HIF-1α在奶牛胎盘滋养层细胞迁移及侵袭变化中的机制研究

doi:10.7668/hbnxb.20193290

摘要/Abstract

摘要：

为探究HIF-1α在CoCl₂影响奶牛胎盘滋养层细胞迁移及侵袭变化中的作用,揭示奶牛胎盘在缺氧条件下的形成机制,从早期妊娠奶牛胎盘中分离纯化奶牛胎盘滋养层细胞(BTCs),用吉姆萨染色法观察细胞形态,免疫荧光法检测上皮细胞标志蛋白角蛋白7(CK7)、Thy/CD90蛋白表达,后将pCI-neo-hTERT质粒转染至BTCs,用qPCR和Western Blot法检测细胞TERT mRNA及蛋白表达,用CCK8法测定细胞增殖率,用ELISA法检测细胞分泌胎盘催乳素(PL)的能力,进一步利用siRNA沉默细胞缺氧诱导因子-1α(HIF-1α)的表达,ELISA法检测细胞分泌血管内皮生长因子(VEGFA)能力,通过划痕试验及Transwell检测细胞迁移及侵袭能力。结果显示,分离纯化的BTCs为典型上皮样细胞,存在一定数量的双核细胞,并表达上皮细胞标志蛋白CK7,未见Thy/CD90表达,转染后的细胞可稳定表达端粒酶(TERT)mRNA及蛋白,连续传代50代未见细胞老化现象,且增殖率显著高于原代BTCs(P<0.05),分泌PL能力较原代BTCs无显著差异(P>0.05)。经CoCl₂处理后发现,BTCs细胞活力随CoCl₂处理浓度及时间的增加而降低,HIF-1α mRNA及蛋白表达随CoCl₂浓度的增加而减少(P<0.05),400 μmol/L CoCl₂作用于BTCs 24 h建立缺氧模型,VEGFA分泌和迁移侵袭能力在BTCs缺氧状态下显著升高(P<0.05),而将HIF-1α基因沉默后显著降低了BTCs分泌VEGFA和迁移侵袭能力(P<0.05)。总之,通过外源性TERT基因转导建立了永生化的BTCs(Bovine trophoblast cells),永生化后的细胞具有与原代细胞相似的生物学特性,BTCs在缺氧条件下具有更强的VEGFA分泌能力及迁移侵袭能力,且与HIF-1α基因表达的上调有关。

关键词: 奶牛, 滋养层细胞, 缺氧, 迁移, 侵袭, HIF-1α

Abstract:

To investigate the effect of HIF-1α on the migration and invasion of trophoblast cells in placenta of dairy cows affected by cobalt dichloride(CoCl₂),and reveal the formation mechanism of placenta of dairy cows under hypoxia conditions,bovine placental trophoblast cells(BTCs)were isolated and purified from the placenta of early pregnancy cows.The cell morphology was observed by giemsa staining method,and the expression of cell markers cytokeratin 7(CK7)and Thy/CD90 were detected by immunofluorescence method.pCI-neo-hTERT plasmid was then transfected into BTCs,qPCR and Western Blot methods were used to detect the TERT mRNA and protein expression in BTCs.Proliferation rate and placental lactogen(PL)secretory ability were analyzed with CCK8 method and ELISA,respectively.siRNA was further used to silence the expression of Hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor A(VEGFA)secretion ability in BTCs was detected by ELISA and the ability of migration and invasion of BTCs was detected by scratch test and transwell.According to the above all results,the isolated and purified BTCs were typical epithelioid cells with a certain number of binucleated cells and expressed epithelial marker protein CK7,but Thy/CD90 expression was not observed.The transfected cells could stably express telomerase reverse transcriptase(TERT)mRNA and protein.No cell aging was observed after 50 generations of continuous passage,and the proliferation rate was significantly higher than these primary BTCs(P<0.05),and the PL secretion ability showed no significantly different from that of primary BTCs(P>0.05).The cell viability of BTCs treated with CoCl₂ decreased with the increase of CoCl₂ concentration and time(P<0.05),and HIF-1α mRNA and protein expression decreased with the increase of CoCl₂ concentration(P<0.05).BTCs was treated with 400 μmol/L CoCl₂ for 24 h to establish a hypoxia model,and VEGFA secretion and invasion ability were significantly increased under BTCs hypoxia(P<0.05),while HIF-1α silencing decreased VEGFA secretion and invasion ability of BTCs(P<0.05).In conclusion,immortalized BTCs was established through exogenous TERT gene transduction.The immortalized BTCs had similar biological characteristics to the primary cells,and have stronger VEGFA secretion and invasion ability under hypoxia condition,and were associated with HIF-1α up-regulation.

Key words: Dairy cows, Trophoblasts, Hypoxia, Migration, Invasion, HIF-1α

苏月, 郭勇, 高玉平, 肖龙菲, 廖晨星, 齐晓龙, 盛熙晖, 邢凯, 王相国, 倪和民. HIF-1α在奶牛胎盘滋养层细胞迁移及侵袭变化中的机制研究[J]. 华北农学报, 2023, 38(1): 202-209. doi: 10.7668/hbnxb.20193290.

SU Yue, GUO Yong, GAO Yuping, XIAO Longfei, LIAO Chenxing, QI Xiaolong, SHENG Xihui, XING Kai, WANG Xiangguo, NI Hemin. The Mechanism of HIF-1α in Migration and Invasion of Dairy Cow Placental Trophoblast Cells[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 202-209. doi: 10.7668/hbnxb.20193290.

http://www.hbnxb.net/CN/Y2023/V38/I1/202

图/表 5

表1 目的基因扩增引物序列

Tab.1 Target gene amplification primer sequences

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	产物长度/bp Product size	用途 Application
TERT	F: TATGCCGTGGTCCAGAAGG	384	qPCR
	R: CAAGAAATCATCCACCAAACG
HIF-1α	F: AGCTTGCTCATCAGTTGCCACTC	110	qPCR
	R: AGCATCCAGAAGTTTCCTCACACG
Glut1	F: TGTCTGGCATCAACGCTGTCTTC	144	qPCR
	R: CCTGCTCGCTCCACCACAAAC
VEGFA	F: CCTTGCCTTGCTGCTCTACCTTC	152	qPCR
	R: AGATGTCCACCAGGGTCTCGATG
GAPDH	F: CGGCACAGTCAAGGCAGAGAAC	116	qPCR
	R: CCACATACTCAGCACCAGCATCAC

图1 奶牛BTCs的分离、纯化与鉴定 A、B分别为显微镜下形态及吉姆萨染色细胞(A.bars=1 mm;B.bars=100 μmol/L);C、D分别为BTC细胞CK7及CD90的表达情况(bars=100 μmol/L)。

Fig.1 Isolation,purification and identification of dairy cows BTCs A and B indicated morphological characteristics and giemsa stain results under a phase-contrast inverted microscope(A.bars=1 mm;B.bars=100 μmol/L),respectively;C and D indicated the expression of CK7 and CD90 in BTCs(bars=100 μmol/L).

图2 永生化BTCs的生物学特性 A.细胞内TERT蛋白相对表达量;B.细胞内TERT mRNA相对表达量;C.细胞生长曲线;D.细胞分泌PL能力;不同小写字母表示差异显著(P<0.05)。图3—4同。

Fig.2 Biological characteristics of immortalized BTCs The relative protein expression level of TERT in cells;B.The relative mRNA expression level of TERT in cells;C.The growth curve of cells;D.The endocrine ability of PL in cells.Different lowercase letters indicate significant difference. The same as Fig.3—4.

图3 BTCs缺氧模型的建立 A.细胞活力;B、C、D分别为细胞HIF-1α蛋白及mRNA相对表达量;E为细胞Glut1和VEGFA mRNA相对表达量。

Fig.3 Establishment of hypoxia models of BTCs A.Cell viability;B,C,D.The relative protein and mRNA expression level of HIF-1α in cells;E.The relative mRNA expression level of Glut1 and VEGFA in cells.

图4 缺氧状态对BTCs HIF-1α、VEGFA表达及其迁移侵袭力的影响 A.细胞内HIF-1α蛋白相对表达量;B.细胞内HIF-1α mRNA相对表达量;C.细胞分泌VEGFA能力;D、E.细胞迁移及侵袭能力(D. bars= 1 mm;E. bars= 100 μm)。

Fig.4 The effects of hypoxic states on the expression of HIF-1α and VEGFA and migration and invasion ability of BTCs A.The relative protein expression of HIF-1α in cells;B.The relative mRNA expression of HIF-1α in cells;C.The endocrine ability of VEGFA in cells;D,E.The migration and invasion ability of cells(D. bars= 1 mm;E. bars= 100 μm).

参考文献 34

[1]	Lonergan P, Sánchez J M, Mathew D J, Passaro C, Fair T. Embryo development in cattle and interactions with the reproductive tract[J]. Reproduction Fertility and Development, 2018, 31(1):118-125.doi:10.1071/RD18409. doi: 10.1071/RD18409 pmid: 32188547
[2]	Wang X G, Li Q R, Xie T T, Yuan M Y, Sheng X H, Qi X L, Xing K, Liu F, Guo Y, Xiao L F, Ni H M. Exosomes from bovine endometrial epithelial cells ensure trophoblast cell development by miR-218 targeting secreted frizzled related protein 2[J]. Journal of Cellular Physiology, 2021, 236(6):4565-4579.doi:10.1002/jcp.30180. doi: 10.1002/jcp.30180 pmid: 33230823
[3]	Horcajo P, Jiménez-Pelayo L, García-Sánchez M, Regidor-Cerrillo J, Collantes-Fernández E, Rozas D, Hambruch N, Pfarrer C, Ortega-Mora L M. Transcriptome modulation of bovine trophoblast cells in vitro by Neospora caninum[J]. International Journal for Parasitology, 2017, 47(12):791-799.doi:10.1016/j.ijpara.2017.08.007. doi: S0020-7519(17)30252-7 pmid: 28899691
[4]	Polei M, G nther J, Koczan D,R. Trophoblast cell differentiation in the bovine placenta:differentially expressed genes between uninucleate trophoblast cells and trophoblast giant cells are involved in the composition and remodeling of the extracellular matrix and O-glycan biosynthesis[J]. BMC Molecular and Cell Biology, 2020, 21(1):1.doi:10.1186/s12860-020-0246-8. doi: 10.1186/s12860-020-0246-8
[5]	董峰. 苦马豆素诱导山羊胎盘滋养层细胞凋亡的信号转导通路研究[D]. 杨凌: 西北农林科技大学, 2014.
	Dong F. Signal transduction of pathways of apoptosis in goat placental trophoblast cells induced by swainsonine[D]. Yangling: Northwest A&F University, 2014.
[6]	Straszewski-Chavez S L, Abrahams V M, Alvero A B, Aldo P B, Ma Y, Guller S, Romero R, Mor G. The isolation andcharacterization of a novel telomerase immortalized first trimester trophoblast cell line,Swan 71[J]. Placenta, 2009, 30(11):939-948.doi:10.1016/j.placenta.2009.08.007. doi: 10.1016/j.placenta.2009.08.007 pmid: 19766308
[7]	Zhang H L, Huang Y, Wang L L, Yu T T, Wang Z G, Chang L L, Zhao X M, Luo X M, Zhang L, Tong D W. Immortalization of porcine placental trophoblast cells through reconstitution of telomerase activity[J]. Theriogenology, 2016, 85(8):1446-1456.doi:10.1016/j.theriogenology.2016.01.006. doi: 10.1016/j.theriogenology.2016.01.006 pmid: 26850465
[8]	Dong F, Huang Y, Li W, Zhao X, Zhang W, Du Q, Zhang H, Song X, Tong D. The isolation and characterization of a telomerase immortalized goat trophoblast cell line[J]. Placenta, 2013, 34(12):1243-1250.doi:10.1016/j.placenta.2013.09.009. doi: 10.1016/j.placenta.2013.09.009 pmid: 24112823
[9]	赵海君, 张园, 高玲玲, 高超, 崔毓桂, 刘嘉茵. 缺氧对胎盘滋养层细胞细胞周期和凋亡的影响[J]. 江苏医药, 2012, 38(19):2236-2239.doi:10.19460/j.cnki.0253-3685.2012.19.002. doi: 10.19460/j.cnki.0253-3685.2012.19.002
	Zhao H J, Zhang Y, Gao L L, Gao C, Cui Y G, Liu J Y. Effects of hypoxia on cell cycle and apoptosis of cultured trophoblastic cells[J]. Jiangsu Medical Journal, 2012, 38(19):2236-2239.
[10]	Bînă A M, Aburel O M, Avram V F, Lelcu T L, Linȶa A V, Chiriac D V, Mocanu A G, Bernad E, Borza C, Craina M L, Popa Z L, Muntean D M, Creȶu O M. Impairment of mitochondrial respiration in platelets and placentas:a pilot study in preeclamptic pregnancies[J]. Molecular and Cellular Biochemistry, 2022, 477(7):1987-2000.doi:10.1007/s11010-022-04415-2. doi: 10.1007/s11010-022-04415-2
[11]	Wu D, Yang N N, Xu Y T, Wang S L, Zhang Y Y, Sagnelli M, Hui B Q, Huang Z Y, Sun L Z. lncRNA HIF1A antisense RNA 2 modulates trophoblast cell invasion and proliferation through upregulating PHLDA1 expression[J]. Molecular Therapy Nucleic Acids, 2019, 16:605-615.doi:10.1016/j.omtn.2019.04.009. doi: 10.1016/j.omtn.2019.04.009 URL
[12]	Wang Z, Zhao G X, Zibrila A I, Li Y B, Liu J J, Feng W Y. Acetylcholine ameliorated hypoxia-induced oxidative stress and apoptosis in trophoblast cells via p38 MAPK/NF-κB pathway[J]. Molecular Human Reproduction, 2021, 27(8):gaab045.doi:10.1093/molehr/gaab045. doi: 10.1093/molehr/gaab045 URL
[13]	Taheem D K, Foyt D A, Loaiza S, Ferreira S A, Ilic D, Auner H W, Grigoriadis A E, Jell G, Gentleman E. Differential regulation of human bone marrow mesenchymal stromal cell chondrogenesis by hypoxia inducible factor-1α hydroxylase inhibitors[J]. Stem Cells, 2018, 36(9):1380-1392.doi:10.1002/stem.2844. doi: 10.1002/stem.2844 pmid: 29726060
[14]	杨丹. 缺氧对山羊胎盘滋养层细胞生物钟和内质网应激信号通路的影响[D]. 杨凌: 西北农林科技大学, 2018.
	Yang D. Effects of hypoxia on the circadian clock and endoplasmic reticulum stress signaling pathways in goat trophoblast cells[D]. Yangling: Northwest A&F University, 2018.
[15]	Haeger J D, Hambruch N, Pfarrer C. The bovine placenta in vivo and in vitro[J]. Theriogenology, 2016, 86(1):306-312.doi:10.1016/j.theriogenology.2016.04.043. doi: 10.1016/j.theriogenology.2016.04.043 pmid: 27155733
[16]	Oliveira L J, Barreto R S N, Perecin F, Mansouri-Attia N, Pereira F T V, Meirelles F V. Modulation of maternal immune system during pregnancy in the cow[J]. Reproduction in Domestic Animals, 2012(S4), 47:384-393.doi:10.1111/j.1439-0531.2012.02102.x. doi: 10.1111/j.1439-0531.2012.02102.x URL
[17]	Igwebuike U M, Ezeasor D N. Morphological assessment of placentomal trophoblastic epithelium in the placenta of West African Dwarf goats:a light and electron microscopic study[J]. Animal Reproduction Science, 2012, 136(1-2):61-68.doi:10.1016/j.anireprosci.2012.10.025. doi: 10.1016/j.anireprosci.2012.10.025 pmid: 23182471
[18]	Male V, Gardner L, Moffett A. Isolation of cells from the feto-maternal interface[J]. Current Protocols in Immunology, 2012, 97(1):1-11.doi:10.1002/0471142735.im0740s97. doi: 10.1002/0471142735.im0740s97
[19]	Kolokol'tsova T D, Saburina I N, Nanovskaya T N, Patrikeeva S L, Vernikovskaya D I, Zurina I M, Gorkun A A, Kosheleva N V, Poltavtseva R A, Sukhikh G T. Characteristics of trophoblasts in long-term culture[J]. Bulletin of Experimental Biology and Medicine, 2017, 164(2):259-265.doi:10.1007/s10517-017-3969-6. doi: 10.1007/s10517-017-3969-6 pmid: 29177904
[20]	Baranyi U, Winter B, Gugerell A, Hegedus B, Brostjan C, Laufer G, Messner B. Primary human fibroblasts in culture switch to a myofibroblast-like phenotype independently of TGF beta[J]. Cells, 2019, 8(7):721.doi:10.3390/cells8070721. doi: 10.3390/cells8070721 URL
[21]	李茜茹, 邢凯, 苏月, 李博宇, 苏运泽, 王春伟, 郭勇, 肖龙菲, 齐晓龙, 盛熙晖, 倪和民, 王相国. 转hTERT基因牛胎盘滋养层细胞生长及分泌特性研究[J]. 中国畜牧杂志, 2021, 57(9):80-83.doi:10.19556/j.0258-7033.20201208-01. doi: 10.19556/j.0258-7033.20201208-01
	Li Q R, Xing K, Su Y, Li B Y, Su Y Z, Wang C W, Guo Y, Xiao L F, Qi X L, Sheng X H, Ni H M, Wang X G. Study on growth and secretion characteristics of hTERT transgenic bovine placental trophoblast cells[J]. Chinese Journal of Animal Science, 2021, 57(9):80-83.
[22]	Pastuschek J, Nonn O, Gutiérrez-Samudio R N, Murrieta-Coxca J M, Müller J, Sanft J, Huppertz B, Markert U R, Groten T, Morales-Prieto D M. Molecular characteristics of established trophoblast-derived cell lines[J]. Placenta, 2021, 108:122-133.doi:10.1016/j.placenta.2021.02.022. doi: 10.1016/j.placenta.2021.02.022 pmid: 33810901
[23]	Shimada A, Nakano H, Takahashi T, Imai K, Hashizume K. Isolation and characterization of a bovine blastocyst-derived trophoblastic cell line,BT-1:development of a culture system in the absence of feeder cell[J]. Placenta, 2001, 22(7):652-662.doi:10.1053/plac.2001.0702. doi: 10.1053/plac.2001.0702 pmid: 11504534
[24]	Hambruch N, Haeger J D, Dilly M, Pfarrer C. EGF stimulates proliferation in the bovine placental trophoblast cell line F3 via Ras and MAPK[J]. Placenta, 2010, 31(1):67-74.doi:10.1016/j.placenta.2009.10.011. doi: 10.1016/j.placenta.2009.10.011 pmid: 19914712
[25]	李鹏云. 缺氧条件下HIF-1α通过调控CXCR4影响滋养细胞侵袭和迁移能力的研究[D]. 郑州: 郑州大学, 2018.
	Li P Y. Effect of HIF-1α on the invasion and migration of trophoblast by regulating CXCR4 in hypoxia[D]. Zhengzhou: Zhengzhou University, 2018.
[26]	Knöfler M, Haider S, Saleh L, Pollheimer J, Gamage T K J B, James J. Human placenta and trophoblast development:key molecular mechanisms and model systems[J]. Cellular and Molecular Life Sciences, 2019, 76(18):3479-3496.doi:10.1007/s00018-019-03104-6. doi: 10.1007/s00018-019-03104-6 pmid: 31049600
[27]	Macklin P S, McAuliffe J, Pugh C W, Yamamoto A. Hypoxia and HIF pathway in cancer and the placenta[J]. Placenta, 2017, 56:8-13.doi:10.1016/j.placenta.2017.03.010. doi: S0143-4004(17)30195-9 pmid: 28330647
[28]	Tomimatsu T, Mimura K, Matsuzaki S, Endo M, Kumasawa K, Kimura T. Preeclampsia:maternal systemic vascular disorder caused by generalized endothelial dysfunction due to placental antiangiogenic factors[J]. International Journal of Molecular Sciences, 2019, 20(17):4246.doi:10.3390/ijms20174246. doi: 10.3390/ijms20174246 URL
[29]	Baumann M U, Schneider H, Malek A, Palta V, Surbek D V, Sager R, Zamudio S, Illsley N P. Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I(IGF-I)[J]. PLoS One, 2014, 9(8):e106037.doi:10.1371/journal.pone.0106037. doi: 10.1371/journal.pone.0106037 URL
[30]	Yang M, Li H, Rong M M, Zhang H Y, Hou L L, Zhang C. Dysregulated GLUT1 may be involved in the pathogenesis of preeclampsia by impairing decidualization[J]. Molecular and Cellular Endocrinology, 2022, 540:111509.doi:10.1016/j.mce.2021.111509. doi: 10.1016/j.mce.2021.111509 URL
[31]	Huang C B, Li Y, Li Z X, Xu Y, Li N, Ge Y, Dong J, Chang A T, Zhao T S, Wang X C, Wang H W, Yang S Y, Xie K P, Hao J H, Ren H. LIMS1 promotes pancreatic cancer cell survival under oxygen-glucose deprivation conditions by enhancing HIF1A protein translation[J]. Clinical Cancer Research, 2019, 25(13):4091-4103.doi:10.1158/1078-0432.CCR-18-3533. doi: 10.1158/1078-0432.CCR-18-3533 pmid: 30679163
[32]	Fan Q P, Li L H, Wang T L, Emerson R E, Yan Xu Y. A novel ZIP4-HDAC4-VEGFA axis in high-grade serous ovarian cancer[J]. Cancers(Basel), 2021, 13(15):3821.doi:10.3390/cancers13153821. doi: 10.3390/cancers13153821 URL
[33]	Liu X, Yamaguchi K, Takane K, Zhu C, Hirata M, Hikiba Y, Maeda S, Furukawa Y, Ikenoue T. Cancer-associated IDH mutations induce Glut1 expression and glucose metabolic disorders through a PI3K/Akt/mTORC1-Hif1α axis[J]. PLoS One, 2021, 16(9):e0257090.doi:10.1371/journal.pone.0257090. doi: 10.1371/journal.pone.0257090 URL
[34]	Yu N, Wu J L, Xiao J, Fan L, Chen S H, Li W. HIF-1α regulates angiogenesis via Notch1/STAT3/ETBR pathway in trophoblastic cells[J]. Cell Cycle, 2019, 18(24):3502-3512.doi:10.1080/15384101.2019.1689481. doi: 10.1080/15384101.2019.1689481 pmid: 31724455

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