大鼠內(nèi)耳高增殖細胞的定量研究
發(fā)布時間：2018-08-13 13:18
【摘要】：目的：探討不同年齡及生長因子對于大鼠內(nèi)耳高增殖細胞體外增殖的影響。 方法：1．分離取出不同年齡即出生后1天(P1)，7天(P7)，14天(P14)，21天(P21)，30天(P30)，60天(P60)的SD大鼠各內(nèi)耳器官：橢圓囊斑，球囊斑和耳蝸Corti器，每個年齡段6只大鼠（12耳），總計72只大鼠（144耳），在無血清條件下進行單細胞懸浮細胞培養(yǎng)，至第七天，在倒置相差顯微鏡下對所形成的細胞球進行計數(shù)，對計數(shù)結(jié)果進行統(tǒng)計學分析；同時描繪出同種器官不同年齡SD大鼠形成細胞球的數(shù)量變化曲線；2．分離取出60只（120耳）出生后1天SD大鼠各內(nèi)耳器官：橢圓囊斑，球囊斑和耳蝸Corti器，隨機分為對照組和實驗組，對照組不添加任何生長因子，選用上階段實驗數(shù)據(jù)，實驗組分別添加EGF，bFGF，IGF-1，LIF，進行單細胞懸浮培養(yǎng)，至第八天，在倒置相差顯微鏡下進行細胞球計數(shù)，對計數(shù)結(jié)果進行統(tǒng)計學分析；3．免疫熒光染色檢測細胞球的表型特征。 結(jié)果：1．P1SD大鼠內(nèi)耳各個器官單細胞nestin表現(xiàn)為陽性，培養(yǎng)7天后，可形成懸浮的細胞球，球體細胞免疫熒光表現(xiàn)為nestin和BrdU陽性；2．⑴各個內(nèi)耳器官形成的細胞球數(shù)量：A.耳蝸Corti器：P1，P7大鼠單個Corti器可形成細胞球數(shù)分別為50±23個和33±15個，其差異有顯著的統(tǒng)計學意義(P㩳0.05)。而在P14、P21、P30、P60大鼠所取細胞在相同條件下培養(yǎng)，未能觀察到有細胞球形成；B.球囊斑：各個年齡段均觀察到有細胞球形成，P1至P60單個球囊斑形成的細胞球數(shù)分別為23±9、21±8、16±6、14±4、13±3、12±3。P1與P7之間差異無統(tǒng)計學意義(P㧐0.05)，P1與其他各年齡段間差異有統(tǒng)計學意義(P㩳0.05)；P7與P14間差異無統(tǒng)計學意義(P㧐0.05)，P7與P21、P30、P60間差異有統(tǒng)計學意義(P㩳0.05)；P14、P21、P30、P60互相之間差異無統(tǒng)計學意義(P㧐0.05)。C.橢圓囊斑：各個年齡段均觀察到有細胞球形成，P1至P60單個橢圓囊斑形成的細胞球數(shù)分別為29±10、24±9、21±6、18±4、15±4、14±5。P1與P7之間差異無統(tǒng)計學意義(P㧐0.05)，P1與其他各年齡段間差異有統(tǒng)計學意義(P㩳0.05)；P7與P14、P21間差異無統(tǒng)計學意義(P㧐0.05)，P7與P30、P60之間差異有統(tǒng)計學意義(P㩳0.05)；P14與P21間差異無統(tǒng)計學意義(P㧐0.05)，，P14與P30、P60之間差異有統(tǒng)計學意義(P㩳0.05)；P21、P30、P60互相之間差異無統(tǒng)計學意義(P㧐0.05)。⑵同年齡段單個不同內(nèi)耳器官所形成細胞球數(shù)之間的統(tǒng)計分析：①P1年齡段：耳蝸Corti器與球囊之間差異有統(tǒng)計學意義(P㩳0.05)，耳蝸Corti器與橢圓囊之間差異有統(tǒng)計學意義(P㩳0.05)，球囊與橢圓囊之間差異無統(tǒng)計學意義(P㧐0.05)；P7年齡段：耳蝸Corti器與球囊之間差異有統(tǒng)計學意義(P㩳0.05)，耳蝸Corti器與橢圓囊之間差異無統(tǒng)計學意義(P㧐0.05)，球囊與橢圓囊之間差異無統(tǒng)計學意義(P㧐0.05)；②P14、P21、P30、P60年齡段：P21大鼠的球囊斑和橢圓囊斑之間差異有統(tǒng)計學意義(P㩳0.05)，P14、P30、P60大鼠的球囊斑和橢圓囊斑之間差異無統(tǒng)計學意義(P㧐0.05)。3．添加了生長因子EGF、bFGF、IGF-1后，單個橢圓囊斑、球囊斑和耳蝸Corti器進行細胞培養(yǎng)，所形成的細胞球數(shù)量明顯增多，與對照組比較，差異有顯著統(tǒng)計學意義，而三組之間無差異；添加LIF組則球團數(shù)量增多不明顯，與對照組比較提示差異無統(tǒng)計學意義。 結(jié)論1．大鼠內(nèi)耳前庭器官（球囊斑，橢圓囊斑）以及耳蝸（Corti器）內(nèi)有高增殖能力細胞，能表達神經(jīng)干細胞的特性；但隨著鼠齡的增長，這種增殖能力總體趨勢表現(xiàn)為減退；2．與前庭器官比較，耳蝸內(nèi)高增殖細胞的增殖能力減退更快，但出生后1天的大鼠耳蝸內(nèi)高增殖細胞的增殖能力更強，出生后14天大鼠耳蝸內(nèi)未能發(fā)現(xiàn)高增殖細胞，同時還發(fā)現(xiàn)。前庭器官內(nèi)高增殖細胞的增殖能力下降緩慢，持續(xù)至出生后60天仍可見高增殖細胞，球囊斑高增殖細胞減少主要集中在出生后第1天至21天，而橢圓囊斑高增殖細胞減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，其他同年齡段兩種前庭器官內(nèi)高增殖細胞的增殖能力無差異；3．EGF、bFGF、IGF-1這三種生長因子均可單獨促進出生后1天大鼠內(nèi)耳高增殖細胞的增殖能力，但三者之間促增殖作用無差異，而LIF的促增殖作用不明顯。
[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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R764
本文編號：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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R764
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