SD大鼠螺旋神经节细胞与骨髓间充质干细胞共培养观察
发布时间：2019-05-17 03:29
【摘要】：目的SD大鼠原代螺旋神经节细胞(Spiral ganglion cells, SGC)与骨髓间充质干细胞(bone marrow mesenchymal stem cells BMSCs)间接共培养,观察BMSCs是否诱导分化为神经细胞,并观察其对SGCs生长影响,为治疗感音神经性耳聋治疗寻求新的治疗方法。 方法1.BMSCs的原代及传代培养：SD大鼠全骨髓直接贴壁法培养BMSCs并进行传代培养,取第3代细胞进行流式细胞仪检测及免疫细胞化学染色。2.SGCs的原代培养,取新生SD大鼠进行离体SGCs离体培养,取第3天细胞用神经元特异性烯醇化酶(NSE)进行免疫细胞化学染色。3.采用间接共培养方法,将SGCs与培养第3代BMSCs按1：1比例进行共培养,每天观察细胞形态。取第7天的BMSCs用NSE一抗进行免疫细胞化学染色,观察是否有染色阳性的神经元样细胞。 结果1.酶消化法SGCs培养24小时后,大部分细胞贴壁,并出现较短的轴突,48-72小时轴突逐渐变长,细胞胞体呈椭圆形或圆形,突起细长,常交织成网状,培养6至14天时,细胞数量明显减少,部分细胞凋亡。组织块法接种24小时后,组织块贴壁。细胞形态与酶消化法相似,但生存时间比它更长。2.原代培养的BMSCs24h-48h后贴壁细胞,呈集落样生长,细胞呈短梭型、多角型等。第6d起细胞迅速增殖,培养11天时,细胞长满瓶底的80-90%左右传代,第二代细胞增殖速度较第一代细胞明显增快。培养至第3代时,细胞形态为均一的“纺锤形”,呈明显的漩涡样生长。流式细胞仪检测,造血干细胞和内皮细胞表面标志抗原CD34表达呈阴性,间充质干细胞表面标志抗原CD44表达阳性,免疫细胞化学染色呈阳性,结合细胞形态分析符合大鼠BMSCs的特征。3.共培养组中BMSCs第5天开始突起增加,有的呈星状。共培养组中SGCs与对照组中SGCs相比,前者SGCs维持时间相对更长,第6天神经细胞数量减少,部分细胞轴突缩短,而后者第4天数量就开始出现减少,15天时,共培养组中SGCs数量已明显减少,而对照组中SGCs接近凋亡。取第7天的BMSCs进行免疫细胞化学染色,可见有细胞着色,染色时间为5分钟,阳性率约为15%,第一对照组BMSCs几乎呈阴性。 结论1,体外成功培养了SD大鼠SGCs,组织块法培养比酶消化法培养SGCs生存时间更长。2.单纯应用贴壁培养法获得SD大鼠BMSCs简单易行,可分离培养出较纯化的BMSCs。3.BMSCs与SGCs共培养过程中,培养第7天,BMSCs部分分化为神经元样细胞,分化率约为15%；同时,与BMSCs共培养,SGCs的生存时间延长,凋亡减慢。
[Abstract]:Objective to observe whether BMSCs is induced to differentiate into nerve cells by indirect co-culture of (Spiral ganglion cells, SGC) from primary spiral ganglion cells of SD rats and (bone marrow mesenchymal stem cells BMSCs) of bone marrow mesenchymal stem cells, and to observe its effect on the growth of SGCs. To seek a new treatment for sensorineural deafness. Methods Primary and subculture of 1.BMSCs: BMSCs was cultured by direct adherent method of whole bone marrow of SD rats and subcultured. The third passage cells were detected by flow cytometry and immunochemical staining. 2.SGCs were cultured in primary culture. Neonatal SD rats were cultured in vitro with SGCs in vitro, and the cells were stained with neuron-specific enolase (NSE) on the 3rd day. The SGCs and the third generation BMSCs were co-cultured at 1:1 by indirect co-culture method, and the cell morphology was observed every day. On the 7th day, BMSCs was stained with NSE antibody to observe whether there were positive neuron-like cells. Result 1. After 24 hours of SGCs culture, most of the cells adhered to the wall and appeared short axons. After 48 hours, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. After 6 to 14 days of culture, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. The number of cells decreased significantly and some cells were apoptotic. After 24 hours of vaccination, the tissue block adheres to the wall. The morphology of cells is similar to that of enzyme digestion, but the survival time is longer than that of enzyme digestion. 2. The adherent cells cultured in primary culture showed colony-like growth, short shuttle type, polyangular type and so on. On the 6th day, the cells proliferated rapidly. on the 11th day of culture, the cells grew about 80% of the bottom of the bottle, and the proliferation rate of the second generation cells was significantly faster than that of the first generation cells. When cultured to the third generation, the morphology of the cells was homogeneous "fusiform", showing obvious whirlpool growth. Flow cytometry showed that the expression of surface marker antigen CD34 of hematopoietic stem cells and endothelial cells was negative, the expression of surface marker antigen CD44 of mesenchymal stem cells was positive, and the expression of surface marker antigen CD44 of mesenchymal stem cells was positive. The combined cell morphology analysis was in accordance with the characteristics of rat BMSCs. 3. In the co-culture group, the protrusions of BMSCs began to increase on the 5th day, some of them were starlike. Compared with SGCs in control group, SGCs in co-culture group lasted longer, the number of nerve cells decreased and some axons decreased on the 6th day, while the number of axons in the latter began to decrease on the 4th day, and on the 15th day. The number of SGCs in co-culture group was significantly decreased, while SGCs in control group was close to apoptosis. On the 7th day, BMSCs was stained with cells, the staining time was 5 minutes, the positive rate was about 15%, and the BMSCs in the first control group was almost negative. Conclusion 1. The survival time of SGCs, tissue block culture of SD rats was longer than that of SGCs cultured by enzyme digestion. 2. BMSCs of SD rats was obtained by adherent culture alone. The purified BMSCs.3.BMSCs and SGCs could be isolated and co-cultured. On the 7th day of culture, BMSCs partially differentiated into neuron-like cells, and the differentiation rate was about 15%. At the same time, co-culture with BMSCs prolonged the survival time of SGCs and slowed down apoptosis.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:Objective to observe whether BMSCs is induced to differentiate into nerve cells by indirect co-culture of (Spiral ganglion cells, SGC) from primary spiral ganglion cells of SD rats and (bone marrow mesenchymal stem cells BMSCs) of bone marrow mesenchymal stem cells, and to observe its effect on the growth of SGCs. To seek a new treatment for sensorineural deafness. Methods Primary and subculture of 1.BMSCs: BMSCs was cultured by direct adherent method of whole bone marrow of SD rats and subcultured. The third passage cells were detected by flow cytometry and immunochemical staining. 2.SGCs were cultured in primary culture. Neonatal SD rats were cultured in vitro with SGCs in vitro, and the cells were stained with neuron-specific enolase (NSE) on the 3rd day. The SGCs and the third generation BMSCs were co-cultured at 1:1 by indirect co-culture method, and the cell morphology was observed every day. On the 7th day, BMSCs was stained with NSE antibody to observe whether there were positive neuron-like cells. Result 1. After 24 hours of SGCs culture, most of the cells adhered to the wall and appeared short axons. After 48 hours, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. After 6 to 14 days of culture, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. The number of cells decreased significantly and some cells were apoptotic. After 24 hours of vaccination, the tissue block adheres to the wall. The morphology of cells is similar to that of enzyme digestion, but the survival time is longer than that of enzyme digestion. 2. The adherent cells cultured in primary culture showed colony-like growth, short shuttle type, polyangular type and so on. On the 6th day, the cells proliferated rapidly. on the 11th day of culture, the cells grew about 80% of the bottom of the bottle, and the proliferation rate of the second generation cells was significantly faster than that of the first generation cells. When cultured to the third generation, the morphology of the cells was homogeneous "fusiform", showing obvious whirlpool growth. Flow cytometry showed that the expression of surface marker antigen CD34 of hematopoietic stem cells and endothelial cells was negative, the expression of surface marker antigen CD44 of mesenchymal stem cells was positive, and the expression of surface marker antigen CD44 of mesenchymal stem cells was positive. The combined cell morphology analysis was in accordance with the characteristics of rat BMSCs. 3. In the co-culture group, the protrusions of BMSCs began to increase on the 5th day, some of them were starlike. Compared with SGCs in control group, SGCs in co-culture group lasted longer, the number of nerve cells decreased and some axons decreased on the 6th day, while the number of axons in the latter began to decrease on the 4th day, and on the 15th day. The number of SGCs in co-culture group was significantly decreased, while SGCs in control group was close to apoptosis. On the 7th day, BMSCs was stained with cells, the staining time was 5 minutes, the positive rate was about 15%, and the BMSCs in the first control group was almost negative. Conclusion 1. The survival time of SGCs, tissue block culture of SD rats was longer than that of SGCs cultured by enzyme digestion. 2. BMSCs of SD rats was obtained by adherent culture alone. The purified BMSCs.3.BMSCs and SGCs could be isolated and co-cultured. On the 7th day of culture, BMSCs partially differentiated into neuron-like cells, and the differentiation rate was about 15%. At the same time, co-culture with BMSCs prolonged the survival time of SGCs and slowed down apoptosis.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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