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目的：探讨同型半胱氨酸（homocysteine,Hcy）对滋养细胞融合过程的影响及潜在机制。方法：在体外用Bewo细胞模拟体内滋养细胞进行培养，利用毛喉素（forskolin,FSK）成功构建细胞融合模型，并使用50µmol/L Hcy处理细胞48 h。运用共聚焦显微镜观察融合情况并计算融合指数；通过ELISA检测人绒毛膜促性腺激素（human chorionic gonadotropin,hCG）含量；通过qPCR技术检测融合基因ERVW‑1、ERVFRD‑1、GCM1、CGB的mRNA表达水平，及与线粒体功能和氧化应激相关基因的mRNA表达情况；通过Western blot测定ERVW-1、ERVFRD-1、GCM1的蛋白表达水平；通过免疫荧光检测细胞活性氧（reactive oxygen species,ROS）水平和膜电位变化；使用化学发光测定ATP水平；采用WST检测超氧化物歧化酶（superoxide dismutase,SOD）的活性。结果：滋养细胞在FSK诱导下成功融合，加入Hcy后，细胞出现明显变化：细胞融合指数明显降低（P<0.001）,hCG分泌浓度大幅减少（P<0.001），细胞融合相关的基因和蛋白表达水平明显降低（P<0.05）；线粒体膜电位下降（P<0.001）,ATP水平明显减少（P<0.01），线粒体融合相关基因表达量明显降低（P<0.001），而线粒体分裂基因的表达量明显升高（P<0.05）；细胞内ROS生成量明显增加（P<0.01）,T-SOD活性明显降低（P<0.001），氧化应激关键调控基因NRF2 mRNA表达水平明显降低（P<0.001）。结论：Hcy可能通过干扰线粒体正常功能，促使ROS大量生成，引发氧化应激反应，阻碍滋养细胞的正常融合与hCG分泌，对胎盘发育和妊娠健康产生负面影响。
Abstract：Objective: To investigate the effect of homocysteine(Hcy) on the fusion process of trophoblast cells and its potential mechanism. Methods: Bewo cells were cultured in vitro to mimic in vivo trophoblasts, successfully constructing a cell fusion model with forskolin(FSK), and treating cells with 50 µmol/L Hcy for 48 hours. A confocal microscopy was used to observe the fusion status and calculate the fusion index, and the human chorionic gonadotropin(hCG) levels were detected by ELISA. The mRNA expression levels of fusion genes ERVW‑1, ERVFRD‑1, GCM1,and CGB, as well as the mRNA expression of genes relate to mitochondrial function and oxidative stress were detected by qPCR. The protein expression levels of ERVW-1, ERVFRD-1, and GCM1 were measured by Western blot. Intracellular reactive oxygen species(ROS) levels and membrane potential were detected by immunofluorescence. ATP levels were measured using chemiluminescence method. The activity of superoxide dismutase(SOD) was determining by WST method. Results: Trophoblast cells were successfully fused under FSK induction, and the cells showed significant changes upon the addition of Hcy; the cell fusion index was significantly reduced(P<0.001); the hCG secretion was greatly reduced(P<0.001), and both gene and protein expression levels relate to cell fusion were significantly down-regulated(P<0.05). Mitochondrial membrane potential declined(P<0.001), the ATP levels significantly decreased(P<0.01), and the expression of mitochondrial fusion-related genes substantially reduced(P<0.001), while the expression of mitochondrial fission genes widely increased(P<0.05). The amount of intracellular ROS production was significantly increased(P<0.01). The activity of T-SOD was visibly declined(P<0.001), and the mRNA expression level of NRF2, a key gene of oxidative stress was significantly decreased(P<0.001). Conclusion: Hcy may have a negative impact on placental development and pregnancy health by interfering with the normal function of mitochondria, promoting the production of ROS, triggering oxidative stress, and hindering the normal fusion of trophoblasts and hCG secretion.
1 Wu D, Yin R, Deng J. Homocysteine, hyperhomocyste-inemia, and H-type hypertension[J]. Eur J Prev Cardi-ol, 2024, 31(9):1092-1103.
2 Kaplan P, Tatarkova Z, Sivonova MK, et al. Homocys-teine and mitochondria in cardiovascular and cerebrovas-cular systems[J]. Int J Mol Sci, 2020, 21(20):7698.
3杨岩婷，霍虎艳，杨欣，等.同型半胱氨酸水平与PCOS患者代谢特征及妊娠结局相关性的研究进展[J].生殖医学杂志，2024, 33(9):1263-1267.Yang YT, Huo HY, Yang X, et al. Research progresson the correlation between homocysteine serum level andmetabolism and pregnancy in polycystic ovary syndrome[J]. J Reprod Med, 2024, 33(9):1263-1267.
4国家卫生健康委医政医管局.罕见病诊疗指南（2019年版）[M].北京：中国协和医科大学出版社，2019:268-273.National Health Commission Medical Administration.Diagnosis and treatment guide for rare diseases(2019edition)[M]. Beijing:Peking Union Medical CollegePress, 2019:268-273.
5刘晓红，张雅，王影. PAI-1、Hcy水平与妊娠合并心脏病患者妊娠结局的相关性分析[J].标记免疫分析与临床，2024, 31(8):1431-1436.Liu XH, Zhang Y, Wang Y. A correlation analysisbetween PAI-1 and Hcy levels and pregnancy outcomesin pregnant women with heart disease[J]. LabelImmunoassays Clin Med, 2024, 31(8):1431-1436.
6 Serrano NC, Quintero-Lesmes DC, Becerra-Bayona S,et al. Association of pre-eclampsia risk with maternal lev-els of folate, homocysteine and vitamin B12 in Colombia:A case-control study[J]. PloS one, 2018, 13(12):e0208137.
7 Gaiday AN, Tussupkaliyev AB, Bermagambetova SK,et al. Effect of homocysteine on pregnancy:A systematicreview[J]. Chem Biol Interact, 2018, 293:70-76.
8 Knöfler M, Haider S, Saleh L, et al. Human placentaand trophoblast development:Key molecular mecha-nisms and model systems[J]. Cell Mol Life Sci, 2019,76(18):3479-3496.
9 Bucher M, Kadam L, Ahuna K, et al. Differences inglycolysis and mitochondrial respiration between cytotro-phoblast and syncytiotrophoblast in‑vitro:Evidence forsexual dimorphism[J]. Int J Mol Sci, 2021, 22(19):10875.
10 Burton GJ, Jauniaux E. The cytotrophoblastic shell andcomplications of pregnancy[J]. Placenta, 2017, 60:134-139.
11张瑶，莫礼志，何璐，等.线粒体损伤诱导细胞焦亡在心血管疾病中的研究进展[J].中南医学科学杂志，2024, 52(6):1069-1073.Zhang Y, Mo LZ, He L, et al. Research progress onmitochondrial damage induced cell pyroptosis incardiovascular diseases[J]. Med Sci J Central SouthChin, 2024, 52(6):1069-1073.
12刘辉.干扰线粒体融合蛋白2表达对滋养细胞株JEG-3侵袭能力影响的作用机制研究[J].中国优生与遗传杂志，2022, 30(4):602-608.Liu H. Mechanism of interfering expression of mitochon-drial fusion protein 2 on invasion ability of trophoblasticcell line JEG-3[J]. Chin J Birth Health Hered, 2022, 30(4):602-608.
13 Ghosh A, Kumar R, Kumar RP, et al. The GATA tran-scriptional program dictates cell fate equilibrium to estab-lish the maternal-fetal exchange interface and fetal devel-opment[J]. Proc Natl Acad Sci U S A, 2024, 121(8):e2310502121.
14 Li X, Li Z, Wang Y, et al. A comprehensive review ofhuman trophoblast fusion models:Recent developmentsand challenges[J]. Cell Death Discov, 2023, 9(1):372.
15 Zhang C, Hu J, Wang X, et al. High level of homocys-teine is associated with pre-eclampsia risk in pregnantwoman:A meta-analysis[J]. Gynecol Endocrinol, 2022,38(9):705-712.
16 He J, Jiang D, Cui X, et al. Vitamin B12 status and folicacid/vitamin B12 related to the risk of gestational diabetesmellitus in pregnancy:A systematic review and meta-analysis of observational studies[J]. BMC PregnancyChildbirth, 2022, 22(1):587.
17 Kassab SE, Abu-Hijleh MF, Al-Shaikh HB, et al. Hy-perhomocysteinemia in pregnant rats:Effects on arterialpressure, kidneys and fetal growth[J]. Eur J Obstet Gy-necol Reprod Biol, 2005, 122(2):177-181.
18 Zhou R, Zhe L, Chen F, et al. Maternal folic acid andvitamin B12 supplementation during medium to late gesta-tion promotes fetal development via improving placentalantioxidant capacity, angiogenesis and amino acid trans-port[J]. J Sci Food Agric, 2024, 104(5):2832-2841.
19 Cesana M, Tufano G, Panariello F, et al. TFEB con-trols syncytiotrophoblast formation and hormone produc-tion in placenta[J]. Cell Death Differ, 2024, 31(11):1439-1451.
20 Liu K, Wu S, Cui Y, et al. Trophoblast fusion in fetalgrowth restriction is inhibited by CTGF in a cell-cycle-dependent manner[J]. J Mol Histol, 2024, 55(5):895-908.
21 Zhao X, Cai X, Zhu H, et al. 27-Hydroxycholesterol in-hibits trophoblast fusion during placenta development byactivating PI3K/AKT/mTOR signaling pathway[J].Arch Toxicol, 2024, 98(3):849-863.
22 Soliman A, Lacasse AA, Lanoix D, et al. Placentalmelatonin system is present throughout pregnancy andregulates villous trophoblast differentiation[J]. J PinealRes, 2015, 59(1):38-46.
23范秀丽，周洁.重度子痫前期合并胎儿宫内生长受限孕妇血清PLGF, Hcy及IGFr1水平变化及其与妊娠结局的相关性研究[J].中国妇幼保健，2020, 35(14):2707-2709.Fan XL, Zhou J. Changes of serum PLGF, Hcy, andIGFr1 levels in pregnant women with severe preeclampsiacomplicated with intrauterine growth restriction and theircorrelation with pregnancy outcomes[J]. Mater ChildHealth Care Chin, 2020, 35(14):2707-2709.
24 Yoshida K, Kusama K, Shinohara G, et al. Quercetinstimulates trophoblast fusion via the mitochondrial func-tion[J]. Sci Rep, 2024, 14(1):287.
25 Walker OS, Ragos R, Wong MK, et al. Reactive oxy-gen species from mitochondria impacts trophoblast fusionand the production of endocrine hormones by syncytiotro-phoblasts[J]. PLoS One, 2020, 15(2):e0229332.
26 Decker ST, Funai K. Mitochondrial membrane lipids inthe regulation of bioenergetic flux[J]. Cell Metab, 2024,36(9):1963-1978.
27 Miwa S, Kashyap S, Chini E, et al. Mitochondrial dys-function in cell senescence and aging[J]. J Clin Invest,2022, 132(13):e158447.
28 Chen H, Detmer SA, Ewald AJ, et al. Mitofusins Mfn1and Mfn2 coordinately regulate mitochondrial fusion andare essential for embryonic development[J]. J Cell Biol,2003, 160(2):189-200.
29 Ren Z, Zhang X, Ding T, et al. Mitochondrial dynamicsimbalance:A strategy for promoting viral infection[J].Front Microbiol, 2020, 11:1992.
30 Amorim JA, Coppotelli G, Rolo AP, et al. Mitochon-drial and metabolic dysfunction in ageing and age-relateddiseases[J]. Nat Rev Endocrinol, 2022, 18(4):243-258.
31 Martinez F, Olvera-Sanchez S, Esparza-Perusquia M,et al. Multiple functions of syncytiotrophoblast mitochon-dria[J]. Steroids, 2015, 103:11-22.
32李亚奇，郑志坚，张美诗，等.利福平通过NRF2/NQO1通路对小鼠肝细胞造成氧化损伤[J].海南医科大学学报，2025, 31(4):248-253.Li YQ, Zheng ZJ, Zhang MS, et al. Oxidative damageto mouse liver cells caused by rifampicin through theNRF2/NQO1 pathway[J]. J Hainan Med Univ, 2025,31(4):248-253.
33 Afrose D, Alfonso-Sánchez S, McClements L. Target-ing oxidative stress in preeclampsia[J]. Hypertens Preg-nancy, 2025, 44(1):2445556.
34 Yang M, Wang M, Li N. Advances in pathogenesis ofpreeclampsia[J]. Arch Gynecol Obstet, 2024, 309(5):1815-1823.
35 Bu C, Wang Z, Ren Y, et al. Syncytin-1 nonfusogenicactivities modulate inflammation and contribute to pre-eclampsia pathogenesis[J]. Cell Mol Life Sci, 2022, 79(6):290.
基本信息:
DOI：10.13210/j.cnki.jhmu.20250520.002
中图分类号:R714
引用信息:
[1]王议平,李铭洋,蔡忠宇,等.同型半胱氨酸干扰滋养细胞线粒体并影响细胞融合功能[J].海南医科大学学报,2026,32(08):570-578.DOI:10.13210/j.cnki.jhmu.20250520.002.
基金信息:
青海省科学技术厅自然科学基金（2024-ZJ-923）; 青海省“昆仑英才”高端创新创业人才项目（QHKLYC-GDCXCY-2022-080）~~
2025-05-21
2025-05-21
2025-05-21