缝隙连接蛋白Connexin26在小鼠耳蜗发育晚期中的作用研究

发布时间：2018-05-29 09:45

本文选题：缝隙连接蛋白 + 转基因小鼠　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分出生后不同时间点Connexin26基因敲除小鼠模型的建立和鉴定目的：建立及鉴定出生后不同时间点Connexin26基因敲除小鼠模型。方法：通过遗传学手段,将Cx26loxp/loxp转基因小鼠与工具鼠Rosa26CreER杂交获得Cx26lop/WT;Rosa26CreER子一代小鼠。子一代小鼠相互交配,获得Cx26loxp/loxp; Rosa26CreER子二代小鼠。分别在上述子二代小鼠出生后第1天(P1),第6天(P6)和第12天(P12)给予颈背部单次皮下他莫昔芬(tamoxifen, TMX)注射,获得出生后不同时间点Connexin26基因敲除小鼠模型。使用基因分型(genotyping)、蛋白印记(Western Blot)和免疫荧光技术检测小鼠的基因背景和蛋白敲除情况。使用听性脑干反应(auditory brainstem responses, ABR)检测小鼠听力。结果：Cx26loxp/loxp;Rosa26CreER小鼠可以稳定繁殖,体重和发育正常,毛色光亮,无明显退化现象。蛋白印记和免疫荧光技术确认3种不同时间点打药的小鼠耳蜗内Connexin26被广泛敲除。ABR结果显示P1和P6敲除组小鼠早期出现中重度耳聋,耳聋随时间推移稍有加重。P12敲除组小鼠早期听力损伤较轻,后期呈进行性加重的听力损伤。结论：我们成功的建立了出生后不同时间点Connexin26基因敲除小鼠的动物模型。第二部分出生后不同时间点Connexin26基因敲除小鼠耳蜗细胞长时程损伤模式的研究目的：研究和比较出生后不同时间点Connexin26小鼠耳蜗毛细胞损伤、Corti器(organ of Corti, OC)形态结构变化和继发性螺旋神经节细胞(spiral ganglion cells, SGCs)死亡的模式。方法：使用基底膜铺片(n=3)和耳蜗轴位切片苏木素-伊红染色(n=3)来观察P1、P6和P12敲除组小鼠出生后1、3和5个月时耳蜗毛细胞凋亡模式和Corti器形态变化。采用螺旋神经节计数(n=3)的方法,比较上述不同实验组和对照组耳蜗螺旋神经节细胞不同时间的损伤程度。结果：基底膜铺片显示,P1和P6敲除组在出生后1个月耳蜗中回出现毛细胞大量死亡,随时间推移毛细胞死亡范围逐渐扩大到底回,中回损伤进一步加重。P12敲除组在出生后3个月出现耳蜗底回的少量毛细胞损伤。切片HE染色提示,P1组中回Corti隧道;tunnel of Corti, TC)不能开放并随时间推移出现支持细胞(supporting cell,SC)大量死亡,而P6和P12组可以正常打开。上述3组光镜下未见血管纹(striae vascularis, SV)、外侧壁和其他部位细胞明显异常。耳蜗螺旋神经节细胞计数提示,在毛细胞损伤对应部位,P1和P6敲除组出现继发性螺旋神经节细胞死亡,P12组未见明显螺旋神经节细胞死亡。结论：出生后Connexin26早期敲除组小鼠毛细胞、支持细胞和螺旋神经节细胞的损伤程度和范围明显大于出生后晚期敲除组,提示Connexin26在出生后耳蜗的早期发育中起到重要作用,而在出生后耳蜗晚期发育中的作用是可替代的。第三部分出生后早期Connexin26敲除小鼠耳蜗细胞损伤与葡萄糖转运障碍的研究目的：探究出生后早期Connexin26敲除小鼠耳蜗细胞损伤与葡萄糖转运子(glucose transporter, GLUT)变化和细胞能量代谢的关系。方法：使用蛋白印记技术检测1月龄出生后第一天(P1)降低Connexin26表达组小鼠(n=5)与对照组(n=5)耳蜗磷酸化AMPK蛋白表达变化情况。采用实时定量PCR技术检测上述小鼠耳蜗不同葡萄糖转运子的表达变化。结果：1月龄P1敲除组小鼠和对照组小鼠耳蜗磷酸化MPK蛋白表达差异无统计学意义。P1敲除组小鼠和对照组小鼠耳蜗GLUT1、GLUT8和GLUT10在mRNA水平上表达无统计学意义。结论：出生后第一天敲除组小鼠耳蜗细胞损伤可能与细胞葡萄糖能量代谢障碍无明显相关性。出生后降低Connexin26表达对耳蜗葡萄糖转运子GLUT1、GLUT8和GLUT10表达无明显调节作用。
[Abstract]:Part one
Establishment and identification of Connexin26 knockout mice at different time points after birth
Objective: to establish and identify a Connexin26 knockout mouse model at different time points after birth.
Methods: through genetic method, the Cx26loxp/loxp transgenic mice were hybridized with the tool mouse Rosa26CreER to obtain Cx26lop/WT; Rosa26CreER offspring mice. The offspring of the offspring were mating each other to obtain Cx26loxp/loxp; the Rosa26CreER son two generation mice. First days after the birth of the above two generation mice (P1), sixth days (P6) and twelfth days (P12) were given to the neck respectively. A single subcutaneous tamoxifen (TMX) injection was used to obtain a Connexin26 knockout mouse model at different time points after birth. The gene typing (genotyping), protein imprint (Western Blot) and immunofluorescence technique were used to detect the gene background and protein knockout of mice. The auditory brainstem response (auditory brainstem responses, auditory) was used. ABR) test the hearing of mice.
Results: Cx26loxp/loxp, Rosa26CreER mice can reproduce steadily, body weight and development are normal, hair color is bright, and there is no obvious degeneration. Protein imprint and immunofluorescence technique confirm that the Connexin26 in the cochlea of mice with 3 different time points is widely knocked out of.ABR, and the results of the early severe deafness in the P1 and P6 knockout mice are in the early stage of the deafness and deafness. Time lapse slightly aggravated the early hearing impairment of.P12 knockout mice.
Conclusion: we successfully established the animal model of Connexin26 knockout mice at different time points after birth.
The second part
Long term damage patterns of cochlear cells in Connexin26 knockout mice at different time points after birth
Objective: To study and compare the damage of the cochlear hair cells in Connexin26 mice, the morphological changes of the Corti (organ of Corti, OC) and the mode of secondary spiral ganglion cells (spiral ganglion cells, SGCs) in different time points at different postnatal points.
Methods: the mode of apoptosis and the morphological changes of the cochlear hair cells in P1, P6 and P12 knockout mice were observed by using basal membrane (n=3) and cochlear axis sectioning with hematoxylin eosin staining (n=3). The spiral ganglion count (n=3) method was used to compare the spiral ganglion of the cochlear cochlear group and the control group with the method of spiral ganglion count (n=3). The degree of damage at different times of the cell.
Results: the basal membrane lay showed that the hair cells in the P1 and P6 knockout groups died in the middle of the cochlea 1 months after birth. The death range of the hair cells gradually expanded to the end as time went on. The middle gyrus damage further aggravated the few hair cell damage in the.P12 knockout group at the 3 month after birth. The slice HE staining suggested that the P1 group returned to C. The Orti tunnel; tunnel of Corti, TC) can not be open and appear to be in large number of death cells (supporting cell, SC) with time, and P6 and P12 groups can be opened normally. The 3 groups of light mirrors have no vascular lines (striae vascularis,), the lateral wall and other parts of the cells are obviously abnormal. Cochlear spiral ganglion cell count suggests, in hair cells In the P1 and P6 knockout group, secondary spiral ganglion cells died, but no obvious spiral ganglion cells were found in P12 group.
Conclusion: the damage degree and scope of mouse hair cells, supporting cells and spiral ganglion cells in the early Connexin26 knockout group were significantly greater than those in the late postnatal knockout group, suggesting that Connexin26 plays an important role in the early development of the cochlea after birth, and the role in the late development of the cochlea after birth is alternative.
The third part
Damage of cochlear cells and impaired glucose transport in Connexin26 knockout mice at early postnatal stage
Objective: To explore the relationship between the damage of the cochlear cells and the changes of the glucose transporter (glucose transporter, GLUT) and the energy metabolism in the cochlear cells of Connexin26 knockout mice.
Methods: the protein imprinting technique was used to detect the changes in the expression of phosphorylated AMPK protein in the cochlea of the Connexin26 expression group (n=5) and the control group (n=5) after 1 month old days of birth (P1). The changes in the expression of different glucose transporters in the cochlea of the mice were detected by real-time quantitative PCR.
Results: there was no significant difference in the expression of Phosphorated MPK protein in the cochlea between the 1 month old P1 knockout mice and the control group. There was no significant difference in the expression of the cochlea GLUT1 in the.P1 knockout mice and the control mice, and the expression of GLUT8 and GLUT10 at the mRNA level was not statistically significant.
Conclusion: the damage of the cochlear cells in the first day knockout group of mice may not be significantly correlated with the glucose energy metabolism disorder. The expression of Connexin26 after birth has no obvious regulating effect on the expression of GLUT1, GLUT8 and GLUT10 in the cochlear glucose transporter.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43

【参考文献】