大鼠耳蜗及下丘GABA_A受体生后表达规律的实验研究
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摘要
前 言
随着对非遗传性及非先天性重度感音神经性聋病因研究以及预防和
临床治疗方面研究的开展,对听觉系统发育规律尤其是在耳蜗发育敏感期
的研究越来越受到重视。其中关于听觉系统内主要的抑制性神经递质 GABA
及其受体 GABAAR 的研究国外开展了一些,但是其在听觉系统内的分布、
亚型构成、作用以及在发育早期的功能和意义还不很清楚。
现在已经清楚耳蜗和下丘分别是听觉系统重要的外周感受器和听觉
上行径路主要神经元的交换站。GABA 不仅是中枢神经系统的主要抑制性神
经递质,也是耳蜗和下丘内关键的快速抑制性神经递质,其受体 GABAAR
存 在 于 耳 蜗 和 下 丘 内 。 在 耳 蜗 发 育 的 敏 感 期 关 于 耳 蜗 损 伤 对 听 觉 中 枢
GABAAR 可塑性等方面影响的研究已经开展的很多。但是,由于所用实验对
象及致耳蜗损伤的方式不同,而导致报告的结果不同。为了进一步明确庆
大霉素所致生后大鼠耳蜗不同程度损伤的动物模型能否给实验造成差异
以及探讨耳蜗发育敏感期听觉系统 GABAAR 亚单位表达规律,我们制做并
选用了庆大霉素耳毒性药物所致耳蜗不同损伤程度的两种大鼠模型来进
行实验研究:一种是使耳蜗受到彻底的损毁;另一种是只使耳蜗受到轻微
损坏。通过 RT-PCR 和原位杂交的方法对 GABAAR 2 和 2 亚单位在发育期大
鼠耳蜗和下丘内的表达规律进行了研究,以期获得耳蜗和下丘生理和疾病
状态下突触可塑性以及为感音神经性耳聋病因研究及其治疗等方面工作
的开展提供参照数据资料及理论根据。
材料与方法
正常成年 Wistar 大鼠分成两组:组一为给药组,组二为对照组。大
乳鼠分成三个实验组,每组设对照组。用听觉脑干反应(ABRs)检测听力
[1-4]。实验组的大乳鼠分别自 P1 至 P8,P7 至 P16 每天经腹腔注射给予氨
1
吉林大学博士学位论文
基糖甙类抗生素庆大霉素 80 mg/kg。另一组于 P9 一次性给予庆大霉素药
200 mg/kg。每组设正常对照。[1-4] 成年大鼠实验对照组经腹腔注射连续 8
天每天给予庆大霉素 80 mg/kg。
通过 ABRs 以及耳蜗基底膜扫描电镜评估动物模型,用半定量 RT-PCR
方法定量,用原位杂交方法定位和定性。将 GABAAR 2 和 2 亚单位的特异
性引物与内参 GAPDH 一起进行 PCR,将产物的电泳结果在紫外分析系统下进
行灰度分析比较差异。用非放射性寡核苷酸探针进行原位杂交,光镜下观察
分析。
结 果
使用 ABR 和扫描电镜观察耳蜗损伤程度对动物模型的进行评估。ABR
显示,实验组二即连续给药组及其正常对照组 P16 大鼠可在较高的阈值引
出 ABR 反应。经统计分析实验组的听阈比正常对照组 P16 大乳鼠的听阈高
有统计学意义。实验组三即一次性大剂量给药的 P16 大鼠即使在 110dBSPL
强度的刺激下仍无峰值。扫描电镜观察显示,实验组中 P7 给药组与正常对
照组、成年大鼠给药组和正常对照组电镜下无明显差别。实验组第三组在
电镜下见 Corti’s 器的严重损伤,同样影响耳蜗的每一回。可见内外毛
细胞的完全毁坏。网状膜表面无细胞头板,内外毛细胞均严重的丢失,以
底回最甚。实验组二 P16 大鼠的基底膜表面在电镜下见毛细胞表面的静纤
毛排列不规则、倒伏、粘连及丢失现象严重,以底回外毛细胞为重。毛细
胞丢失现象少见。
GABAAR 2 和 2 亚单位在生后正常大鼠耳蜗和下丘内表达的总体趋势
是非线性增加的,P7 时表达最低,P13 时为高峰,P16 时下降到与成年时
的水平相当。耳蜗内无γ2 亚单位的 PCR 产物。原位杂交结果显示,只在
螺旋神经节处可见γ2 亚单位的微弱标记。下丘 GABAAR 2 和 2 亚单位的
2
吉林大学博士学位论文
表达模式相似。与对照组相比,P7 至 P16 给药组动物下丘 2 和 2 两种亚
单位表达的改变最明显。
讨 论
耳蜗和下丘(IC)分别是听觉系统重要的外周感受器和听觉上行径路主
要神经元的交换站。GABA 是耳蜗和 IC 的主要抑制性神经递质,而且受体介
导哺乳动物大脑中大量快速抑制神经递质的 GABAAR 存在于耳蜗和 IC 内。[4-6,
10,17,44]
但是 GABAAR 表达的规律及其亚型构成仍不很清楚。为了进一步明确
不同耳蜗损伤动物模型给实验带来差异的原因以及耳蜗发育敏感期听觉系
统 GABAAR 亚单位表达规律,我们制做并选用了耳毒性药物所致耳蜗损伤的
两种大鼠模型来进行实验研究。一种动物是彻底的去除耳蜗的听觉;另一种
模型是只使耳蜗受到轻微损坏,毛细胞未丢失或很少丢失。本研究通过
RT-PCR 和原位杂交的方法对发育期大鼠耳蜗和下丘 GABAAR 2 和 2 亚单位的
表达规律进行了研究,获得了 GABAAR 在耳蜗和下丘分布和表达的相关资料。
我们的研究发现耳蜗及下丘 GABAAR 2 和 2 两种亚单位的表达模式是在
P7 时是降低的,而后直到 P13 到达高峰后又下降,到 P16 以后表达逐渐平
稳。我们考虑此时表达的下调正是耳蜗及下丘内部发育的一种表现。因为
P7 处下降的这个转折点正好是耳蜗发育的敏感期。这一阶段无论从形态上
还是功能上的研究[5-6]都表明耳蜗的发育是阶段性的,是不同步的。而且传
出 OC 神经元在耳蜗组织的生长和终末连接有多阶段性。[7-10]。听觉中枢神经
系统对外周听觉感受器耳蜗的发育的影
Introduction
With the progress of the investigation on the etiology, prophylaxis and
clinical treatment of non-genetic congenital profound deafness, the laws of
development in the auditory system, especially during the developmental
sensitive period, are paid more and more attention. And the relations between
the inhibitory GABAergic system and the auditory have been studied by some
researchers oversea, but the distribution, subunits and role of the GABAA
receptor and its functions and significance during the early developmental
period is unclear in the auditory system..
Now it is obvious that the cochlea is the peripheroceptor and inferior
colliculus is the key exchange station through the auditory way. And GABA
is the main inhibitory neurotransmitter not only in the vertebrate central
neural system but also in the cochlea and IC. And one of its receptors GABAA
receptor is present in them. Although the effects of cochlear lesion on
plasticity of the auditory central system during the cochlea developmental
sensitive period have been explored, the results are different due to the
different subjects and different auditory deprivation methods in the different
experiments. To investigate the cause of the different results and explore the
expression rules of GABAAR subunits in the cochleas and IC in the rats
during the cochlea developmental sensitive period and also to investigate the
influence of early auditory deprivation on the receptor, two gentamicin
ototoxic animal models were developed in the newborn rat: the cochleas in
one group were thoroughly ruined, and another just softly damaged. Using
9
吉林大学博士学位论文
RT-PCR and in situ hybridization, the expression rules of GABAAR 2 and
2 subunits in the cochleas and I C in postnatal rats during the early
developmental period, so as to get study information of synaptic plasticity
and to provide the reference data and theory basis of the investigation on the
etiology, prophylaxis and clinical treatment of non-genetic congenital
profound deafness.
Material and Methods
The adult Wister rats were divided into two groups: group I:
experimental group, group II: controls. The newborn rats were divided into
three experimental groups and accordingly the other three control groups:
experimental groups animals were intraperitoneally administered daily
gentamicin 80 mg/kg respectively from P1to P8,P7to P16,another group
animals were administered once gentamicin 200 mg/kg in P9. [1-4]Hearing
loss was evaluated by the pinna reflex and by click-evoked auditory
brainstem responses (ABRs). And the adult Wister rats were intraperitoneally
administered daily gentamicin 80 mg/kg for eight days.
The animal models were evaluated by ABRs and scanning electron
microscope(SEM). And the expressions were quantitated by half quantitation
PT-PCR and located by in situ hybridization. The PCR were done using the
special primers of GABAAR 2and 2 subunits together with interior control
GAPDH. The gray scales of the electrophoresis products of PCR were
observed and analyzed under ultraviolet analytical system. In situ
hybridization were done by non-radioactivity oligonucleotide probe and results
were observed by light microscope.
10
吉林大学博士学位论文
Results
ABRs were recorded in all the animals to evaluate the hearing thresholds.
The responses were obtained at the higher thresholds in the experimental group
administered gentamicin from P7 to P16 and its control group. And the
difference between them is significant.No peaks could be identified at
intensities as high as 110 dB SPL at P16 given gentamicin at P9.
SEM showed that no obviously difference between the group given
gentamicin from P1 to P7 and its control group, the same in the adult group.
The results in the group administered gentamicin at P9 demonstrated that
severe lesions of the organ of Corti, affecting similarly each turn of the
cochlea. The complete loss of the inner and outer hair cells was observed.
There were no cuticular plate of IHCs and OH
随着对非遗传性及非先天性重度感音神经性聋病因研究以及预防和
临床治疗方面研究的开展,对听觉系统发育规律尤其是在耳蜗发育敏感期
的研究越来越受到重视。其中关于听觉系统内主要的抑制性神经递质 GABA
及其受体 GABAAR 的研究国外开展了一些,但是其在听觉系统内的分布、
亚型构成、作用以及在发育早期的功能和意义还不很清楚。
现在已经清楚耳蜗和下丘分别是听觉系统重要的外周感受器和听觉
上行径路主要神经元的交换站。GABA 不仅是中枢神经系统的主要抑制性神
经递质,也是耳蜗和下丘内关键的快速抑制性神经递质,其受体 GABAAR
存 在 于 耳 蜗 和 下 丘 内 。 在 耳 蜗 发 育 的 敏 感 期 关 于 耳 蜗 损 伤 对 听 觉 中 枢
GABAAR 可塑性等方面影响的研究已经开展的很多。但是,由于所用实验对
象及致耳蜗损伤的方式不同,而导致报告的结果不同。为了进一步明确庆
大霉素所致生后大鼠耳蜗不同程度损伤的动物模型能否给实验造成差异
以及探讨耳蜗发育敏感期听觉系统 GABAAR 亚单位表达规律,我们制做并
选用了庆大霉素耳毒性药物所致耳蜗不同损伤程度的两种大鼠模型来进
行实验研究:一种是使耳蜗受到彻底的损毁;另一种是只使耳蜗受到轻微
损坏。通过 RT-PCR 和原位杂交的方法对 GABAAR 2 和 2 亚单位在发育期大
鼠耳蜗和下丘内的表达规律进行了研究,以期获得耳蜗和下丘生理和疾病
状态下突触可塑性以及为感音神经性耳聋病因研究及其治疗等方面工作
的开展提供参照数据资料及理论根据。
材料与方法
正常成年 Wistar 大鼠分成两组:组一为给药组,组二为对照组。大
乳鼠分成三个实验组,每组设对照组。用听觉脑干反应(ABRs)检测听力
[1-4]。实验组的大乳鼠分别自 P1 至 P8,P7 至 P16 每天经腹腔注射给予氨
1
吉林大学博士学位论文
基糖甙类抗生素庆大霉素 80 mg/kg。另一组于 P9 一次性给予庆大霉素药
200 mg/kg。每组设正常对照。[1-4] 成年大鼠实验对照组经腹腔注射连续 8
天每天给予庆大霉素 80 mg/kg。
通过 ABRs 以及耳蜗基底膜扫描电镜评估动物模型,用半定量 RT-PCR
方法定量,用原位杂交方法定位和定性。将 GABAAR 2 和 2 亚单位的特异
性引物与内参 GAPDH 一起进行 PCR,将产物的电泳结果在紫外分析系统下进
行灰度分析比较差异。用非放射性寡核苷酸探针进行原位杂交,光镜下观察
分析。
结 果
使用 ABR 和扫描电镜观察耳蜗损伤程度对动物模型的进行评估。ABR
显示,实验组二即连续给药组及其正常对照组 P16 大鼠可在较高的阈值引
出 ABR 反应。经统计分析实验组的听阈比正常对照组 P16 大乳鼠的听阈高
有统计学意义。实验组三即一次性大剂量给药的 P16 大鼠即使在 110dBSPL
强度的刺激下仍无峰值。扫描电镜观察显示,实验组中 P7 给药组与正常对
照组、成年大鼠给药组和正常对照组电镜下无明显差别。实验组第三组在
电镜下见 Corti’s 器的严重损伤,同样影响耳蜗的每一回。可见内外毛
细胞的完全毁坏。网状膜表面无细胞头板,内外毛细胞均严重的丢失,以
底回最甚。实验组二 P16 大鼠的基底膜表面在电镜下见毛细胞表面的静纤
毛排列不规则、倒伏、粘连及丢失现象严重,以底回外毛细胞为重。毛细
胞丢失现象少见。
GABAAR 2 和 2 亚单位在生后正常大鼠耳蜗和下丘内表达的总体趋势
是非线性增加的,P7 时表达最低,P13 时为高峰,P16 时下降到与成年时
的水平相当。耳蜗内无γ2 亚单位的 PCR 产物。原位杂交结果显示,只在
螺旋神经节处可见γ2 亚单位的微弱标记。下丘 GABAAR 2 和 2 亚单位的
2
吉林大学博士学位论文
表达模式相似。与对照组相比,P7 至 P16 给药组动物下丘 2 和 2 两种亚
单位表达的改变最明显。
讨 论
耳蜗和下丘(IC)分别是听觉系统重要的外周感受器和听觉上行径路主
要神经元的交换站。GABA 是耳蜗和 IC 的主要抑制性神经递质,而且受体介
导哺乳动物大脑中大量快速抑制神经递质的 GABAAR 存在于耳蜗和 IC 内。[4-6,
10,17,44]
但是 GABAAR 表达的规律及其亚型构成仍不很清楚。为了进一步明确
不同耳蜗损伤动物模型给实验带来差异的原因以及耳蜗发育敏感期听觉系
统 GABAAR 亚单位表达规律,我们制做并选用了耳毒性药物所致耳蜗损伤的
两种大鼠模型来进行实验研究。一种动物是彻底的去除耳蜗的听觉;另一种
模型是只使耳蜗受到轻微损坏,毛细胞未丢失或很少丢失。本研究通过
RT-PCR 和原位杂交的方法对发育期大鼠耳蜗和下丘 GABAAR 2 和 2 亚单位的
表达规律进行了研究,获得了 GABAAR 在耳蜗和下丘分布和表达的相关资料。
我们的研究发现耳蜗及下丘 GABAAR 2 和 2 两种亚单位的表达模式是在
P7 时是降低的,而后直到 P13 到达高峰后又下降,到 P16 以后表达逐渐平
稳。我们考虑此时表达的下调正是耳蜗及下丘内部发育的一种表现。因为
P7 处下降的这个转折点正好是耳蜗发育的敏感期。这一阶段无论从形态上
还是功能上的研究[5-6]都表明耳蜗的发育是阶段性的,是不同步的。而且传
出 OC 神经元在耳蜗组织的生长和终末连接有多阶段性。[7-10]。听觉中枢神经
系统对外周听觉感受器耳蜗的发育的影
Introduction
With the progress of the investigation on the etiology, prophylaxis and
clinical treatment of non-genetic congenital profound deafness, the laws of
development in the auditory system, especially during the developmental
sensitive period, are paid more and more attention. And the relations between
the inhibitory GABAergic system and the auditory have been studied by some
researchers oversea, but the distribution, subunits and role of the GABAA
receptor and its functions and significance during the early developmental
period is unclear in the auditory system..
Now it is obvious that the cochlea is the peripheroceptor and inferior
colliculus is the key exchange station through the auditory way. And GABA
is the main inhibitory neurotransmitter not only in the vertebrate central
neural system but also in the cochlea and IC. And one of its receptors GABAA
receptor is present in them. Although the effects of cochlear lesion on
plasticity of the auditory central system during the cochlea developmental
sensitive period have been explored, the results are different due to the
different subjects and different auditory deprivation methods in the different
experiments. To investigate the cause of the different results and explore the
expression rules of GABAAR subunits in the cochleas and IC in the rats
during the cochlea developmental sensitive period and also to investigate the
influence of early auditory deprivation on the receptor, two gentamicin
ototoxic animal models were developed in the newborn rat: the cochleas in
one group were thoroughly ruined, and another just softly damaged. Using
9
吉林大学博士学位论文
RT-PCR and in situ hybridization, the expression rules of GABAAR 2 and
2 subunits in the cochleas and I C in postnatal rats during the early
developmental period, so as to get study information of synaptic plasticity
and to provide the reference data and theory basis of the investigation on the
etiology, prophylaxis and clinical treatment of non-genetic congenital
profound deafness.
Material and Methods
The adult Wister rats were divided into two groups: group I:
experimental group, group II: controls. The newborn rats were divided into
three experimental groups and accordingly the other three control groups:
experimental groups animals were intraperitoneally administered daily
gentamicin 80 mg/kg respectively from P1to P8,P7to P16,another group
animals were administered once gentamicin 200 mg/kg in P9. [1-4]Hearing
loss was evaluated by the pinna reflex and by click-evoked auditory
brainstem responses (ABRs). And the adult Wister rats were intraperitoneally
administered daily gentamicin 80 mg/kg for eight days.
The animal models were evaluated by ABRs and scanning electron
microscope(SEM). And the expressions were quantitated by half quantitation
PT-PCR and located by in situ hybridization. The PCR were done using the
special primers of GABAAR 2and 2 subunits together with interior control
GAPDH. The gray scales of the electrophoresis products of PCR were
observed and analyzed under ultraviolet analytical system. In situ
hybridization were done by non-radioactivity oligonucleotide probe and results
were observed by light microscope.
10
吉林大学博士学位论文
Results
ABRs were recorded in all the animals to evaluate the hearing thresholds.
The responses were obtained at the higher thresholds in the experimental group
administered gentamicin from P7 to P16 and its control group. And the
difference between them is significant.No peaks could be identified at
intensities as high as 110 dB SPL at P16 given gentamicin at P9.
SEM showed that no obviously difference between the group given
gentamicin from P1 to P7 and its control group, the same in the adult group.
The results in the group administered gentamicin at P9 demonstrated that
severe lesions of the organ of Corti, affecting similarly each turn of the
cochlea. The complete loss of the inner and outer hair cells was observed.
There were no cuticular plate of IHCs and OH
引文