B细胞淋巴瘤/白血病11B在小鼠耳蜗螺旋器发育中的表达研究

doi:10.3969/j.issn.1674-8115.2022.10.001

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (10): 1361-1374.doi: 10.3969/j.issn.1674-8115.2022.10.001

• 创新团队成果专栏 •

B细胞淋巴瘤/白血病11B在小鼠耳蜗螺旋器发育中的表达研究

姜正麟¹^,²^,³()(), 高云鸽¹^,²^,³, 吴皓¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海 200125
^3.上海市耳鼻疾病转化医学重点实验室，上海 200125

收稿日期:2022-05-07 接受日期:2022-08-09 出版日期:2022-10-28 发布日期:2022-12-02
通讯作者: 吴皓 E-mail:jiangzhenglin@sjtu.edu.cn;wuhao@shsmu.edu.cn
作者简介:姜正麟（1993—），男，硕士生；电子信箱：jiangzhenglin@sjtu.edu.cn。
基金资助:
国家自然科学基金(81730028);上海市科学技术委员会科技创新行动计划(21JC1404000);上海市耳鼻疾病转化医学重点实验室(14DZ2260300)

Study on the expression of B-cell leukemia/lymphoma 11B in the organ of Corti during mice cochlea development

JIANG Zhenglin¹^,²^,³()(), GAO Yunge¹^,²^,³, WU Hao¹^,²^,³()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
^3.Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200125, China

Received:2022-05-07 Accepted:2022-08-09 Online:2022-10-28 Published:2022-12-02
Contact: WU Hao E-mail:jiangzhenglin@sjtu.edu.cn;wuhao@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(81730028);Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission(21JC1404000);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)

摘要/Abstract

摘要：

目的·探索B细胞淋巴瘤/白血病11B（B-cell leukemia/lymphoma 11B，BCL11B）在小鼠耳蜗螺旋器（organ of Corti）发育过程中的表达。方法·选择出生前后不同时期/年龄［胚胎期14.5 d（E14.5）、E15.5、E16.5、E18.5，出生后0日龄（P0）、P2、P4、P7］、随机性别的野生型C57BL/6J小鼠24只，每个年龄3只。将其处死后，取出小鼠双侧耳蜗并固定，对左侧耳蜗解剖获得完整基底膜，右侧耳蜗经蔗糖脱水后进行包埋并制作冰冻切片。采用BCL11B抗体与肌球蛋白6（MYOSIN 6）抗体对小鼠耳蜗基底膜解剖样品、冰冻切片耳蜗样品进行免疫组化分析。采用共聚焦荧光显微镜对上述染色后的不同年龄点的组织样品进行观察，并对BCL11B阳性表达的毛细胞的计数及百分比进行统计。结果·成功制备了8个年龄点的小鼠耳蜗基底膜解剖样品及冰冻切片耳蜗样品。共聚焦荧光显微镜下显示，最先于E15.5胎鼠耳蜗螺旋器底中圈发现BCL11B的表达，其表达特异性出现在MYOSIN6阳性的外侧3排外毛细胞中；随着耳蜗发育，BCL11B在外毛细胞内的表达逐渐增强，至P2时达到顶峰，于P4时逐渐减弱，直至P7时已无法检测到。在BCL11B于外毛细胞表达的整个时期内，螺旋器中的其他细胞均未有BCL11B的表达。分别对BCL11B阳性表达毛细胞计数及百分比进行统计分析，结果显示E16.5、P0和P4这3个年龄点间的差异均无统计学意义（均P>0.05）。结论·通过免疫组化共染BCL11B与MYOSIN6证实，BCL11B是一个在耳蜗螺旋器中外毛细胞发育早期的特异性表达蛋白。

关键词: 螺旋器, 表达模式, 外毛细胞, B细胞淋巴瘤/白血病11B

Abstract:

Objective ·To explore the expression of B-cell leukemia/lymphoma 11B (BCL11B) in the organ of Corti during mice cochlea development. Methods ·A total of 24 wild-type C57BL/6J mice of random sex at different ages before and after birth [embryonic 14.5 days (E14.5), E15.5, E16.5 and E18.5, and postnatal 0 day (P0), P2, P4 and P7] were selected, with 3 at each age point. After the mice were killed, the bilateral cochleae of the mice were sampled and fixed. The left cochleae were dissected to obtain complete basement membranes, and right cochleae were dehydrated by sucrose solution following cross section. BCL11B antibody was combined with MYOSIN 6 antibody were used for immunohistochemical analysis of whole mount samples of mouse cochlea basement membrane and cross section samples. Confocal fluorescence microscope was used for observing the tissue samples at different age points after staining, and counting the numbers to calculate the percentage of BCL11B-expressing hair cells. Results ·Whole mount samples of mouse cochlear basement membrane and cross section samples at eight age points were successfully prepared. Confocal fluorescence microscope showed that BCL11B expression was first detected specifically in outer three rolls of MYOSIN6 positive hair cells in E15.5 middle-basal turn of mouse cochlea; with the development of mouse cochlea, BCL11B expression gradually strengthened until P2, at which time point BCL11B reached top expression level and started to weaken in P4, with almost no BCL11B expression detected in P7. During the whole period of BCL11B expression in outer hair cells, no BCL11B expression was detected in other cell types in the organ of Corti. The results of the number and percentage of BCL11B-expressing hair cells showed that there was no significant difference among the three age points of E16.5, P0, and P4 (both P>0.05). Conclusion ·It is confirmed that BCL11B is a specifically expressed protein in the early development of outer hair cells in the organ of Corti by co-immunostaining of BCL11B and MYOSIN6.

Key words: organ of Corti, expression pattern, outer hair cell, B-cell leukemia/lymphoma 11B (BCL11B)

中图分类号:

R764.4

姜正麟, 高云鸽, 吴皓. B细胞淋巴瘤/白血病11B在小鼠耳蜗螺旋器发育中的表达研究[J]. 上海交通大学学报（医学版）, 2022, 42(10): 1361-1374.

JIANG Zhenglin, GAO Yunge, WU Hao. Study on the expression of B-cell leukemia/lymphoma 11B in the organ of Corti during mice cochlea development[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(10): 1361-1374.

图/表 9

图1 共聚焦显微镜下观察E14.5时BCL11B与MYOSIN6在胎鼠耳蜗螺旋器中的表达Note：A. Expression and distribution of BCL11B and MYOSIN6 in inner hair cells under the low-magnification microscope (×10). It shows the merged image of the apical turn, middle turn and basal turn of the same cochlea, bar=200 μm. B/B'. Expression of BCL11B (B) and MYOSIN6 (B') in the hair cells of basal turn of whole mount samples. B". Merged image of B, B' and nuclear staining (×10). Bar=200 μm. C/C'. Expression of BCL11B (C) and MYOSIN6 (C') in the apical turn of whole mount samples under the high-magnification microscope (×60). C". Merged image of C, C' and nuclear staining (×60). Bar=20 μm. D/D'. Expression of BCL11B (D) and MYOSIN6 (D') in the apical turn of cross section samples under the high-magnification microscope (×60). D". Merged image of D, D' and nuclear staining (×60). Bar=20 μm. E/E'. Expression of BCL11B (E) and MYOSIN6 (E') in the middle-basal turn of whole mount samples under the high-magnification microscope (×60). E". Merged image of E, E' and nuclear staining (×60). Bar=20 μm. F/F'. Expression of BCL11B (F) and MYOSIN6 (F') in the middle-basal turn of cross section samples under the high-magnification microscope (×60). F". Merged image of F, F' and nuclear staining (×60). Bar=20 μm. Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; white box represents prosensory region; white arrow represents IHC; OC—organ of Corti; OHC—outer hair cell; IHC—inner hair cell.

Fig 1 Expression of BCL11B and MYOSIN6 in the organ of Corti at E14.5 mouse cochlea by confocal imaging

图2 共聚焦显微镜下观察E15.5时BCL11B与MYOSIN6在胎鼠耳蜗螺旋器中的表达Note：A: Expression and distribution of BCL11B and MYOSIN6 in the organ of Corti under the low-magnification microscope (×10). It shows the merged image of the apical turn, middle turn and basal turn of the same cochlea, bar=200 μm. B. Expression of BCL11B in the basal turn of organ of Corti of whole mount samples. B'. Merged image of B, MYOSIN6 and nuclear staining (×10). Bar=200 μm. C/C'. Expression of BCL11B (C) and MYOSIN6 (C') in the apical turn of whole mount samples under the high-magnification microscope (×60). C". Merged image of C, C' and nuclear staining (×60). Bar=20 μm. D/D'. Expression of BCL11B (D) and MYOSIN6 (D') in the apical turn of cross section samples under the high-magnification microscope (×60). D". Merged image of D, D' and nuclear staining (×60). Bar=20 μm. E/E'. Expression of BCL11B (E) and MYOSIN6 (E') in the middle turn of whole mount samples under the high-magnification microscope (×60). E". Merged image of E, E' and nuclear staining (×60). Bar=20 μm. F/F'. Expression of BCL11B (F) and MYOSIN6 (F') in the middle turn of cross section samples under the high-magnification microscope (×60). F". Merged image of F, F' and nuclear staining (×60). Bar=20 μm. G/G'. Expression of BCL11B (G) and MYOSIN6 (G') in the basal turn of whole mount samples under the high-magnification microscope (×60). G". Merged image of G, G' and nuclear staining (×60). Bar=20 μm. H/H'. Expression of BCL11B (H) and MYOSIN6 (H') in the basal turn of cross section samples under the high-magnification microscope (×60). H". Merged image of H, H' and nuclear staining (×60). Bar=20 μm. Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; white box represents prosensory region; white arrow represents IHC; black vertical line and yellow arrow represent OHC; OC—organ of Corti; IHC—inner hair cell; OHC—outer hair cell.

Fig 2 Expression of BCL11B and MYOSIN6 in the organ of Corti at E15.5 mouse cochlea by confocal imaging

图3 共聚焦显微镜下观察E16.5时BCL11B与MYOSIN6在胎鼠耳蜗螺旋器中的表达Note：A/A'. Expression of BCL11B (A) and MYOSIN6 (A') in the apical turn of whole mount samples under the high-magnification microscope (×60). A''. Merged image of A, A' and nuclear staining (×60). B/B'. Expression of BCL11B (B) and MYOSIN6 (B') in the apical turn of cross section samples under the high-magnification microscope (×60). B''. Merged image of B, B' and nuclear staining (×60). C/C'. Expression of BCL11B (C) and MYOSIN6 (C') in the middle-basal turn of whole mount samples under the high-magnification microscope (×60). C''. Merged image of C, C' and nuclear staining (×60). D/D'. Expression of BCL11B (D) and MYOSIN6 (D') in the middle-basal turn of cross section samples under the high-magnification microscope (×60). D''. Merged image of D, D' and nuclear staining (×60). Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; white arrow represents IHC; black vertical line and yellow arrow represent OHC; OHC—outer hair cell; IHC—inner hair cell. Bar=20 μm.

Fig 3 Expression of BCL11B and MYOSIN6 in the organ of Corti at E16.5 mouse cochlea by confocal imaging

图4 共聚焦显微镜下观察E18.5时BCL11B与MYOSIN6在胎鼠耳蜗螺旋器中的表达Note：A/A'. Expression of BCL11B (A) and MYOSIN6 (A') in the apical turn of whole mount samples under the high-magnification microscope (×60). A". Merged image of A, A' and nuclear staining (×60). B/B'. Expression of BCL11B (B) and MYOSIN6 (B') in the apical turn of cross section samples under the high-magnification microscope (×60). B". Merged image of B, B' and nuclear staining (×60). C/C'. Expression of BCL11B (C) and MYOSIN6 (C') in the middle-basal turn of whole mount samples under the high-magnification microscope (×60). C". Merged image of C, C' and nuclear staining (×60). D/D'. Expression of BCL11B (D) and MYOSIN6 (D') in the middle-basal turn of cross section samples under the high-magnification microscope (×60). D". Merged image of D, D' and nuclear staining (×60). Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; white arrow represents IHC; black vertical line and yellow arrow represent OHC; OHC—outer hair cell; IHC—inner hair cell. Bar=20 μm.

Fig 4 Expression of BCL11B and MYOSIN6 in the organ of Corti at E18.5 mouse cochlea by confocal imaging

图5 共聚焦显微镜下观察P0时BCL11B与MYOSIN6在乳鼠耳蜗螺旋器中的表达Note：A. Expression and distribution of BCL11B and MYOSIN6 in inner hair cells under the low-magnification microscope (×10). It shows the merged image of the apical turn, middle turn and basal turn of the same cochlea, bar=200 μm. B/B'. Expression of BCL11B (B) and MYOSIN6 (B') in the hair cells of middle turn of whole mount samples. B". Merged image of B, B' and nuclear staining (×10). Bar=200 μm. C/C'. Expression of BCL11B (C) and MYOSIN6 (C') in the middle turn of whole mount samples under the high-magnification microscope (×60). C". Merged image of C, C' and nuclear staining (×60). Bar=20 μm. D/D'. Expression of BCL11B (D) and MYOSIN6 (D') in the middle turn of cross section samples under the high-magnification microscope (×60). D". Merged image of D, D' and nuclear staining (×60). Bar=20 μm. Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; white arrow represents IHC; black vertical line and yellow arrow represent OHC; OHC—outer hair cell; IHC—inner hair cell.

Fig 5 Expression of BCL11B and MYOSIN6 in the organ of Corti at P0 mouse cochlea by confocal imaging

图6 共聚焦显微镜下观察P2时BCL11B与MYOSIN6在乳鼠耳蜗螺旋器中的表达Note：A/A'. Expression of BCL11B (A) and MYOSIN6 (A') in the middle turn of whole mount samples under the high-magnification microscope (×60). A". Merged image of A, A' and nuclear staining (×60). B/B'. Expression of BCL11B (B) and MYOSIN6 (B') in the middle turn of cross section samples under the high-magnification microscope (×60). B". Merged image of B, B' and nuclear staining (×60). Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; white arrow represents IHC; black vertical line and yellow arrow represent OHC; OHC—outer hair cell; IHC—inner hair cell. Bar=20 μm.

Fig 6 Expression of BCL11B and MYOSIN6 in the organ of Corti at P2 mouse cochlea by confocal imaging

图7 共聚焦显微镜下观察P4时BCL11B与MYOSIN6在乳鼠耳蜗螺旋器中的表达Note：A/A'. Expression of BCL11B (A) and MYOSIN6 (A') in the apical turn of whole mount samples under the high-magnification microscope (×60). A". Merged image of A, A' and nuclear staining (×60). B/B'. Expression of BCL11B (B) and MYOSIN6 (B') in the middle turn of whole mount samples under the high-magnification microscope (×60). B". Merged image of B, B' and nuclear staining (×60). C/C'. Expression of BCL11B (C) and MYOSIN6 (C') in the basal turn of whole mount samples under the high-magnification microscope (×60). C". Merged image of C, C' and nuclear staining (×60). Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; black vertical line represents OHC; IHC—inner hair cell; OHC—outer hair cell. Bar=20 μm. As whole mount samples and cross section samples showed the same conclusion, only whole mount samples are presented here.

Fig 7 Expression of BCL11B and MYOSIN6 in the organ of Corti at P4 mouse cochlea by confocal imaging

图8 共聚焦显微镜下观察P7时BCL11B与MYOSIN6在乳鼠耳蜗螺旋器中的表达Note：A/A'. Expression of BCL11B (A) and MYOSIN6 (A') in the apical turn of whole mount samples under the high-magnification microscope (×60). A". Merged image of A, A' and nuclear staining (×60). B/B'. Expression of BCL11B (B) and MYOSIN6 (B') in the middle turn of whole mount samples under the high-magnification microscope (×60). B". Merged image of B, B' and nuclear staining (×60). C/C'. Expression of BCL11B (C) and MYOSIN6 (C') in the basal turn of whole mount samples under the high-magnification microscope (×60). C". Merged image of C, C' and nuclear staining (×60). Green area at the bottom right of figure A is the overexposed image of the white dotted box area. Green signal represents BCL11B, red signal represents MYOSIN6, and blue signal represents nucleus; black vertical line represents OHC; IHC—inner hair cell; OHC—outer hair cell. Bar=20 μm. As whole mount samples and cross section samples showed the same conclusion, only whole mount samples are presented here.

Fig 8 Expression of BCL11B and MYOSIN6 in the organ of Corti at P7 mouse cochlea by confocal imaging

图9 E16.5、P0及P4时小鼠螺旋器中BCL11B阳性表达的毛细胞的计数及百分比统计Note：A. Statistics on the count of BCL11B-expressing HCs in mice. HCs—hair cells. B. Percentage statistics of BCL11B-expressing HCs in mice.

Fig 9 Count and percentage statistics of BCL11B-expressing HCs in the organ of Corti of mice at E16.5, P0 and P4

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B细胞淋巴瘤/白血病11B在小鼠耳蜗螺旋器发育中的表达研究

Study on the expression of B-cell leukemia/lymphoma 11B in the organ of Corti during mice cochlea development

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