K14阳性细胞在下颌下腺损伤修复中的分布特征

doi:10.3969/j.issn.1005-4979.2021.06.001

摘要/Abstract

摘要：

目的：角蛋白14（cytokeratin 14, K14）是唾液腺干/祖细胞的标志物，本研究探讨K14⁺细胞在下颌下腺损伤修复中表达分布的特征。方法：构建8周龄小鼠双侧下颌下腺主导管结扎-开放模型，分别于结扎后0、14 d及开放后3、28、56 d收样，利用组织学染色法评估下颌下腺损伤及再生状况，免疫荧光染色法观察K14⁺细胞增殖，实时荧光定量PCR检测唾液腺功能相关基因表达的变化。结果：在下颌下腺发育过程中K14⁺细胞随着年龄增长逐渐减少；成功构建小鼠下颌下腺主导管结扎-开放模型，导管结扎后，腺体萎缩，导管扩张；开放导管后，腺体修复早期K14⁺细胞数目增多，增殖活跃，但是唾液腺功能相关基因表达较低。结论：在下颌下腺损伤修复早期K14⁺细胞表达增加，提示K14⁺细胞参与了下颌下腺损伤修复的过程。

关键词: 下颌下腺, 损伤, 再生, 角蛋白14

Abstract:

Objective: Cytokeratin 14(K14) is the marker of salivary gland stem/progenitor cells. This study aimed to explore the expression and role of K14⁺ during the injury and regeneration process of the submandibular gland (SMG). Methods: The main salivary gland ducts of 8-week-old mice were ligated for 14-d and then deligated. The mice were sacrificed at 0-d, 14-d after ligation and 3-d, 28-d, 56-d after deligation. The injury and regeneration of SMG were observed by histological staining, and the proliferation of K14⁺ cells was observed by immunofluorescence staining. The mRNA expression level and changes of genes related to salivary gland function were detected by real-time PCR. Results: Mouse SMG duct ligation-deligation model was successfully established. The number of K14⁺ cells gradually decreased over time during the development period of the SMG. The glands showed atrophy and the duct showed dilatation after the main duct was ligated; the number of K14⁺ cells increased in the early stage of SMG regeneration, and most of them were in a proliferating state; but the expression level of genes related to salivary gland function was low. Conclusion: During the injury and regeneration process of the SMG, the expression of K14⁺ cells increased in the early stage during regeneration, suggesting that K14⁺ cells are involved in the regeneration process of the SMG.

Key words: submandibular gland, injury, regeneration, cytokeratin 14

周济, 张雪明, 耿江予, 牛萍萍, 孙瑶. K14阳性细胞在下颌下腺损伤修复中的分布特征[J]. 《口腔颌面外科杂志》, 2021, 31(6): 335-341.

ZHOU Ji, ZHANG Xueming, GENG Jiangyu, NIU Pingping, SUN Yao. Distribution characteristics of K14 positive cells during the injury and regeneration of the submandibular gland[J]. 《Journal of Oral and Maxillofacial Surgery》, 2021, 31(6): 335-341.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2021/V31/I6/335

图/表 6

图1 下颌下腺主导管结扎示意图 A. 小鼠下颌下腺解剖示意图；B. 小鼠下颌下腺主导管结扎示意图。

Figure 1 The schematic model of mouse SMG duct ligation-deligation

表1 qPCR引物序列

Table 1 Primer sequences of qPCR

基因	正向引物序列（5’-3’）	反向引物序列（5’-3’）
甘油醛-3-磷酸脱氢酶（GAPDH）	TGTTCCTACCCCCAATGTGT	GGTCCTCAGTGTAGCCCAAG
K14	AGCGGCAAGAGTGAGATTTCT	CCTCCAGGTTATTCTCCAGGG
角蛋白5（cytokeratin 5, K5）	GGAGCAGATCAAGACCCTCA	CGGATCCAGGTTCTGCTTTA
表皮生长因子（epidermal growth factor, EGF）	CATCATGGTGGTGGCTGTCTG	CACTTCCGCTTGGCTCATCA
神经生长因子（nerve growth factor, NGF）	CCAGTGAAATTAGGCTCCCTG	CCTTGGCAAAACCTTTATTGGG
水通道蛋5（aquaporin 5, AQP5）	CCTTCTTCAAGGCCGTGTTC	AGGCTATGGAGATCTGCAGAATG

图2 下颌下腺发育过程中K14⁺细胞表达特点（×180） A~D. 胚胎16.5 d、出生后1、3、8周小鼠下颌下腺中K14表达特点；E. 小鼠下颌下腺发育过程中K14阳性细胞百分比；**表示与胚胎16.5 d比较，P<0.01。

Figure 2 Expression characteristics of K14⁺ cells during the development of SMG（×180）

图3 下颌下腺在主导管结扎-开放模型中形态学变化 A. 下颌下腺导管结扎及开放后收样时间点；B~D. 结扎0、14 d及开放56 d的小鼠腺体大体图片；E. 结扎0、14 d及开放3、28、56 d的小鼠腺体质量/体质量变化情况；F~J. 结扎0、14 d及开放3、28、56 d的小鼠下颌下腺HE染色（×40）；*表示两组相比，P<0.05，**表示两组相比，P<0.01。

Figure 3 Comparison of morphological variation of SMG in duct ligation-deligation model

图4 小鼠下颌下腺损伤修复过程中分泌功能相关蛋白AQP5和NGF的表达 A~E. 下颌下腺损伤修复过程中NGF表达变化；F~J. 下颌下腺损伤修复过程中AQP5表达变化；K~N. 下颌下腺损伤修复过程中K5、Egf、NGF、AQP5 mRNA表达量变化（×60）；*表示两组相比，P<0.05；**表示两组相比，P<0.01。

Figure 4 Expression of secretory function-related proteins and genes during the injury and repair of mouse SMG

图5 小鼠下颌下腺损伤修复过程中K14⁺细胞分布特征 A~E. 结扎0、14 d及开放3、28、56 d的小鼠下颌下腺中K14和Ki67表达变化（×120）；F. K14和Ki67双阳性细胞比率；G.结扎0、14 d及开放3、28、56 d的小鼠下颌下腺K14 mRNA表达水平；*表示两组比较，P<0.05，**表示两组比较，P<0.001。

Figure 5 Expression and distribution characteristics of K14⁺ cells during the injury and regeneration process of mouse SMG

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