity nerve growth factor receptor P75NTR and glial fibrillary acidic protein (GFAP). Results:OECs from the olfactory nerve layer of adult SD rats were highly purified based on the differing rates of attachment of the various cell types. Cultured OECs displayed a typical spindle-shaped soma with long and slim processes. The addition of bFGF and forskolin two days after plating can activate the growth of olfactory ensheathing cells and single layer of OECs was seen seven days after plating.92 percent of the cells cultured were P75NTR immunoreactive, and about 12 percent of the cells positive for GFAP. Conclusion:The high pure of olfactory ensheathing cells could be harvested using special rate of attachment which was different from the other harvested cell types during culture and facilitate the research on the olfactory ensheathing cells in vitro. Addition of basic fibroblast growth factor and forskolin into the medium two days after plating could promote the growth of olfactory ensheathing cells. Objective: To explore whether olfactory ensheathing cells (OECs) can promote newborn rat spiral ganglion cells (SGCs) survival. Methods:Co-cultures of OECs from adult rat olfactory bulb with SGCs from newborn rat cochlea were established and single culture of SGCs acted as control. In addition, OECs conditioned medium (OEC-CM) was employed to culture SGCs in contrast with the co-cultures. OECs were obtained and purified based on their special rate of attachment which was different from the other harvested cell types during culture. OECs and SGCs were immunocytochemically characterized and confirmed by expression of low-affinity nerve growth factor receptor P75NTR and neuron-specificβⅢ-tubulin, respectively. For statistic analyses, randomized block ANOVA was performed. Results: Single layer of OECs (92% pure) was seen seven days after plating. Surviving SGCs, that extended their primary neurites, were found on the surface of the layer in the co-cultures. When OECs and SGCs were co-cultured, the number of surviving SGCs was significantly greater than that in other two groups (P<0.01). Nine days after culture, there was even no change in the number of surviving SGCs in co-cultures while the number of SGCs in single culture dramatically reduced. The number of surviving SGCs in control group significantly decreased from 14.6±1.1 SGCs/ field on day 3 to 3.3±0.3 SGCs/ field on day 14. By contrast, OEC-CM had a positive effect on SGCs survival, with an overall decrease of surviving SGCs from 35.3±2.1 SGCs/field on day 3 to 12.9±0.3 SGCs/ field on day 14 of culture. Surprisingly, the number of surviving SGCs co-cultured with OECs slightly decreased from 66.9±1.2 SGCs/ field on day 3 to 38.9±1.0 SGCs/ field on day 14. Conclusion:Olfactory ensheathing cells can promote the survival of newborn rat spiral ganglion cell in vitro. Objective:To explore the underlying mechanisms that OECs promote the survival of SGCs in vitro. Methods:Co-cultures of OECs from adult rat olfactory bulb with SGCs from newborn rat cochlea were established. In the present study, there were three groups:co-cultures, co-cultures with addition of BDNF (500 pg/ml), and co-cultures with addition of anti-BDNF antibody (50μg/ml). The cultures were fixed three days after cultured. SGCs were immunocytochemically characterized and confirmed by the expression of neuron-specificβⅢ-tubulin. One-way ANOVA and Bonferroni were employed to investigate the differences in the number of SGCs in different cultures. Results:In comparison with co-cultures without treatment, addition of BDNF (500 pg/mL) into the medium had no obvious survival-promoting on SGCs. The number of surviving SGCs reduced significantly when anti-BDNF antibody(50μg/mL) was applied into co-cultures (P<0.01). The number of SGCs between co-cultures and co-cultures+BDNF has no significant difference (P>0.05); the difference between co-cultures+anti-BDNF and the others was significant (P<0.01). Conclusion:BDNF expressed by OECs is in part responsible for the survival-promoting of olfactory ensheathing cells on spiral ganglion cells.更多还原