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浙江大学学报（医学版）
浙江大学学报（医学版）  2022, Vol. 51 Issue (2): 204-214    DOI: 10.3724/zdxbyxb-2021-0354
原著     
高脂饮食诱导的肥胖早孕小鼠子宫内膜蜕膜反应受损
陈子轩,李伟轲,陈雪梅,刘学庆,丁裕斌,李方方,何俊琳,王应雄,高茹菲()
重庆医科大学公共卫生与管理学院生殖生物学研究室 教育部生殖与发育国际合作联合 实验 室，重庆 400016
Impairment of endometrial decidual reaction in early pregnant mice fed with high fat diet
CHEN Zixuan,LI Weike,CHEN Xuemei,LIU Xueqing,DING Yubin,LI Fangfang,HE Junlin,WANG Yingxiong,GAO Rufei()
Laboratory of Reproductive Biology, Joint International Research Laboratory of Reproduction & Development, School of Public Health and Management, Chongqing Medical University, Chongqing 400016, China
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摘要：

目的： 探讨高脂饮食诱导的肥胖对小鼠子宫内膜蜕膜反应的影响。 方法： 将12只4周龄健康C57BL/6J雌性小鼠随机分为高脂组和对照组（每组6只），分别饲喂高脂 （22?kJ/g）、 正常 （16?kJ/g） 饲料12周。每周测量小鼠体重，喂养12周后测量小鼠体长、体宽并检测空腹血清三酰甘油和总胆固醇水平。将雌鼠与健康C57BL/6J雄鼠合笼交配，收集妊娠第7天子宫组织，采用苏木精-伊红染色观察子宫内膜细胞形态，Masson染色观察子宫内膜胶原纤维；采用免疫组织化学法、蛋白质印迹法检测子宫内膜蜕膜反应相关蛋白表达。分离原代小鼠子宫内膜基质细胞，油酸、棕榈酸模拟高脂环境进行干预，雌二醇、孕酮诱导细胞发生蜕膜反应后，油 红O、 Bodipy染色观察脂滴累积情况，鬼笔环肽染色观察细胞骨架，实时荧光定量PCR、蛋白质印迹法检测蜕膜反应相关标志物的基因和蛋白表达。 结果 ：饲养12周后，高脂组体重明显高于对照组（ P <0.01），两组体长无明显差异（ P >0.05），但高脂组体宽显著大于对照组（ P <0.01），血清三酰甘油和总胆固醇水平均显著高于对照组（均 P <0.05）。高脂组胚胎着床数显著少于对照组（ P <0.01），高脂组子宫内膜基质细胞向蜕膜细胞转换缓慢且形态异常，蜕膜反应标志物骨形成蛋白（BMP）2、同源异形框A10（HOXA10）表达水平均低于对照组，其中两组HOXA10表达水平差异有统计学意义（ P <0.01）。油红O、Bodipy染色结果显示，原代小鼠子宫内膜基质细胞经高脂处理后，脂滴堆积明显增多，鬼笔环肽染色发现其细胞骨架形态异常，蜕膜反应相关基因 dtprp 、 HOXA10 以及蛋白BMP2、HOXA10、环氧合酶2的表达水平均明显低于对照组（均 P <0.05）。 结论 ：高脂饮食诱导的肥胖会损伤小鼠子宫内膜的蜕膜反应。
关键词： 高脂饮食;  肥胖;  胚胎着床;  蜕膜反应;  小鼠;  子宫内膜基质细胞    
Abstract:

Objective: To investigate the effect of obesity induced by high fat diet on decidual reaction of endometrium in mice, and the effect of high fat treatment on decidual reaction of endometrial stromal cells. Methods: Twelve 4-week-old healthy C57BL/6J female mice were randomly divided into high fat diet group and control group with 6 mice in each group. They were fed with high fat diet (22?kJ/g) or normal diet (16?kJ/g) for 12 weeks, respectively. The body weight of mice was measured every week. After feeding for 12 weeks, the body length and width of mice were measured, and the levels of fasting serum triglyceride and total cholesterol were determined. Then the mice were mated with healthy C57BL/6J male mice, and the uterine tissues were collected on the seventh day of pregnancy. The decidual cells and collagen fibers in mouse endometrium was observed by HE staining and Masson staining respectively. The expression of decidual reaction related proteins in mouse endometrium were detected by immunohistochemistry and Western blotting. Mouse endometrial stromal cells (mESCs) were isolated and treated with the oleic acid and palmitic acid in vitro , and the decidual reaction was induced with estradiol and progesterone. The accumulation of lipid droplets in mESCs was observed by oil red O and Bodipy staining. The cytoskeleton of mESCs was observed by phalloidin staining. The levels of decidual reaction related genes and proteins were detected by real-time fluorescence quantitative PCR and Western blotting. Results: After feeding for 12 weeks, the body weight of mice in the high fat group was significantly higher than that in the control group ( P <0.01), and there was no significant difference in body length between two groups ( P >0.05), but the body width of mice in the high fat group was significantly larger than that in the control group ( P <0.01), and the levels of serum triglyceride and total cholesterol were significantly higher than those in the control group (Both P <0.05). The number of embryo implantation in the high fat group was significantly less than that in the control group ( P <0.01). The differentiation of mESCs to decidual cells in high fat group was slow and abnormal. The expression levels of decidual reaction markers bone morphogenetic protein (BMP)2 and homeobox A10 (HOXA10) were lower than those in the control group, and there was significant difference in the expression level of HOXA10 ( P <0.01). The results of oil red O and Bodipy staining in mESCs showed that after high fat treatment, the accumulation of lipid droplets increased significantly, phalloidin staining showed abnormal cytoskeleton morphology. The expression levels of decidual reaction related genes dtprp , HOXA10 and proteins BMP2, HOXA10 and cyclooxygenase (COX)2 were significantly lower than those in the control group ( P <0.05). Conclusion: Obesity induced by high fat diet and high fat treatment can impair the decidual reaction of endometrium and endometrial stromal cells in mice.
Key words: High fat diet    Obesity    Embryo implantation    Decidual reaction    Mouse    Endometrial stromal cells
收稿日期: 2021-11-20 出版日期: 2022-08-02
CLC:  R714.14  
基金资助: 重庆市研究生科研创新项目（CYS20207）；重庆市教育委员会科学技术计划项目（KJQN201900413）
通讯作者: 高茹菲     E-mail: gao_ru_fei@cqmu.edu.cn
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陈子轩
李伟轲
陈雪梅
刘学庆
丁裕斌
李方方
何俊琳
王应雄
高茹菲

引用本文:

陈子轩,李伟轲,陈雪梅,刘学庆,丁裕斌,李方方,何俊琳,王应雄,高茹菲. 高脂饮食诱导的肥胖早孕小鼠子宫内膜蜕膜反应受损[J]. 浙江大学学报（医学版）, 2022, 51(2): 204-214.

CHEN Zixuan,LI Weike,CHEN Xuemei,LIU Xueqing,DING Yubin,LI Fangfang,HE Junlin,WANG Yingxiong,GAO Rufei. Impairment of endometrial decidual reaction in early pregnant mice fed with high fat diet. J Zhejiang Univ (Med Sci), 2022, 51(2): 204-214.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0354    或    https://www.zjujournals.com/med/CN/Y2022/V51/I2/204

基因

序列（5′→3′）

dtprp

正向：AGCCAGAAATCACTGCCACT

反向：TGATCCATGCACCCATAAAA

HOXA10

正向：GGCAGTTCCAAAGGCGAAAAT

反向：GTCTGGTGCTTCGTGTAAGGG

β-actin

正向：GGCTGTATTCCCCTCCATCG

反向：CCAGTTGGTAACAATGCCATGT
表 1  实时荧光定量PCR引物序列
图 1  高脂组与对照组体重增长曲线及体型比较 A：小鼠体重增长曲线. 与对照组比较， <0.01；B：小鼠妊娠第7天体型外观.标尺=2 cm.
图 2  高脂饮食诱导的肥胖早孕小鼠子宫组织病理学变化
图 3  高脂饮食诱导的肥胖早孕小鼠子宫内膜蜕膜反应标志物表达变化 A：小鼠妊娠第7天子宫内膜蜕膜反应标志物BMP2的表达.BMP2蛋白主要定位在初级蜕膜区、次级蜕膜区，且高脂组BMP2表达强度显著低于对照组（黑色圈指示蜕膜区），左图标尺 =500 μm， 右图标尺=100 μm.B：小鼠妊娠第7天子宫内膜蜕膜反应标志物HOXA10、BMP2的表达. HOXA10：同源异形框A10；BMP：骨形成蛋白；β-actin：β肌动蛋白.

组 别

n

HOXA10

BMP2

对照组

3

1.45±0.13

1.49±0.45

高脂组

3

0.38±0.21 **

1.13±0.11 **

t 值

—

6.012

1.104

P 值

—

<0.01

>0.05
表 2  小鼠妊娠第7天子宫内膜蜕膜反应标志物HOXA10、BMP2表达水平比较
图 4  高脂处理诱导蜕膜反应细胞模型构建成功 A：普通显微镜下观察细胞状态.标尺=100 μm；B：油红O染色观察细胞脂滴堆积情况，高脂处理后蜕膜细胞中红色的脂滴累积增加，标尺=20 μm；C：Bodipy染色观察细胞脂滴堆积情况，高脂处理后蜕膜细胞中出现大量绿色荧光脂滴堆积.标尺 =20 μm.
图 5  鬼笔环肽染色法观察细胞骨架形态 鬼笔环肽染色法观察细胞骨架形态，对照组细胞呈明显成纤维样梭形，胞体小，扁平星形，呈放射状生长；蜕膜组细胞核明显变大变圆，细胞质丰富，骨架大而圆，呈现明显的蜕膜样变化；高脂组细胞呈不规则多边形，细胞核较小，未能达到正常蜕膜细胞形态. 标尺 =20 μm.
图 6  各组细胞蜕膜反应相关蛋白COX2、HOXA10、BMP2表达比较 A：各组细胞蜕膜反应相关蛋白电泳图；B：各组细胞蜕膜反应相关蛋白灰度值比较. COX：环氧合酶；HOXA10：同源异形框A10；BMP：骨形态发生蛋白；β-actin：β-肌动蛋白. 与对照组比较， <0.05， <0.01；与蜕膜组比较， <0.01.

组别

n

dtprp

HOXA10

对照组

3

0.75±0.11

0.77±0.06

蜕膜组

3

138.48±6.94 **

1.28±0.11 *

高脂组

3

25.83±15.44 **##

0.39±0.14 **##

F 值

—

113.27

32 .42

P 值

—

<0.01

<0.01
表 3  各组细胞蜕膜反应相关基因 、 表达比较
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