体外缺氧诱导子痫前期滋养细胞模型优化及代谢组学鉴定

发布时间：2018-12-20 11:54

【摘要】：目的:探讨滋养细胞体外缺氧不同处理方法模拟子痫前期模型的优化研究。方法:HTR8/SVneo滋养细胞分别在1%O2(缺氧环境)和21%O2(常氧环境)培养箱中培养1 h(记为H1、N1)或2 h(记为H2、N2),缺氧1 h后复氧1 h(记为H1R1)和缺氧2 h后复氧2 h(记为H2R2),H1R1后再缺氧1 h(记为H1R1H1),H2R2后再缺氧2 h(记为H2R2H2),H1R12个循环(记为H1R1H1R1)和H2R22个循环(记为H2R2H2R2),缺氧2 h后复氧6 h(记为H2R6),持续缺氧24 h,以及持续常氧4 h(记为N4)、8 h(记为N8)和24 h(记为Normoxia),然后提取蛋白以Western blot检测腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)磷酸化水平,以验证各组处理对于诱导滋养细胞能量应激的效果;HTR8/SVneo滋养细胞在常氧或者缺氧环境培养24 h后经气相质谱技术(gas chromatography mass spectrum,GS-MS)进行细胞代谢组学分析,以验证AMPK磷酸化水平上调是否与滋养细胞代谢组变化具有一致性。结果:缺氧组AMPK的活性(1.615±0.111)明显高于常氧组(1.000±0.107)和缺氧复氧组(1.277±0.113);缺氧时显著增加的代谢物有4-甲基-2-戊酮酸(log22.597为1.377)、水合乙醛酸(log22.483为1.312)、组氨酸(log21.188为0.248)、苯丙氨酸(log21.262为0.335)、缬氨酸(log21.518为0.602)、正亮氨酸(log21.519为0.603)、乙酰丝氨酸(log21.691为0.758)、丝氨酸(log21.783为0.834)、半胱氨酸(log21.851为0.889)、蛋氨酸(log22.072为1.051)、鸟氨酸(log22.251为1.170)等;而棕榈反油酸(log20.127为-2.983)、11,14,17-廿碳三烯酸(log20.334为-1.583)、二十四单烯酸(log20.600为-0.738)、共轭亚油酸(log20.680为-0.557)、顺式十八碳烯酸(log20.711为-0.492)、9-十七碳烯酸(log20.782为-0.355)、二十二碳六烯酸(log20.829为-0.271)、二十碳五烯酸(log20.841为-0.250)、芥酸(log20.844为-0.244)、二十二碳五烯酸(log20.898为-0.156)、柠檬酸(log20.279为-1.842)、苹果酸(log20.208为-2.264)、琥珀酸(log20.254为-1.980)、顺乌头酸(log20.260为-1.946)、β-柠檬酸-左旋谷氨酸(log20.093为-3.430)、β-丙氨酸(log20.139为-2.851)、胱硫醚(log20.267为-1.904)、顺式-4-羟脯氨酸(log20.500为-1.000)等在缺氧时显著降低。代谢途径结果分析显示缺氧时滋养细胞中核苷酸代谢[log2(1.811、1.149)分别为0.857、0.201]、能量代谢[log2(1.510、1.173、1.149)分别为0.595、0.230、0.201]、维生素代谢[log2(1.045、1.052、1.125)分别为0.064、0.073、0.170]、氨基酸代谢[log2(1.245、1.020、1.027、1.123、1.127、1.076)分别为0.316、0.028、0.039、0.167、0.173、0.106]、信号转导(log21.046为0.065)、蛋白翻译(log21.026为0.037)等途径被激活,而碳水化合物[log2(0.857、0.857、0.799)分别为-0.222、-0.222、-0.323]、脂肪酸[log2(0.944、0.912、0.826)分别为-0.083、-0.133、-0.276]、内分泌代谢[log2(0.885、0.799、0.799)分别为-0.176、-0.323、-0.323]和其他第二代谢物生物合成[log2(0.947、0.871、0.743)分别为-0.079、-0.199、-0.428]等代谢途径显著受到抑制。结论:单纯缺氧模型更适合用于子痫前期病理生理机制的体外研究。
[Abstract]:Objective: to study the optimization of hypoxia-induced preeclampsia model with different treatments of trophoblastic cells in vitro. Methods: HTR8/SVneo trophoblast cells were cultured in 1%O2 (hypoxic environment) and 21%O2 (normoxic environment) incubators for 1 h (H _ 1N _ 1) or 2 h (H _ 2N _ 2), respectively. One hour after hypoxia (H1R1), 2 hours after hypoxia (H2R2), 1 hour after H1R1 (H1R1H1), 2 hours after H2R2 (H2R2H2), H1R12 (H1R1H1R1) and H2R22 (H2R2H2R2). Reoxygenation 6 h (H2R6) after 2 h hypoxia, 24 h continuous hypoxia, 4 h normoxic (N4), 8 h (N8) and 24 h (Normoxia),) Then Western blot was used to detect the phosphorylation of adenylate activated protein kinase (AMP-activated protein kinase,AMPK) in order to verify the effect of each group on the induction of trophoblastic energy stress. HTR8/SVneo trophoblastic cells were cultured in normoxic or anoxic environment for 24 hours, and then were analyzed by GC-MS (gas chromatography mass spectrum,GS-MS). To verify whether the up-regulation of AMPK phosphorylation is consistent with the changes of trophoblast metabolism. Results: the activity of AMPK in hypoxia group (1.615 卤0.111) was significantly higher than that in normoxic group (1.000 卤0.107) and hypoxia reoxygenation group (1.277 卤0.113). The metabolites increased significantly during hypoxia were 4-methyl-2-pentanoic acid (log22.597 1.377), hydrated glyoxylic acid (log22.483 1.312), histidine (log21.188 0.248), phenylalanine (log21.262 0.335). Valine (log21.518 = 0.602), L-leucine (log21.519 = 0.603), acetylserine (log21.691 = 0.758), serine (log21.783 = 0.834), cysteine (log21.851 = 0.889), valine (log21.518 = 0.602), L-leucine (log21.519), acetylserine (log21.691), serine (log21.783), cysteine (log21.851), Methionine (log22.072 1.051), ornithine (log22.251 1.170), etc. However, log20.127 was-2.983, log20.334 was-1.583, log20.600 was-0.738, conjugated linoleic acid was-0.557, and palmitoic acid was-2.983, log20.334 was-1.583, log20.600 was-0.738, conjugated linoleic acid was-0.557, and palmitoic acid was-2.983, log20.334 was-1.583, log20.600 was-0.738, conjugated linoleic acid was-0.557. Cis-octadecenoic acid (log20.711 = -0.492), 9-heptadecenoic acid (log20.782 = -0.355), 22 carbohexaenoic acid (log20.829 = -0.271), eicosapentaenoic acid (log20.841 = -0.250), Erucic acid (log20.844 =-0.244), 22 carbapentaenoic acid (log20.898 = -0.156), citric acid (log20.279 = -1.842), malic acid (log20.208 = -2.264), succinic acid (log20.254 = -1.980), Cis aconitic acid (log20.260 =-1.946), 尾 -citrate-L-glutamic acid (log20.093), 尾 -alanine (log20.139 = -2.851), cystathione (log20.267 = -1.904), 尾 -citrate-L-glutamic acid (log20.093), 尾 -alanine (-2.851), cystathion (-1.904). Cis-4-hydroxyproline (log20.500 =-1.000) decreased significantly during hypoxia. Metabolic pathway analysis showed that nucleotide metabolism [log2 (1.8111.149) was 0.857U 0.201] and energy metabolism (log2 (1.510U 1.173U 1.149) was 0.5950.2300.201, respectively) in trophoblastic cells under hypoxia. Vitamin metabolism [log2 (1.045 ~ 1.052 ~ 1.125) = 0.064 ~ 0.073 ~ 0.170] and amino acid metabolism [log2 (1.245 ~ 1.020 ~ 1.027 ~ 1.127 ~ 1.127 ~ 1.076) = 0.316 ~ 0.028 ~ 0.039 ~ 0.1670.1730.106], respectively. Signal transduction (log21.046 = 0.065) and protein translation (log21.026 = 0.037) were activated, while carbohydrate [log2 (0.857N) 0.857U 0.799) was -0.222kg-0.222kg-0.323]. Fatty acids [log2 (0.944 ~ 0.912 ~ (0.826) were -0.083 ~ (-0.133) ~ 0.276] and endocrine metabolism [log2 _ (0.885) ~ (0.799) ~ (0.799) were -0.176 ~ (-0.323), respectively. The metabolic pathways such as-0.323 and other secondary metabolites biosynthesis [log2 (0. 947 0. 871%) were-0. 079-0. 199-0. 428] were significantly inhibited. Conclusion: hypoxia model alone is more suitable for in vitro study of preeclampsia pathophysiology.
【作者单位】：重庆医科大学附属第二医院妇产科重庆医科大学"中国-加拿大-新西兰"联合母胎医学实验室;
【基金】：国家自然科学基金面上资助项目(编号:81671488) 重庆市教育委员会资助项目(编号:KJ1500223) 重庆医科大学资助项目(编号:CYYQ201507)
【分类号】：R714.244

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