Human chorionic gonadotropin promotes in vitro embryo implantation by up-regulating the expression of integrin beta3 and osteopontin

doi:10.3877/cma.j.issn.1673-5250.2019.06.008

Objective

To explore the molecular regulatory mechanism of human chorionic gonadotropin (hCG) effect on embryo implantation in vitro.

Methods

Human trophoblast cell line BeWo and human endometrial cell line RL95-2 were selected as study cell lines. BeWo cells and RL95-2 cells were co-cultured to establish an in vitro embryo implantation model. Based on this model, the following research was carried out. ① The expression locations of integrin β₃ and its ligand osteopontin (OPN) in BeWo cells were observed by indirect immunofluorescence; ② The effects of different contents of hCG on the relative expressions of integrin β₃ and OPN in BeWo cells were analyzed through reverse transcription polymerase chain reaction (RT-PCR) technique; ③ The effects of hCG on the adhesion and implantation of BeWo spheroids to RL95-2 monolayer were observed by BeWo spheroids adhesion and outgrowth assays. The differences of relative expressions of integrin β₃ and OPN mRNA were compared among different contents of hCG treated to BeWo cell, also the adhesion rates and expansion times of BeWo spheroids were compared between before and after hCG treatment by one-way ANOVA or t test.

Results

① Integrin β₃ and OPN were both expressed on the membrane surface of BeWo cells. ② The relative mRNA expression levels of integrin β₃ and OPN were (2.07±0.57) and (1.95±0.48), respectively, after BeWo cells being treated with 1 000 IU/mL hCG, which were both higher than those of (1.12±0.31) and —(8.44±0.18), respectively, after BeWo cells being treated with 10 IU/mL hCG; Also, the relative mRNA expression level of integrin β₃ after BeWo cells being treated with 1 000 IU/mL hCG was higher than that of (1.24±0.19) after BeWo cells being treated with 100 IU/mL hCG, and the differences above were statistically significant (P=0.007, 0.009, 0.014). ③ After BeWo cells being treated with 1 000 IU/mL hCG, the adhesion rate of BeWo globules co-cultrued with RL95-2 monolayers for 0.5 h was (89.51±6.47)%, which was significantly higher than that of (50.24±7.73)% of BeWo cells without being treated with hCG, and the difference was statistically significant (t=90.900, P<0.001). ④ After BeWo spheroids and RL95-2 monolayers being treated with 1 000 IU/mL hCG for 24 hours, the expansion times of BeWo spheroids was (2.28±0.52), which was higher than that of (1.93±0.36) without being treated with hCG, and the difference was statistically significant (t=6.864, P=0.012).

Conclusion

Being treated with hCG may promote embryo implantation in vitro by up-regulating the expression of integrin β₃ and OPN on trophoblast surface.

Key words: Chorionic gonadotropin, Integrin beta3, Osteopontin, Trophoblasts, Embryo implantation, In vitro, BeWo cell line, RL95-2 cell line

表1 本研究采用的主要试剂、抗体及试剂盒等材料

材料名称	批号	生产厂家
DMEM/F12培养基	07116003	美国Corning公司
胎牛血清	2036230	美国GIBCO公司
0.25%胰酶(含EDTA)	2062475	美国GIBCO公司
hCG	20170809	以色列Prospec公司
胰岛素干粉	SLCD4698	美国Sigma公司
PVP干粉	SLCD1677	美国Sigma公司
BSA	SLCD6786	美国Sigma公司
DABCO封片剂	SLCD3612	美国Sigma公司
TRIzol	S8012	美国Invitrogen公司
RNase free dH₂O	2121661	美国Invitrogen公司
4%多聚甲醛	20161118	中国Solarbio公司
Tween-20	041816160917	中国碧云天公司
DAPI	041816160917	中国碧云天公司
羊抗人整合素β₃一抗(1∶100)	sc-6627	美国Santa Cruz公司
鼠抗人OPN一抗(1∶100)	sc-21742	美国Santa Cruz公司
驴抗羊IgG-TR二抗(1∶100)	sc-2783	美国Santa Cruz公司
羊抗鼠IgG-FITC二抗(1∶100)	sc-2010	美国Santa Cruz公司
逆转录试剂盒PrimeScript? RT Master Mix(cat.# RR036)	AI20777A	中国大连TaKaRa公司
荧光定量试剂盒SYBR Premix Ex Taq(Tli RNaseH Plus)(cat.# RR420)	AI12393A	中国大连TaKaRa公司
70 μm、100 μm细胞滤网	BD20161211	美国BD公司
24孔板专用细胞爬片	20171112	中国Solarbio公司

表1 本研究采用的主要试剂、抗体及试剂盒等材料

材料名称	批号	生产厂家
DMEM/F12培养基	07116003	美国Corning公司
胎牛血清	2036230	美国GIBCO公司
0.25%胰酶(含EDTA)	2062475	美国GIBCO公司
hCG	20170809	以色列Prospec公司
胰岛素干粉	SLCD4698	美国Sigma公司
PVP干粉	SLCD1677	美国Sigma公司
BSA	SLCD6786	美国Sigma公司
DABCO封片剂	SLCD3612	美国Sigma公司
TRIzol	S8012	美国Invitrogen公司
RNase free dH₂O	2121661	美国Invitrogen公司
4%多聚甲醛	20161118	中国Solarbio公司
Tween-20	041816160917	中国碧云天公司
DAPI	041816160917	中国碧云天公司
羊抗人整合素β₃一抗(1∶100)	sc-6627	美国Santa Cruz公司
鼠抗人OPN一抗(1∶100)	sc-21742	美国Santa Cruz公司
驴抗羊IgG-TR二抗(1∶100)	sc-2783	美国Santa Cruz公司
羊抗鼠IgG-FITC二抗(1∶100)	sc-2010	美国Santa Cruz公司
逆转录试剂盒PrimeScript? RT Master Mix(cat.# RR036)	AI20777A	中国大连TaKaRa公司
荧光定量试剂盒SYBR Premix Ex Taq(Tli RNaseH Plus)(cat.# RR420)	AI12393A	中国大连TaKaRa公司
70 μm、100 μm细胞滤网	BD20161211	美国BD公司
24孔板专用细胞爬片	20171112	中国Solarbio公司

表2 本研究3种引物的相关信息

基因	基因库(Gene Bank)序号	正向引物	反向引物	扩增片段长度(bp)
整合素β₃	NM_000212	5′-CATCCTGGTGGTCCTGCTCT-3′	5′-GCCTCTTTATACAGTGGGTTGTT-3′	247
OPN	NM_001040058.1	5′-TGGCCGAGGTGATAGTGTG-3′	5′-CGGGGATGGCCTTGCATG-3′	205
β-actin	NM_001101	5′-AAGACCTGTACGCCAACACAGT-3′	5′-AGAAGCATTTGCGGTGGACGAT-3′	348

表2 本研究3种引物的相关信息

基因	基因库(Gene Bank)序号	正向引物	反向引物	扩增片段长度(bp)
整合素β₃	NM_000212	5′-CATCCTGGTGGTCCTGCTCT-3′	5′-GCCTCTTTATACAGTGGGTTGTT-3′	247
OPN	NM_001040058.1	5′-TGGCCGAGGTGATAGTGTG-3′	5′-CGGGGATGGCCTTGCATG-3′	205
β-actin	NM_001101	5′-AAGACCTGTACGCCAACACAGT-3′	5′-AGAAGCATTTGCGGTGGACGAT-3′	348

图1 整合素β₃和骨桥蛋白在无处理的BeWo单层细胞中定位表达的免疫荧光染色图(图1A：DAPI染成蓝色的细胞核；图1B：显示为红色荧光的整合素β₃；图1C：显示为绿色荧光的骨桥蛋白；图1D：图1A、1B、1C的合成效果图，显示细胞核部分基本为蓝色，而绿色和红色荧光聚集于细胞核周围)(荧光显微镜，×200)

图2 整合素β₃和骨桥蛋白在制备的对照组BeWo球状体中定位表达的免疫荧光染色图(图2A：DAPI染成蓝色的细胞核；图2B：显示为红色荧光的整合素β₃；图2C：显示为绿色荧光的骨桥蛋白；图2D：图2A、2B、2C的合成效果图，显示细胞核部分基本为蓝色，而绿色和红色荧光聚集于细胞膜)(荧光显微镜，×200)

表3 不同含量人绒毛膜促性腺激素作用于BeWo细胞后BeWo细胞整合素β₃及骨桥蛋白mRNA相对表达量比较(±s)

hCG含量		孔数	mRNA相对表达量
hCG含量		孔数	整合素β₃	OPN
①10 IU/mL		5	1.12±0.31	—(8.44±0.18)
②100 IU/mL		5	1.24±0.19	1.50±0.65
③1 000 IU/mL		5	2.07±0.57	1.95±0.48
总体比较		?	?	?
?	F值	?	7.131	5.481
?	P值	?	0.014	0.028
多重比较的P值		?	?	?
?	① vs ②	?	0.682	0.081
?	① vs ③	?	0.007	0.009
?	② vs ③	?	0.014	0.217

表3 不同含量人绒毛膜促性腺激素作用于BeWo细胞后BeWo细胞整合素β₃及骨桥蛋白mRNA相对表达量比较(±s)

hCG含量		孔数	mRNA相对表达量
hCG含量		孔数	整合素β₃	OPN
①10 IU/mL		5	1.12±0.31	—(8.44±0.18)
②100 IU/mL		5	1.24±0.19	1.50±0.65
③1 000 IU/mL		5	2.07±0.57	1.95±0.48
总体比较		?	?	?
?	F值	?	7.131	5.481
?	P值	?	0.014	0.028
多重比较的P值		?	?	?
?	① vs ②	?	0.682	0.081
?	① vs ③	?	0.007	0.009
?	② vs ③	?	0.014	0.217

图3 细胞培养孔内BeWo球状体铺展情况照片(图3A、3B：分别为BeWo球状体＋RL95-2单层细胞共培养1 h时BeWo球状体起始状态及24 h时BeWo球状体铺展状态；图3C、3D：分别为hCG＋BeWo球状体＋RL95-2单层细胞共培养1 h时BeWo球状体起始状态及24 h时BeWo球状体铺展状态)(倒置荧光显微镜，×200)

[1]	吕波，薛金锋，祝献民，等. 哺乳动物胚胎着床分子机制综述的研究现状[J]. 中华生殖与避孕杂志，2018, 38(1): 76-82.
[2]	包丝雨，于洋，乔杰. 胚胎着床中的分子机制研究进展[J]. 生殖与避孕，2015, 35(5): 328-332.
[3]	顾方乐，张晓梅. 整合素αvβ3、骨桥蛋白对子宫内膜容受性的影响[J]. 中国生育健康杂志，2013, 24(2): 174-176.
[4]	Lessey BA. Assessment of endometrial receptivity[J]. Fertil Steril, 2011, 96(3): 522-529.
[5]	Johnson GA, Burghardt RC, Bazer FW, et al. Osteopontin: roles in implantation and placentation[J]. Biol Reprod, 2003, 69(5): 1458-1471.
[6]	田丽槟，方妍萍，李隽. 骨桥蛋白在哺乳动物生殖中的作用[J]. 医学分子生物学杂志，2015, 37(5): 306-310.
[7]	黄晶. 孕酮和人绒毛膜促性腺激素测定在早期妊娠结局预测中的应用价值分析[J/CD]. 实用妇科内分泌杂志(电子版), 2018, 5(21): 49-50.
[8]	李洁，陈晓燕，徐少元. 人绒毛膜促性腺激素与胚胎种植[J]. 生殖医学杂志，2013, 22(4): 255-258.
[9]	Ho H, Singh H, Aljofan M, et al. A high-throughput in vitro model of human embryo attachment[J]. Fertil Steril, 2012, 97(4): 974-978.
[10]	Kaneko Y, Day ML, Murphy CR. Integrin β3 in rat blastocysts and epithelial cells is essential for implantation in vitro: studies with Ishikawa cells and small interfering RNA transfection[J]. Hum Reprod, 2011, 26(7): 1665-1674.
[11]	Yang H, Taylor HS, Lei CX, et al. Hormonal regulation of galectin 3 in trophoblasts and its effects on endometrium[J]. Reprod Sci, 2011, 18(11): 1118-1127.
[12]	Xiong T, Zhao YQ, Hu D, et al. Administration of calcitonin promotes blastocyst implantation in mice by up-regulating integrin β3 expression in endometrial epithelial cells[J]. Hum Reprod, 2012, 27(12): 3540-3551.
[13]	王涵，赵盼盼，张坤. 哺乳动物着床前胚胎中细胞系的分化及调控机制[J]. 浙江大学学报(农业与生命科学版), 2018, 44(2): 125-130.
[14]	Lessey BA, Castelbaum AJ. Integrins and implantation in the human[J]. Rev Endocr Metab Disord, 2002, 3(2): 107-117.
[15]	刘娜娜，张秋芳，李蓉，等. 控制性超促排卵在FSH/LH搭配中添加hCG又如何？[J]. 生殖医学杂志，2010, 19(4): 359-361.
[16]	黄嘉昱，杜伯涛. 联合雌、孕激素、β-hCG水平对早期妊娠结局的预测及治疗指导[J]. 中国生育健康杂志，2018, 29(3): 274-276.
[17]	林娜，马艳萍，武泽，等. 超排卵周期中高雌、孕激素水平对着床期子宫内膜整合素αvβ3、骨桥蛋白表达及子宫内膜容受性的影响[J]. 中国妇幼保健，2015, 30(9): 1438-1442.
[18]	顾方乐，张晓梅. 人绒毛膜促性腺激素对胚胎着床相关黏附分子的影响[J/CD]. 中华妇幼临床医学杂志(电子版), 2017, 13(2): 231-233.

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[2]	Juan Gu, Qingqing Sun, Fangfang Hu, Yijuan Cao, Yujuan Qi. Clinical application of endometrial receptivity array to improve pregnancy outcomes in women with repeated embryo implantation failure[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2023, 19(05): 582-587.
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[8]	Lei Li, Peng Liu, Ming Li, Wanni Zhao, Qi Pan, Mingwei Zhu, Junmin Wei. Experimental study on the stability of insulin in parenteral nutrition in multi-cavity bag[J]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2020, 15(03): 231-234.
[9]	Lijia Niu, Yixin Xia, Qi Song, Xiaoli Zhang. Correlations of changes of β-human chorionic gonadotropin, matrix metalloproteinase 9, and nucleotide-binding oligomerization domain receptor 2 levels in the amniotic fluid with amniotic membrane infection of pregnant women with term premature rupture of membranes[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2021, 15(05): 311-316.
[10]	Yanni Cui, Yan Ren, Fengting Liang, Yanhong Tan, Hongwei Wang. Clinical research and development progress of cord blood-derived product Omidubicel[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(03): 178-182.
[11]	Yuan Gao, Wei Sheng, Guoying Huang. Application of pluripotent stem cells in establishing heart model in vitro[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(05): 314-318.
[12]	Yingqian Zhou, Zhenxing Wang, Yifan Zhang, Jiaming Sun, Yilin Cao. Modular and vascularized tissue engineering techniques[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(03): 161-166.
[13]	Hua Li, Xiaofan Li, Nainong Li. Research advances ofumbilical cord blood stem cells proliferation promoted by mesenchymal stem cells in vitro[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(01): 34-38.
[14]	Wanshan Zheng, Yin Yin, Zhaowei Tu, Bin Cao. Selection of internal references gene for studying trophoblast syncytialization[J]. Chinese Journal of Obstetric Emergency(Electronic Edition), 2022, 11(01): 46-52.
[15]	Pengzhu Li, Xia Zhan, Gui Xie, Xin Liu, Yujie Qian, Zhuoying Li, Lihua Huang, Zuocheng Yang. Osteopontin gene locus 708C/T polymorphism in children with Kawasaki disease[J]. Chinese Journal of Diagnostics(Electronic Edition), 2020, 08(02): 107-111.

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