豚鼠耳蜗外侧壁中MDR_1的表达及对ADM应激能力的研究
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摘要
目的：研究不同年龄段豚鼠耳蜗外侧壁中MDR1的表达情况及ADM对正常豚鼠耳蜗外侧壁中MDR1表达的影响，从而进一步明确不同年龄段豚鼠耳蜗外侧壁中MDR1对ADM的应激能力，为某些耳毒性药物的年龄敏感性机制提供理论依据。
方法：采用成组实验、分层随机抽样法，分别从三个年龄段封闭群豚鼠：出生后1-2周龄(75~100g)、3周龄(200~250g)和4周龄(300~350g) 中，选取Preyer反射正常、健康豚鼠各40只，雌雄不拘；每个年龄段分成ADM组(12只)、生理盐水组(12只)和对照组(16只)。用ADM（30mg/Kg,iv）处理ADM组动物24小时造成模型；用生理盐水（30mg/Kg,iv,余条件相同）代替ADM，作为生理盐水组；对照组为不用药组。静注ADM 24小时时相点快速处死动物，解剖出骨性耳蜗，自耳蜗底回吸取内淋巴液后剥离耳蜗外侧壁；用电极法检测内淋液纯度。利用ISHH技术、RT-PCR技术和HPLC技术，分别检测不同年龄段豚鼠耳蜗外侧壁中MDR1mRNA的表达，ADM对其表达的影响及内淋巴液中ADM的积聚浓度。
结果：(1) 原位杂交结果提示不同年龄段豚鼠耳蜗外侧壁中都有MDR1mRNA阳性细胞的不均匀分布。(2)豚鼠耳蜗外侧壁中MDR1基因的表达随年龄增长而增加，各年龄组间MDR1mRNA相对量差异有显著性意义（P<0.05）,其中4周龄组MDR1mRNA相对量最大，1-2周龄组最小。(3)同一年龄段，ADM组较对照组及生理盐水组MDR1mRNA相对值高，并有显著性意义（P<0.05）；生理盐水组与对照组间MDR1mRNA相对值差异没有显著性意义（P>0.05），提示正常豚鼠耳蜗外侧壁中的MDR1是一种应激基因，可被ADM诱导。（4）静脉注射ADM（30mg/Kg）24小时后，1-2周龄组MDR1mRNA相对值改变量较3周龄、4周龄小，并有显著性意义（P<0.05），而3周龄与4周龄组间改变量差异没有显著性意义（P>0.05）。(5) HPLC检测内淋巴液中ADM的积聚浓度发现1-2周龄组内淋巴液中ADM积聚浓度较3周龄、4周龄组高，并有显著性意义（P<0.05）；而3周龄与4周龄组间差异无显著性意义（P>0.05）。
结论：（1）不同年龄段豚鼠耳蜗外侧壁细胞中均有MDR1mRNA的表达，其表达相对值随年龄而增高，耳蜗外侧壁外向运输随年龄而成熟。（2）MDR1基因是一种应激基因，可被ADM诱导；正常豚鼠耳蜗外侧壁中MDR1基因的应激能力随年龄增长而增高，并于一定时期趋于稳定。（3）MDR1基因及所编码的P-gp参与耳蜗外侧壁的外向性运输，构成血迷路屏障一种新的自我防护机制，其生理意义可能在于通过外排耳毒性物质，减少其在内耳中的积聚，防止某些耳毒性物质的内耳损害。
本研究初步提示：由MDR1/P-gp介导的耳蜗外侧壁外向性运输随年龄而成熟，MDR1对某些耳毒性物质的应激能力存在年龄差异，导致其在内耳中的积聚浓度不同，这可能是某些耳毒性物质年龄敏感性的原因之一。
Objective To study the expression of MDR1 gene and the influence of adriamycin on it in cochlea lateral wall of guinea pig, to make sure the capability of stress to adriamycin, and hence to provide the theoretical basis for the mechanism of susceptivity to some ototoxicity drugs in infant.
Methods White guinea pigs with red eyes were choosed randomly from 1-2 weeks,3 weeks and 4 weeks guinea pigs,respectively divided into three groups:control group,N.S group and adriamycin group. Guinea pigs were administered an intravenous injection of 30mg/kg adriamycin or N.S. 24 hours later, the animals were sacrificed ,and the cochlea lateral wall were then immediately removed after the endolymph was drawn by the siphon from the basal turn of cochlea. The expression and the quantity changes of the MDR1 mRNA in the cochlea lateral wall of guinea pigs were observed by in situ hybridization and RT-PCR. Furthermore, the ADM concentration in endolymph were measured by high performance liquid chromatography.
Results (1) The results of in situ hybridization showed that the MDR1mRNA asymmetrically distributed in cells from cochlea lateral wall of guinea pigs.(2) The expression of MDR1 gene enhanced during aging.The density ratio of MDR1mRNA to β-actin was significantly different among three age groups(P<0.05).In addition, the density ratio of MDR1mRNA to β-actin of 4-week-group is largest,and that of 3-week-group is smallest.(3) Regardless of group, the density ratio of MDR1mRNA to β-actin of the adriamycin group was significantly different from the control group (P<0.05). In contrast , there was no significant difference between the N.S group and the control group (P>0.05).(4) After 24 hours administered injection of 30mg/Kg adriamycin,the gain of MDR1mRNA in 1-2 weeks group was obviously lower than that of 3-week-group and 4-week-group(P<0.05),yet there appeared no significant difference between 3-week-group and 4-week-group(P>0.05). (5) According to HPLC,it was shown that extrusion of adriamycin by cochlea lateral wall was age-dependent.The concentration of adriamycin in endolymph of 3-week-group was approximately similar to 4-week-group (P>0.05). Compared with 3-week-group or 4-week-group,that of 1-2 weeks group was
significantly higher(P<0.05).
Conclusions (1) The expression of MDR1mRNA in cochlea lateral wall alter along with the growth of guinea pig, extrusive transport of which mediates may come to be mature with aging. (2) MDR1 gene may be a stress gene which can be induced by adriamycin.The stress capability of MDR1 gene in normal cochlea lateral wall of guinea pig may be increasing with it's growing,and level off when reached some stage.(3) MDR1 gene and P-gp may take part in the extrusive transport.The physiological function as an efflux pump can decrease the accumulation of some ototoxicity drugs in inner ear,and avoid the inner ear damage.
In conclusion,the present study demonstrated that the expression and the capability of stress to some ototoxicity substance of MDR1 gene in the cells from cochlea lateral wall of guinea pig is age-dependent,It appeared to contribute to a new theoretical basis for the mechanism of susceptivity to some ototoxicity drugs in infant.
方法：采用成组实验、分层随机抽样法，分别从三个年龄段封闭群豚鼠：出生后1-2周龄(75~100g)、3周龄(200~250g)和4周龄(300~350g) 中，选取Preyer反射正常、健康豚鼠各40只，雌雄不拘；每个年龄段分成ADM组(12只)、生理盐水组(12只)和对照组(16只)。用ADM（30mg/Kg,iv）处理ADM组动物24小时造成模型；用生理盐水（30mg/Kg,iv,余条件相同）代替ADM，作为生理盐水组；对照组为不用药组。静注ADM 24小时时相点快速处死动物，解剖出骨性耳蜗，自耳蜗底回吸取内淋巴液后剥离耳蜗外侧壁；用电极法检测内淋液纯度。利用ISHH技术、RT-PCR技术和HPLC技术，分别检测不同年龄段豚鼠耳蜗外侧壁中MDR1mRNA的表达，ADM对其表达的影响及内淋巴液中ADM的积聚浓度。
结果：(1) 原位杂交结果提示不同年龄段豚鼠耳蜗外侧壁中都有MDR1mRNA阳性细胞的不均匀分布。(2)豚鼠耳蜗外侧壁中MDR1基因的表达随年龄增长而增加，各年龄组间MDR1mRNA相对量差异有显著性意义（P<0.05）,其中4周龄组MDR1mRNA相对量最大，1-2周龄组最小。(3)同一年龄段，ADM组较对照组及生理盐水组MDR1mRNA相对值高，并有显著性意义（P<0.05）；生理盐水组与对照组间MDR1mRNA相对值差异没有显著性意义（P>0.05），提示正常豚鼠耳蜗外侧壁中的MDR1是一种应激基因，可被ADM诱导。（4）静脉注射ADM（30mg/Kg）24小时后，1-2周龄组MDR1mRNA相对值改变量较3周龄、4周龄小，并有显著性意义（P<0.05），而3周龄与4周龄组间改变量差异没有显著性意义（P>0.05）。(5) HPLC检测内淋巴液中ADM的积聚浓度发现1-2周龄组内淋巴液中ADM积聚浓度较3周龄、4周龄组高，并有显著性意义（P<0.05）；而3周龄与4周龄组间差异无显著性意义（P>0.05）。
结论：（1）不同年龄段豚鼠耳蜗外侧壁细胞中均有MDR1mRNA的表达，其表达相对值随年龄而增高，耳蜗外侧壁外向运输随年龄而成熟。（2）MDR1基因是一种应激基因，可被ADM诱导；正常豚鼠耳蜗外侧壁中MDR1基因的应激能力随年龄增长而增高，并于一定时期趋于稳定。（3）MDR1基因及所编码的P-gp参与耳蜗外侧壁的外向性运输，构成血迷路屏障一种新的自我防护机制，其生理意义可能在于通过外排耳毒性物质，减少其在内耳中的积聚，防止某些耳毒性物质的内耳损害。
本研究初步提示：由MDR1/P-gp介导的耳蜗外侧壁外向性运输随年龄而成熟，MDR1对某些耳毒性物质的应激能力存在年龄差异，导致其在内耳中的积聚浓度不同，这可能是某些耳毒性物质年龄敏感性的原因之一。
Objective To study the expression of MDR1 gene and the influence of adriamycin on it in cochlea lateral wall of guinea pig, to make sure the capability of stress to adriamycin, and hence to provide the theoretical basis for the mechanism of susceptivity to some ototoxicity drugs in infant.
Methods White guinea pigs with red eyes were choosed randomly from 1-2 weeks,3 weeks and 4 weeks guinea pigs,respectively divided into three groups:control group,N.S group and adriamycin group. Guinea pigs were administered an intravenous injection of 30mg/kg adriamycin or N.S. 24 hours later, the animals were sacrificed ,and the cochlea lateral wall were then immediately removed after the endolymph was drawn by the siphon from the basal turn of cochlea. The expression and the quantity changes of the MDR1 mRNA in the cochlea lateral wall of guinea pigs were observed by in situ hybridization and RT-PCR. Furthermore, the ADM concentration in endolymph were measured by high performance liquid chromatography.
Results (1) The results of in situ hybridization showed that the MDR1mRNA asymmetrically distributed in cells from cochlea lateral wall of guinea pigs.(2) The expression of MDR1 gene enhanced during aging.The density ratio of MDR1mRNA to β-actin was significantly different among three age groups(P<0.05).In addition, the density ratio of MDR1mRNA to β-actin of 4-week-group is largest,and that of 3-week-group is smallest.(3) Regardless of group, the density ratio of MDR1mRNA to β-actin of the adriamycin group was significantly different from the control group (P<0.05). In contrast , there was no significant difference between the N.S group and the control group (P>0.05).(4) After 24 hours administered injection of 30mg/Kg adriamycin,the gain of MDR1mRNA in 1-2 weeks group was obviously lower than that of 3-week-group and 4-week-group(P<0.05),yet there appeared no significant difference between 3-week-group and 4-week-group(P>0.05). (5) According to HPLC,it was shown that extrusion of adriamycin by cochlea lateral wall was age-dependent.The concentration of adriamycin in endolymph of 3-week-group was approximately similar to 4-week-group (P>0.05). Compared with 3-week-group or 4-week-group,that of 1-2 weeks group was
significantly higher(P<0.05).
Conclusions (1) The expression of MDR1mRNA in cochlea lateral wall alter along with the growth of guinea pig, extrusive transport of which mediates may come to be mature with aging. (2) MDR1 gene may be a stress gene which can be induced by adriamycin.The stress capability of MDR1 gene in normal cochlea lateral wall of guinea pig may be increasing with it's growing,and level off when reached some stage.(3) MDR1 gene and P-gp may take part in the extrusive transport.The physiological function as an efflux pump can decrease the accumulation of some ototoxicity drugs in inner ear,and avoid the inner ear damage.
In conclusion,the present study demonstrated that the expression and the capability of stress to some ototoxicity substance of MDR1 gene in the cells from cochlea lateral wall of guinea pig is age-dependent,It appeared to contribute to a new theoretical basis for the mechanism of susceptivity to some ototoxicity drugs in infant.
引文
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