嗅鞘细胞对螺旋神经节细胞离体培养的影响及其机制的研究
发布时间：2018-08-03 13:13
【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(P0.01)；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(P0.05)；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(P0.01)。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。
[Abstract]:Part one: culture, purification and identification of olfactory ensheathing cells in vitro
Objective: to purify and cultivate the olfactory olfactory ensheathing cells (olfactory ensheathing cells OECs) of adult rats, and to prepare for the follow-up experiment. Methods: the olfactory bulb nerve layer of adult rats was taken and the mixed cell suspension containing OECs was obtained by enzyme digestion. OECs was purified by the method of multiple long time differential adherence, and DF12 culture containing 10%FBS was used. In the culture medium, bFGF (20ng/ml) and forskolin (2uM) were added to the medium of 2D to promote the proliferation of OECs. The culture medium was replaced every 3D and a half quantity. The growth state of OECs was observed under the inverted phase contrast microscope. P75NTR and GFAP cell immunohistochemistry were used to identify the OECs. The morphology of OECs was observed under the fluorescence microscope and the purity of the OECs was observed. Most of the contaminated cells, including fibroblasts and astrocytes, were removed, including fibroblasts and astrocytes. The OECs inoculated for about 24 hours was attached to the wall. The cell bodies of most cells were spindle shaped and some cells were polygons and protuberances. The cells grew slowly in the 2D before inoculation. After 2D, bFGF (20ng/ml) and forskolin (2uM) were added in the culture medium to accelerate the proliferation of cell proliferation, and the cell protruded long. After 7d, 90% of OECs covered dish bottom, and the cell monolayer.P75NTR and GFAP staining showed that about 92% of the cells were P75NTR (+), distributed in the protuberance, the nucleus was deeply dyed; about 12% of the cells were GFAP (+). Conclusion: the use of more (+). Conclusion: adopt more (+). "Conclusion: adopt more (+)." conclusion: adopt more (+). "Conclusion: adopt many (+). It is possible to obtain the purity of OECs for the experiment, and the addition of bFGF (20ng/ml) and forskolin (2uM) in the medium (DF12+10%FBS) can significantly promote the proliferation of OECs.
The effect of olfactory ensheathing cells on cochlear spiral ganglion cells in vitro culture in second parts
Objective: To explore the effect of OECs on the survival of the cochlear auditory afferent neurons (spiral ganglion cells SGCs) in vitro. Methods: the cultured rat olfactory bulb and the newborn rat worm axis tissue were cultured for OECs and SGCs, and the OECs. experiment was purified by multiple long time differential adherence methods. S and SGCs co culture group, SGCs in OECs conditioned medium (OEC-CM) culture group and SGCs single culture group. The growth state of OECs and SGCs was observed under the inverted phase contrast microscope. P75NTR immunohistochemical method was used to identify OECs, the neuron specific marker beta III -tubulin labeling SGCs. results: 7 days after the adherence wall culture, the cell monolayer was formed. In the culture system, SGCs grows on the surface of single cell monolayer formed by OECs and protruded long protrusions, showing a typical bipolar neuron form. In the pre culture 6D, with the prolongation of the incubation time, the SGCs is less than before the inoculation, but the survival number of SGCs in the co culture group is significantly higher than that of the SGCs single culture group (P0.01), and compared with SGCs in OEC-CM, The co culture group was more able to promote SGCs survival, and the number of SGCs in the individual culture group decreased significantly in the culture of 6D; in the cultured 9D, there was little SGCs growth in the individual culture group; but in the co culture group, the number of SGCs was not significantly changed (P0.05). The statistics found that the survival SGCs in the individual culture group was 14.6 + 1.1SGCs/ of 3D. The visual field (x 200) was reduced to the 3.3 + 0.3SGCs/ vision of 14d; the SGCs in group OEC-CM decreased from 35.3 + 2.1SGCs/ to 12.9 + 0.3SGCs/, and the surviving SGCs in the co culture group was significantly increased from the 66.9 + 1.2SGCs/ vision of 3D (x 200) to the 38.9 * 1 SGCs/ vision of 14d. Conclusion: OECs and co culture can promote The newborn rat SGCs survived.
The third part of the molecular mechanism of olfactory ensheathing cells promoting the survival of spiral ganglion cells.
Objective: to preliminarily study the molecular mechanism of OECs to promote the survival of SGCs in vitro. Methods: a co culture system of OECs and SGCs was established, and the experiment was divided into three groups: co culture + brain derived neurotrophic factor (BDNF, 500 pg/ml) group; co culture +BDNF antibody (IgY, 50 ug/ml); the control group was co cultured with OECs and SGCs. SGCs was identified by histochemical staining, 4 fields were taken randomly in each culture dish, and the SGCs of beta III -tubulin marked under fluorescence microscope was counted, and the results of SGCs. in each culture group were statistically analyzed. A large number of SGCs in the co culture +BDNF group grew on the surface of monolayer blanket formed by OECs, but the number of SCG was not statistically significant compared with the control group, and BDNF antibody (IgY) was added (IgY). The number of SGCs in the co culture group decreased significantly (P0.01). Conclusion: the addition of BDNF to the co culture of OECs and SGCs has no obvious effect on the survival of SGCs, but the survival of SGCs decreasing.OECs secreted BDNF after BDNF antibody (IgY) may be one of the molecular mechanisms for promoting the survival of SGCs in vitro culture.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329;R764
本文编号：2161853
[Abstract]:Part one: culture, purification and identification of olfactory ensheathing cells in vitro
Objective: to purify and cultivate the olfactory olfactory ensheathing cells (olfactory ensheathing cells OECs) of adult rats, and to prepare for the follow-up experiment. Methods: the olfactory bulb nerve layer of adult rats was taken and the mixed cell suspension containing OECs was obtained by enzyme digestion. OECs was purified by the method of multiple long time differential adherence, and DF12 culture containing 10%FBS was used. In the culture medium, bFGF (20ng/ml) and forskolin (2uM) were added to the medium of 2D to promote the proliferation of OECs. The culture medium was replaced every 3D and a half quantity. The growth state of OECs was observed under the inverted phase contrast microscope. P75NTR and GFAP cell immunohistochemistry were used to identify the OECs. The morphology of OECs was observed under the fluorescence microscope and the purity of the OECs was observed. Most of the contaminated cells, including fibroblasts and astrocytes, were removed, including fibroblasts and astrocytes. The OECs inoculated for about 24 hours was attached to the wall. The cell bodies of most cells were spindle shaped and some cells were polygons and protuberances. The cells grew slowly in the 2D before inoculation. After 2D, bFGF (20ng/ml) and forskolin (2uM) were added in the culture medium to accelerate the proliferation of cell proliferation, and the cell protruded long. After 7d, 90% of OECs covered dish bottom, and the cell monolayer.P75NTR and GFAP staining showed that about 92% of the cells were P75NTR (+), distributed in the protuberance, the nucleus was deeply dyed; about 12% of the cells were GFAP (+). Conclusion: the use of more (+). Conclusion: adopt more (+). "Conclusion: adopt more (+)." conclusion: adopt more (+). "Conclusion: adopt many (+). It is possible to obtain the purity of OECs for the experiment, and the addition of bFGF (20ng/ml) and forskolin (2uM) in the medium (DF12+10%FBS) can significantly promote the proliferation of OECs.
The effect of olfactory ensheathing cells on cochlear spiral ganglion cells in vitro culture in second parts
Objective: To explore the effect of OECs on the survival of the cochlear auditory afferent neurons (spiral ganglion cells SGCs) in vitro. Methods: the cultured rat olfactory bulb and the newborn rat worm axis tissue were cultured for OECs and SGCs, and the OECs. experiment was purified by multiple long time differential adherence methods. S and SGCs co culture group, SGCs in OECs conditioned medium (OEC-CM) culture group and SGCs single culture group. The growth state of OECs and SGCs was observed under the inverted phase contrast microscope. P75NTR immunohistochemical method was used to identify OECs, the neuron specific marker beta III -tubulin labeling SGCs. results: 7 days after the adherence wall culture, the cell monolayer was formed. In the culture system, SGCs grows on the surface of single cell monolayer formed by OECs and protruded long protrusions, showing a typical bipolar neuron form. In the pre culture 6D, with the prolongation of the incubation time, the SGCs is less than before the inoculation, but the survival number of SGCs in the co culture group is significantly higher than that of the SGCs single culture group (P0.01), and compared with SGCs in OEC-CM, The co culture group was more able to promote SGCs survival, and the number of SGCs in the individual culture group decreased significantly in the culture of 6D; in the cultured 9D, there was little SGCs growth in the individual culture group; but in the co culture group, the number of SGCs was not significantly changed (P0.05). The statistics found that the survival SGCs in the individual culture group was 14.6 + 1.1SGCs/ of 3D. The visual field (x 200) was reduced to the 3.3 + 0.3SGCs/ vision of 14d; the SGCs in group OEC-CM decreased from 35.3 + 2.1SGCs/ to 12.9 + 0.3SGCs/, and the surviving SGCs in the co culture group was significantly increased from the 66.9 + 1.2SGCs/ vision of 3D (x 200) to the 38.9 * 1 SGCs/ vision of 14d. Conclusion: OECs and co culture can promote The newborn rat SGCs survived.
The third part of the molecular mechanism of olfactory ensheathing cells promoting the survival of spiral ganglion cells.
Objective: to preliminarily study the molecular mechanism of OECs to promote the survival of SGCs in vitro. Methods: a co culture system of OECs and SGCs was established, and the experiment was divided into three groups: co culture + brain derived neurotrophic factor (BDNF, 500 pg/ml) group; co culture +BDNF antibody (IgY, 50 ug/ml); the control group was co cultured with OECs and SGCs. SGCs was identified by histochemical staining, 4 fields were taken randomly in each culture dish, and the SGCs of beta III -tubulin marked under fluorescence microscope was counted, and the results of SGCs. in each culture group were statistically analyzed. A large number of SGCs in the co culture +BDNF group grew on the surface of monolayer blanket formed by OECs, but the number of SCG was not statistically significant compared with the control group, and BDNF antibody (IgY) was added (IgY). The number of SGCs in the co culture group decreased significantly (P0.01). Conclusion: the addition of BDNF to the co culture of OECs and SGCs has no obvious effect on the survival of SGCs, but the survival of SGCs decreasing.OECs secreted BDNF after BDNF antibody (IgY) may be one of the molecular mechanisms for promoting the survival of SGCs in vitro culture.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329;R764
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