大鼠内耳高增殖细胞的定量研究
发布时间：2018-08-13 13:18
【摘要】：目的：探讨不同年龄及生长因子对于大鼠内耳高增殖细胞体外增殖的影响。 方法：1．分离取出不同年龄即出生后1天(P1)，7天(P7)，14天(P14)，21天(P21)，30天(P30)，60天(P60)的SD大鼠各内耳器官：椭圆囊斑，球囊斑和耳蜗Corti器，每个年龄段6只大鼠（12耳），总计72只大鼠（144耳），在无血清条件下进行单细胞悬浮细胞培养，至第七天，在倒置相差显微镜下对所形成的细胞球进行计数，对计数结果进行统计学分析；同时描绘出同种器官不同年龄SD大鼠形成细胞球的数量变化曲线；2．分离取出60只（120耳）出生后1天SD大鼠各内耳器官：椭圆囊斑，球囊斑和耳蜗Corti器，随机分为对照组和实验组，对照组不添加任何生长因子，选用上阶段实验数据，实验组分别添加EGF，bFGF，IGF-1，LIF，进行单细胞悬浮培养，至第八天，在倒置相差显微镜下进行细胞球计数，对计数结果进行统计学分析；3．免疫荧光染色检测细胞球的表型特征。 结果：1．P1SD大鼠内耳各个器官单细胞nestin表现为阳性，培养7天后，可形成悬浮的细胞球，球体细胞免疫荧光表现为nestin和BrdU阳性；2．⑴各个内耳器官形成的细胞球数量：A.耳蜗Corti器：P1，P7大鼠单个Corti器可形成细胞球数分别为50±23个和33±15个，其差异有显著的统计学意义(P㩳0.05)。而在P14、P21、P30、P60大鼠所取细胞在相同条件下培养，未能观察到有细胞球形成；B.球囊斑：各个年龄段均观察到有细胞球形成，P1至P60单个球囊斑形成的细胞球数分别为23±9、21±8、16±6、14±4、13±3、12±3。P1与P7之间差异无统计学意义(P㧐0.05)，P1与其他各年龄段间差异有统计学意义(P㩳0.05)；P7与P14间差异无统计学意义(P㧐0.05)，P7与P21、P30、P60间差异有统计学意义(P㩳0.05)；P14、P21、P30、P60互相之间差异无统计学意义(P㧐0.05)。C.椭圆囊斑：各个年龄段均观察到有细胞球形成，P1至P60单个椭圆囊斑形成的细胞球数分别为29±10、24±9、21±6、18±4、15±4、14±5。P1与P7之间差异无统计学意义(P㧐0.05)，P1与其他各年龄段间差异有统计学意义(P㩳0.05)；P7与P14、P21间差异无统计学意义(P㧐0.05)，P7与P30、P60之间差异有统计学意义(P㩳0.05)；P14与P21间差异无统计学意义(P㧐0.05)，，P14与P30、P60之间差异有统计学意义(P㩳0.05)；P21、P30、P60互相之间差异无统计学意义(P㧐0.05)。⑵同年龄段单个不同内耳器官所形成细胞球数之间的统计分析：①P1年龄段：耳蜗Corti器与球囊之间差异有统计学意义(P㩳0.05)，耳蜗Corti器与椭圆囊之间差异有统计学意义(P㩳0.05)，球囊与椭圆囊之间差异无统计学意义(P㧐0.05)；P7年龄段：耳蜗Corti器与球囊之间差异有统计学意义(P㩳0.05)，耳蜗Corti器与椭圆囊之间差异无统计学意义(P㧐0.05)，球囊与椭圆囊之间差异无统计学意义(P㧐0.05)；②P14、P21、P30、P60年龄段：P21大鼠的球囊斑和椭圆囊斑之间差异有统计学意义(P㩳0.05)，P14、P30、P60大鼠的球囊斑和椭圆囊斑之间差异无统计学意义(P㧐0.05)。3．添加了生长因子EGF、bFGF、IGF-1后，单个椭圆囊斑、球囊斑和耳蜗Corti器进行细胞培养，所形成的细胞球数量明显增多，与对照组比较，差异有显著统计学意义，而三组之间无差异；添加LIF组则球团数量增多不明显，与对照组比较提示差异无统计学意义。 结论1．大鼠内耳前庭器官（球囊斑，椭圆囊斑）以及耳蜗（Corti器）内有高增殖能力细胞，能表达神经干细胞的特性；但随着鼠龄的增长，这种增殖能力总体趋势表现为减退；2．与前庭器官比较，耳蜗内高增殖细胞的增殖能力减退更快，但出生后1天的大鼠耳蜗内高增殖细胞的增殖能力更强，出生后14天大鼠耳蜗内未能发现高增殖细胞，同时还发现。前庭器官内高增殖细胞的增殖能力下降缓慢，持续至出生后60天仍可见高增殖细胞，球囊斑高增殖细胞减少主要集中在出生后第1天至21天，而椭圆囊斑高增殖细胞减少主要集中在出生后第1天至30天。除外出生后21天的大鼠，其他同年龄段两种前庭器官内高增殖细胞的增殖能力无差异；3．EGF、bFGF、IGF-1这三种生长因子均可单独促进出生后1天大鼠内耳高增殖细胞的增殖能力，但三者之间促增殖作用无差异，而LIF的促增殖作用不明显。
[Abstract]:Objective: To investigate the effects of different ages and growth factors on proliferation of rat inner ear hyperproliferative cells in vitro.
Methods: 1. Seventy-two SD rats (144 ears) were cultured with single cell suspension cells in serum-free condition. The inner ear organs of SD rats at different ages (1 day, 7 days, 14 days, 21 days (P21), 30 days (P30) and 60 days (P60) were isolated: oval cystic plaque, balloon plaque and cochlear Corti apparatus. Seven days later, the number of spheres was counted under inverted phase contrast microscope, and the results were statistically analyzed; meanwhile, the number of spheres in SD rats of different ages of the same organ was plotted; 2. 60 (120 ears) of SD rats were separated and taken out on the first day after birth. I apparatus, randomly divided into control group and experimental group, the control group did not add any growth factors, selected the experimental data of the previous stage, experimental group added EGF, bFGF, IGF-1, LIF, respectively, for single cell suspension culture, to the eighth day, under the inverted phase contrast microscope counting, the counting results were statistically analyzed; Phenotypic characteristics of cell spheres were measured.
Results: 1. Single cell nestin was positive in all organs of the inner ear of P1SD rats. After 7 days of culture, suspended cell spheres were formed, and immunofluorescence showed nestin and BrdU positive in the spheres. 2. Number of cell spheres formed in each organ of the inner ear: A. Cochlear Corti: P1, P7 rat single Corti organ could form cell spheres 50 (+) respectively. There was no cell sphere formation in the cultured cells of P14, P21, P30, P60 rats under the same conditions; B. Balloon plaque: Cell sphere formation was observed in all age groups, and the number of cell spheres formed in single balloon plaque of P1 to P60 was 23 + 9, 21 + 8, 16 + 6, 14 + 4, respectively. There was no significant difference between P1 and other age groups (P? 0.05); there was no significant difference between P7 and P14 (P? 0.05); there was no significant difference between P7 and P21, P30, P60 (P? 0.05); there was no significant difference between P14, P21, P30, P60 (P? 0.05); there was no significant difference between P14, P21, P30, P60 (P? 0.05). C. Oval cystic plaque: each There was no significant difference in the number of cell spheres between P1 and P60. There was no significant difference between P1 and other age groups (P?0.05). There was no significant difference between P1 and P14, P21 (P?0.05), P7 and P30 (P?0.05). There was no significant difference between P14 and P21 (P? 0.05), P14 and P30, P60 (P? 0.05); P21, P30, P60 had no significant difference between each other (P? 0.05). _Statistical analysis of the number of cell spheres formed by different inner ear organs in the same age group: 1) P1 age group There were significant differences between the cochlear Corti apparatus and the balloon (P?0.05), between the cochlear Corti apparatus and the elliptical sac (P?0.05), and between the balloon and the elliptical sac (P?0.05), and between the cochlear Corti apparatus and the elliptical sac (P?0.05). There was no significant difference (P? 0.05), there was no significant difference between the balloon and the oval cyst (P? 0.05); 2 P14, P21, P30, P60 age group: There was significant difference between the balloon and the oval cyst plaque in P21 rats (P? 0.05), P14, P30, P60 rats, there was no significant difference between the balloon and the oval cyst plaque (P? 0.05). 3. Addition of growth. After EGF, bFGF, and IGF-1, the number of cell spheres formed by single oval cystic plaque, balloon plaque and cochlear Corti apparatus was significantly increased, which was statistically significant compared with the control group, but there was no significant difference among the three groups. The number of cell spheres in the LIF group was not significantly increased, and there was no significant difference compared with the control group.
Conclusion 1. There are high proliferative cells in vestibular organs (balloon plaque, oval plaque) and cochlea (Corti organ) of rats, which can express the characteristics of neural stem cells; but the general trend of this proliferative capacity decreased with the growth of rats; 2. Compared with vestibular organs, the proliferative capacity of high proliferative cells in the cochlea decreased faster, but the proliferative capacity of high proliferative cells in the cochlea decreased. The proliferative ability of high proliferative cells in the rat cochlea on the first day after birth was stronger than that in the rat cochlea on the 14th day after birth. From the 1st day to the 21st day after birth, the decrease of hyperproliferative cells was mainly concentrated in the 1st day to the 30th day after birth. The proliferative ability of the cells was not different among the three groups, but LIF had no obvious effect.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764
本文编号：2181114
[Abstract]:Objective: To investigate the effects of different ages and growth factors on proliferation of rat inner ear hyperproliferative cells in vitro.
Methods: 1. Seventy-two SD rats (144 ears) were cultured with single cell suspension cells in serum-free condition. The inner ear organs of SD rats at different ages (1 day, 7 days, 14 days, 21 days (P21), 30 days (P30) and 60 days (P60) were isolated: oval cystic plaque, balloon plaque and cochlear Corti apparatus. Seven days later, the number of spheres was counted under inverted phase contrast microscope, and the results were statistically analyzed; meanwhile, the number of spheres in SD rats of different ages of the same organ was plotted; 2. 60 (120 ears) of SD rats were separated and taken out on the first day after birth. I apparatus, randomly divided into control group and experimental group, the control group did not add any growth factors, selected the experimental data of the previous stage, experimental group added EGF, bFGF, IGF-1, LIF, respectively, for single cell suspension culture, to the eighth day, under the inverted phase contrast microscope counting, the counting results were statistically analyzed; Phenotypic characteristics of cell spheres were measured.
Results: 1. Single cell nestin was positive in all organs of the inner ear of P1SD rats. After 7 days of culture, suspended cell spheres were formed, and immunofluorescence showed nestin and BrdU positive in the spheres. 2. Number of cell spheres formed in each organ of the inner ear: A. Cochlear Corti: P1, P7 rat single Corti organ could form cell spheres 50 (+) respectively. There was no cell sphere formation in the cultured cells of P14, P21, P30, P60 rats under the same conditions; B. Balloon plaque: Cell sphere formation was observed in all age groups, and the number of cell spheres formed in single balloon plaque of P1 to P60 was 23 + 9, 21 + 8, 16 + 6, 14 + 4, respectively. There was no significant difference between P1 and other age groups (P? 0.05); there was no significant difference between P7 and P14 (P? 0.05); there was no significant difference between P7 and P21, P30, P60 (P? 0.05); there was no significant difference between P14, P21, P30, P60 (P? 0.05); there was no significant difference between P14, P21, P30, P60 (P? 0.05). C. Oval cystic plaque: each There was no significant difference in the number of cell spheres between P1 and P60. There was no significant difference between P1 and other age groups (P?0.05). There was no significant difference between P1 and P14, P21 (P?0.05), P7 and P30 (P?0.05). There was no significant difference between P14 and P21 (P? 0.05), P14 and P30, P60 (P? 0.05); P21, P30, P60 had no significant difference between each other (P? 0.05). _Statistical analysis of the number of cell spheres formed by different inner ear organs in the same age group: 1) P1 age group There were significant differences between the cochlear Corti apparatus and the balloon (P?0.05), between the cochlear Corti apparatus and the elliptical sac (P?0.05), and between the balloon and the elliptical sac (P?0.05), and between the cochlear Corti apparatus and the elliptical sac (P?0.05). There was no significant difference (P? 0.05), there was no significant difference between the balloon and the oval cyst (P? 0.05); 2 P14, P21, P30, P60 age group: There was significant difference between the balloon and the oval cyst plaque in P21 rats (P? 0.05), P14, P30, P60 rats, there was no significant difference between the balloon and the oval cyst plaque (P? 0.05). 3. Addition of growth. After EGF, bFGF, and IGF-1, the number of cell spheres formed by single oval cystic plaque, balloon plaque and cochlear Corti apparatus was significantly increased, which was statistically significant compared with the control group, but there was no significant difference among the three groups. The number of cell spheres in the LIF group was not significantly increased, and there was no significant difference compared with the control group.
Conclusion 1. There are high proliferative cells in vestibular organs (balloon plaque, oval plaque) and cochlea (Corti organ) of rats, which can express the characteristics of neural stem cells; but the general trend of this proliferative capacity decreased with the growth of rats; 2. Compared with vestibular organs, the proliferative capacity of high proliferative cells in the cochlea decreased faster, but the proliferative capacity of high proliferative cells in the cochlea decreased. The proliferative ability of high proliferative cells in the rat cochlea on the first day after birth was stronger than that in the rat cochlea on the 14th day after birth. From the 1st day to the 21st day after birth, the decrease of hyperproliferative cells was mainly concentrated in the 1st day to the 30th day after birth. The proliferative ability of the cells was not different among the three groups, but LIF had no obvious effect.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764
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