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北京大学学报（医学版） ›› 2022, Vol. 54 ›› Issue (5): 846-852. doi: 10.19723/j.issn.1671-167X.2022.05.012

• 论著 • 上一篇    下一篇

Kindlin-2通过mTOR和Hippo信号通路调节小鼠子宫内膜发育

张京1,宋佳桂1,2,王振斌1,龚玉清1,王天卓1,周津羽1,战军1,*(),张宏权1,*()   

  1. 1. 北京大学基础医学院人体解剖与组织胚胎学系，北京 100191
    2. 北京大学第三医院医学创新研究院基础医学研究中心，北京 100191
  • 收稿日期:2022-06-16 出版日期:2022-10-18 发布日期:2022-10-14
  • 通讯作者: 战军,张宏权 E-mail:Zhanjun@bjmu.edu.cn;Hongquan.zhang@bjmu.edu.cn
  • 作者简介:张宏权，分子遗传学博士，博士生导师，北京大学基础医学院新体制长聘教授。北京大学学术委员会委员，北京大学医学部学术委员会委员，北京大学基础医学院、药学院、前沿交叉和生物技术学术委员会委员及北京大学第三临床学院学术委员会委员。北京大学国际癌症研究院副院长，基础医学院肿瘤、细胞和衰老学科群主任，人体解剖与组织胚胎学系主任。北京大学天然药物及仿生药物国家重点实验室、北京大学国际癌症研究院、北京大学基础医学院课题组长(principal investigator, PI)。担任国家科技部973项目、重点基础研究专项课题、国家自然科学基金及北京市自然科学基金的负责人。
    在欧美从事肿瘤生物学科研与教学15年，在国内从事组织学与胚胎学及肿瘤生物学的教学与科研17年。主要研究兴趣是基质生物学、肿瘤的发生、侵袭和转移的分子细胞生物学机制、肿瘤干细胞的重编程机制及生殖细胞的调控等。发现了一系列与跨膜受体整合素胞内结构域互作的蛋白，并阐明了其生理和病理意义; 揭示了整合素在细胞膜上运输的机制; 发现乳腺癌干细胞诱导的新机制和干预靶点; 发现乳腺癌转移的表观遗传调控机制和干预靶点; 建立了自发产生雌激素受体阳性或三阴乳腺癌的小鼠模型; 发现并鉴定了一系列组蛋白的新修饰类型并阐明其在生理和肿瘤发生发展中的意义。目前担任中国解剖学会副理事长、组织学与胚胎学专业委员会副主任委员，中国生理学会基质生物学专业委员会前任主任委员，中国抗癌协会肿瘤转移专业委员会主任委员，中国抗癌协会血液病转化医学专业委员会副主任委员，中国抗癌协会肺癌专业委员会委员，中国病理生理学会蛋白质修饰与疾病专业委员会委员，国际上皮间质转化(Epithelial Mesenchymal Transition, EMT)专家委员会唯一中国委员。担任《解剖学报》和《中国组织化学与细胞化学杂志》副主编，Cellular Signalling、Frontier in Oncology和《北京大学学报(医学版)》编委。在Cell、Nature Cell Biology、Molecular Cell、Nature Communications、Cell Research、Cancer Research、Journal of Cell Biology、Cell Reports、Nucleic Acids Research和 Journal of American Society of Nephrology等国际一流期刊发表130余篇论文。获得2项国家发明专利和1项国际专利并转化，获军队科技进步一等奖1项
  • 基金资助:
    国家自然科学基金(81730071);国家自然科学基金(82172972);国家自然科学基金(31170711);国家自然科学基金(81670626);国家自然科学基金(81902840);北京大学双一流建设经费(PKU2021LCXQ023);北京大学双一流建设经费(BMU2022XKQ004)

Kindlin-2 regulates endometrium development via mTOR and Hippo signaling pathways in mice

Jing ZHANG1,Jia-gui SONG1,2,Zhen-bin WANG1,Yu-qing GONG1,Tian-zhuo WANG1,Jin-yu ZHOU1,Jun ZHAN1,*(),Hong-quan ZHANG1,*()   

  1. 1. Department of Human Anatomy, Histology and Embryology, Peking University School of Basic Medical Sciences, Beijing 100191, China
    2. Center of Basic Medical Research, Institute of Medical Innovation and Research, Cancer Center, Peking University Third Hospital, Beijing 100191, China
  • Received:2022-06-16 Online:2022-10-18 Published:2022-10-14
  • Contact: Jun ZHAN,Hong-quan ZHANG E-mail:Zhanjun@bjmu.edu.cn;Hongquan.zhang@bjmu.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(81730071);the National Natural Science Foundation of China(82172972);the National Natural Science Foundation of China(31170711);the National Natural Science Foundation of China(81670626);the National Natural Science Foundation of China(81902840);Peking University Double First-Class Construction Funds(PKU2021LCXQ023);Peking University Double First-Class Construction Funds(BMU2022XKQ004)

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摘要：

目的: 探讨Kindlin-2对小鼠子宫发育及雌鼠生育能力的影响及其作用机制。方法: 利用Cdh16-Cre工具鼠和Kindlin-2flox/flox小鼠构建在子宫内膜中特异性敲除Kindlin-2的小鼠模型，观察敲除Kindlin-2对雌鼠子宫内膜发育和生殖力的影响。在子宫内膜癌细胞系HEC-1和Ish中分别进行高表达和敲低Kindlin-2的实验，检测雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)信号通路的激活变化，并且提取特异性敲除Kindlin-2的雌鼠(实验组，基因型为Cdh16-Cre; Kindlin-2flox/flox)和未特异性敲除Kindlin-2的雌鼠(对照组，基因型为Kindlin-2flox/flox)子宫蛋白，每组包含6~8只小鼠，重复3次独立实验，检测mTOR信号通路和Hippo信号通路关键分子的蛋白水平。结果: 成功构建了子宫内膜特异性敲除Kindlin-2的小鼠模型，通过鼠尾聚合酶链式反应(polymerase chain reaction，PCR)、Western blot、免疫组织化学染色(immunohistochemistry，IHC)等方法鉴定和验证Kindlin-2在小鼠子宫中的敲除效率。子宫内膜特异性敲除Kindlin-2的雌鼠与对照组相比体质量减轻、生殖能力严重受损、出生仔鼠数量减少，但出生仔鼠中雌鼠和雄鼠的比例未发生改变，通过苏木精-伊红染色实验观察表明实验组子宫内膜发育不完整、子宫壁厚度变薄。机制方面，子宫内膜癌细胞系HEC-1和Ish中敲除Kindlin-2能够下调mTOR、磷酸化mTOR、腺嘌呤核糖核苷酸激活蛋白激酶(adenosine monophosphate-activated protein kinase，AMPK)、磷酸化的AMPK和磷酸化的核糖体蛋白(ribosomal protein S6，S6)的蛋白水平，在雌鼠子宫中发现特异性敲除Kindlin-2能够上调Mps结合1(Mps one binding 1，MOB1)、磷酸化的Yes相关蛋白(Yes-associated protein，YAP)的蛋白水平。结论: Kindlin-2通过抑制mTOR信号通路、激活Hippo信号通路抑制子宫内膜的发育，进而抑制雌鼠的生育能力。

关键词: Kindlin-2, 小鼠，特异性敲除, 子宫内膜, mTOR信号通路, Hippo信号通路

Abstract:

Objective: To investigate the effects and mechanisms of Kindlin-2 on uterus development and reproductive capacity in female mice. Methods: Cdh16-Cre tool mice and Kindlin-2flox/flox mice were used to construct the mouse model of uterus specific knockout of Kindlin-2, and the effects of Kindlin-2 deletion on uterine development and reproduction capacity of female mice were observed. High expression and knockdown of Kindlin-2 in endometrial cancer cell lines HEC-1 and Ish were used to detect the regulation of mammalian target of rapamycin (mTOR) signaling pathway. In addition, uterine proteins of the female mice with specific knockout of Kindlin-2 and female mice in the control group were extracted to detect the protein levels of key molecules of mTOR signaling pathway and Hippo signaling pathway. Results: The mouse model of uterine specific knockout of Kindlin-2 was successfully constructed. The knockout efficiency of Kindlin-2 in mouse uterus was identified and verified by mouse tail polymerase chain reaction (PCR), Western blot protein identification, immunohistochemical staining (IHC) and other methods. Compared with the control group, the female mice with uterus specific deletion of Kindlin-2 lost weight, seriously impaired reproductive ability, and the number of newborn mice decreased, but the proportion of the female mice and male mice in the newborn mice did not change. Hematoxylin eosin staining (HE) experiment showed that the endometrium of Kindlin-2 knockout group was incomplete and the thickness of uterine wall became thinner. In terms of mechanism, the deletion of Kindlin-2 in endo-metrial cancer cell lines HEC-1 and Ish could downregulate the protein levels of mTOR, phosphorylated mTOR, adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK and phosphorylated ribosomal protein S6 (S6), and the mTOR signal pathway was inhibited. It was found that the specific deletion of Kindlin-2 could upregulate the protein levels of Mps one binding 1 (MOB1) and phosphorylated Yes-associated protein (YAP) in the uterus of the female mice, and the Hippo signal pathway was activated. Conclusion: Kindlin-2 inhibits the development of uterus by inhibiting mTOR signal pathway and activating Hippo signal pathway, thereby inhibiting the fertility of female mice.

Key words: Kindlin-2, Mouse model, specific knockout, Endometrium, mTOR signaling pathway, Hippo signaling pathway

中图分类号: 

  • R34

图1

鉴定子宫内膜特异性敲除Kindlin-2小鼠模型"

图2

子宫内膜特异性敲除Kindlin-2引起雌鼠生育力下降"

表1

实验组与对照组出生仔鼠雌雄数量"

Items Female of newborn mice, n Male of newborn mice, n Total, n
Ctrl 56 58 114
Kindlin-2 KO 28 25 53
Total 84 83 167

图3

子宫内膜特异性敲除Kindlin-2导致雌鼠子宫发育异常"

图4

子宫内膜特异性敲除Kindlin-2抑制mTOR信号通路、激活Hippo信号通路"

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ISSN 1671-167X
CN 11-4691/R
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