小鼠输卵管上皮类器官的构建及表型验证

doi:10.3969/j.issn.1674-8115.2023.07.007

摘要/Abstract

摘要：

目的·构建野生型（wild type，WT）小鼠和miR-34b/c^-/-且miR-449^-/-双敲除（double knockout，dKO）小鼠输卵管上皮类器官的培养体系，并进行表型验证。方法·利用酶消化法和差速贴壁法分离纯化WT小鼠和dKO小鼠输卵管上皮细胞，并利用免疫荧光法鉴定得到的输卵管上皮细胞的纯度；通过计数和直径测量比较WT小鼠和dKO小鼠输卵管上皮类器官数量、生长速度和生长大小；利用苏木精-伊红（hematoxylin-eosin，H-E）染色和透射电子显微镜（透射电镜）对输卵管上皮类器官进行形态和结构观察；采用免疫荧光法观察并统计纤毛细胞和分泌细胞在WT小鼠和dKO小鼠输卵管上皮类器官中的比例；采用免疫组织化学法、实时荧光定量PCR（RT-qPCR）和Western blotting检测分泌细胞、纤毛细胞相关基因在输卵管上皮类器官中的表达。结果·分离纯化得到的输卵管上皮细胞纯度较高。与WT小鼠相比，dKO小鼠输卵管上皮类器官生长更快，体积更大，数量也更多；但dKO小鼠输卵管上皮类器官发育较慢，于培养28 d时才出现上皮内陷，而WT小鼠的类器官于培养16 d时已经出现。H-E染色和透射电镜结果显示输卵管上皮类器官呈现出与在体输卵管类似的结构。免疫荧光检测显示dKO小鼠输卵管上皮类器官的纤毛细胞显著减少而分泌细胞显著增多（均P<0.05）。免疫组织化学法检测结果显示，dKO小鼠输卵管上皮类器官的分子表达模式与在体输卵管组织基本一致，即纤毛细胞标志物乙酰化微管蛋白α（Ac-α-tubulin）、叉头框J1（forkhead box J1，FOXJ1）表达减少，分泌细胞标志物配对盒8（paired box 8，PAX8）表达增加；RT-qPCR结果显示dKO小鼠输卵管上皮类器官中Foxj1和微管蛋白β4A（tubulin β class Ⅳa，Tubb4a）的mRNA水平均降低（均P<0.05），而Pax8 mRNA水平升高（P<0.05）；Western blotting结果显示，dKO小鼠类器官中FOXJ1的蛋白表达水平显著降低，PAX8表达显著升高（均P<0.05）。结论·研究成功构建WT小鼠和dKO小鼠的输卵管上皮类器官培养体系，该体系可模拟小鼠在体输卵管的表型。

关键词: 类器官, 输卵管, 上皮细胞, miR-34, miR-449, 基因敲除

Abstract:

Objective ·To establish a culture system of oviductal epithelial organoids from wild type (WT) mice and miR-34b/c^-/- and miR-449^-/- double knockout (dKO) mice, and verify the phenotypes. Methods ·The oviduct epithelial cells of WT mice and dKO mice were isolated and purified by enzyme digestion and differential adhesion method, and the purity of the isolated oviduct epithelial cells was identified by immunofluorescence staining. The numbers, growth rates and sizes of oviductal epithelial organoids between WT mice and dKO mice were compared by counting and diameter measurement. Hematoxylin-eosin (H-E) staining and transmission electron microscope (TEM) were used to observe the morphology and structure of the oviductal epithelial organoids. The proportions of ciliated cells and secretory cells in the oviductal epithelial organoids from WT mice and dKO mice were observed and counted by immunofluorescence staining. Immunohistochemistry (IHC), real-time quantitative PCR (RT-qPCR) and Western blotting were used to observe the expression levels of marker genes of ciliated cells and secretory cells in the oviductal epithelial organoids. Results ·The purity of the isolated and purified oviduct epithelial cells was high. Compared with the organoids from WT mice, the oviductal epithelial organoids from dKO mice grew faster and larger, and were more in number. But they developed more slowly than those from WT mice, as the invaginations of the dKO mice organoids appeared on the 28th day of culture, while the WT mice organoids exhibited the same structures on the 16th day. The oviductal epithelial organoids showed similar structures as those of the oviduct in vivo under hematoxylin-eosin (H-E) staining and TEM. Immunofluorescence staining showed that the ciliated cells of oviductal epithelial organoids from dKO mice were significantly reduced and the secretory cells were significantly increased (both P<0.05). IHC showed that the molecular expression patterns of the oviductal epithelial organoids were consistent with those of the oviducts in vivo, i.e. the expression levels of ciliated cell markers acetylated α-tubulin (Ac-α-tubulin) and forkhead box J1 (FOXJ1) decreased, and the expression level of the secretory cell marker paired box 8 (PAX8) increased. RT-qPCR showed that the mRNA levels of Foxj1 and tubulin β class Ⅳa (Tubb4a) decreased (both P<0.05), while Pax8 increased in the oviductal epithelial organoids of dKO mice (P<0.05). Western blotting results showed that the protein expression level of FOXJ1 in the organoids of dKO mice significantly decreased, while the expression of PAX8 significantly increased (both P<0.05). Conclusion ·The culture system of oviductal epithelial organoids of WT mice and dKO mice are successfully constructed, which can simulate the phenotypes of mouse oviduct in vivo.

Key words: organoid, oviduct, epithelial cell, miR-34, miR-449, gene knockout

中图分类号:

R-332

吴其谦, 胡燕琴, 陈迦勒, 李牧辰, 赵有淦, 伍静文. 小鼠输卵管上皮类器官的构建及表型验证[J]. 上海交通大学学报（医学版）, 2023, 43(7): 848-859.

WU Qiqian, HU Yanqin, CHEN Jiale, LI Muchen, ZHAO Yougan, WU Jingwen. Establishment and phenotype verification of mouse oviductal epithelial organoids[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(7): 848-859.

图/表 7

表1 实时荧光定量PCR引物序列

Tab 1 Primer sequences in RT-qPCR

Primer	Sequence (5′→3′)
Foxj1-F	AAGGAGGCAGAAATCCGGTG
Foxj1-R	TTGTAGCCTCCCTTGTGCAG
Pax8-F	CCCTTCGCCATAAAGCAGGA
Pax8-R	AGCATGGGGAAAGGCATTGA
Tubb4a-F	AACCCGGCACCATGGACTCTGT
Tubb4a-R	TGCCTGCTCCGGATTGACCAAATA
Ovgp1-F	TGGACCCCTTTCTTTGTACG
Ovgp1-R	TGGACAGCAGTGTTTTCAGC
Gapdh-F	TGGAAAGCTGTGGCGTGAT
Gapdh-R	GGGTAGGAACACGGAAGGC

图1 WT小鼠和dKO小鼠输卵管上皮类器官形态学比较Note： A. Different genotypes of mice detected by PCR. Triple (miR-34b/c+/- and miR-449-/-), miR-449-/-, and dKO (miR-34b/c-/- and miR-449-/-). B. Primary culture of the oviductal epithelial cells (×400, scale bar=50 μm). C. Identification of the purity of the isolated oviductal epithelial cells using immunofluorescent staining. Ac-α-tubulin for showing ciliated cells, PAX8 for showing secretory cells, ACTA2 for showing smooth muscle cells, and vimentin for showing fibroblasts (×400, scale bar=50 μm). D. Phase contrast images of organoids formation and growth from WT mice and dKO mice (×100, scale bar=200 μm). E. The numbers of the organoids with different sizes were counted after 8 d. F. The numbers of the organoids with different sizes were counted after 16 d. G. The total numbers of organoids were counted after 8 d and 16 d. ①P=0.002, ②P=0.007, ③P=0.004, ④P=0.006, ⑤P=0.001, ⑥P=0.011, ⑦P=0.013, compared with the WT group.

Fig 1 Morphological comparison of oviductal epithelial organoids from WT mice and dKO mice

图2 输卵管上皮类器官的长期培养及传代Note： A/B. Representative mature organoids with epithelial invaginations and foldings (arrows) after 16 d of WT mice (A) and after 28 d of dKO mice (B) (×100, scale bar=200 μm). C. Oviductal epithelial organoids culture of WT mice and dKO mice could be maintainted at least for 4 months and 17 passages (P17) (×100, scale bar=200 μm).

Fig 2 Long-term culture and passage of oviductal epithelial organoids

图3 光学显微镜下输卵管上皮类器官的结构(H-E染色, ×400)Note： Comparison of the histology structure of the oviducts and the oviductal epithelial organoids from WT mice and dKO mice after 2 months of culture (scale bar=50 μm).

Fig 3 Structure of the oviductal epithelial organoids under light microscopy (H-E staining, ×400)

图4 输卵管上皮类器官中纤毛细胞和分泌细胞的微观结构Note： A. Transmission electron microscopic images of the ciliated cells of oviductal epithelial organoids of WT mice and dKO mice after 2 months of culture (×10 000, scale bar=1 μm). The images on the right are the magnification ones in the dotted box on the left (×25 000, scale bar=500 nm). The white line showing the arrangement of the basal bodies, and the white arrows showing multiple disorganized centrioles. B. Transmission electron microscopic images of the secretory cells of oviductal epithelial organoids of WT mice and dKO mice after 2 months of culture (×10 000, scale bar=1 μm). The images on the right are the magnification ones in the dotted box on the left (×25 000, scale bar=500 nm). The white arrows showing the exocytosis granules by the secretory cells.

Fig 4 Microstructures of ciliated cells and secretory cells in the oviductal epithelial organoids

图5 输卵管上皮类器官的细胞构成Note： A/B. Co-immunofluorescent staining of Ac-α-tubulin (showing ciliated cells) and PAX8 (showing secretory cells) in the organoid sections (A) and the oviducts (B) of WT mice and dKO mice (×400, scale bar=50 μm). C/D. Quantification of ciliated cells (C) and secretory cells (D) of the organoids from WT mice and dKO mice across different culture time. ①P=0.004, ②P=0.005, ③P=0.001, ④P=0.000, compared with the WT group.

Fig 5 Cell composition of the oviductal epithelial organoids

图6 纤毛细胞与分泌细胞相关基因在输卵管上皮类器官中的表达Note： A. Expression and localization of Ac-α-tubulin, FOXJ1, and PAX8 in the oviductal tissues and oviductal epithelial organoids of WT mice and dKO mice using immunohistochemical staining (×400, scale bar=50 μm). B. The expression levels of ciliated cell- and secretory cell-related genes of the oviductal epithelial organoids from WT mice and dKO mice detected by RT-qPCR. Tubb4a and Foxj1 for ciliated cells. Pax8 and Ovgp1 for secretory cells. C?E. Western blotting analysis of FOXJ1 and PAX8 protein levels of oviductal epithelial organoids from WT mice and dKO mice (C) with quantitative analysis of FOXJ1 (D) and PAX8 (E). ①P=0.002, ②P=0.001, ③P=0.000, ④P=0.018, compared with the WT group..

Fig 6 Expression levels of ciliated cell- and secretory cell-related genes in oviductal epithelial organoids

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