中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (6): 2245-2254.doi: 10.16431/j.cnki.1671-7236.2023.06.008

• 生理生化 • 上一篇    下一篇

低氧对牦牛肺动脉平滑肌细胞增殖及氧化应激损伤作用的影响

李睿1,2,3, 张伊阳1,2,3, 罗雪慧1, 周熳琳1,2,3, 吴绍米1, 杨琨1,2,3, 乔自林1,2,3   

  1. 1. 西北民族大学生命科学与工程学院, 兰州 730030;
    2. 西北民族大学生物医学研究中心, 甘肃省动物细胞技术创新中心, 兰州 730030;
    3. 国家民族事务委员会生物技术与生物工程重点实验室, 兰州 730030
  • 收稿日期:2022-10-19 出版日期:2023-06-05 发布日期:2023-05-30
  • 通讯作者: 杨琨 E-mail:186152592@xbmu.edu.cn
  • 作者简介:李睿,E-mail:1171183168@qq.com。
  • 基金资助:
    国家自然科学基金(31860687);甘肃省自然科学基金(21JR11RA024);中央高校创新团队(31920200004);教育部动物医学生物工程创新团队(IRT-17R88);西北民族大学校级大学生创新创业训练计划(X202310742257)

Effect of Hypoxia on Proliferation and Oxidative Stress Injury of Pulmonary Artery Smooth Muscle Cells in Yaks

LI Rui1,2,3, ZHANG Yiyang1,2,3, LUO Xuehui1, ZHOU Manlin1,2,3, WU Shaomi1, YANG Kun1,2,3, QIAO Zilin1,2,3   

  1. 1. Life Science and Engineering College, Northwest Minzu University, Lanzhou 730030, China;
    2. Gansu Technology Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China;
    3. Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Lanzhou 730030, China
  • Received:2022-10-19 Online:2023-06-05 Published:2023-05-30

摘要: 【目的】通过研究低氧环境对牦牛(Bos grunniens)肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)增殖及氧化应激损伤的影响,初步探究牦牛肺动脉平滑肌对低氧的适应性特点。【方法】分离纯化并利用α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)鉴定牦牛PASMCs。试验分为常氧组和低氧组,采用CCK-8法检测牦牛PASMCs在低氧和常氧状态下0、2、4、6、12、24、48、72、96、120 h的增殖效率;采用ELISA法检测常氧组和低氧组培养24、48、72、96 h细胞培养上清液中乳酸脱氢酶(lactate dehydrogenase,LDH)、超氧化物歧化酶(superoxide dismutase,SOD)、肌酸激酶(creatine kinase,CK)和丙二醛(malondialdehyde,MDA)的蛋白浓度。【结果】细胞鉴定结果表明,95%以上的细胞均能表达α-SMA,即分离纯化所得细胞为牦牛PASMCs。CCK-8结果表明,低氧组在72、96、120 h细胞增殖倍数极显著低于常氧组(P<0.01);低氧组和常氧组在72 h的细胞增殖倍数均极显著高于48 h (P<0.01),表明牦牛PASMCs在72 h出现增殖高峰,且长时间的低氧环境能够降低牦牛PASMCs增殖效率。ELISA检测结果表明,与常氧组相比,低氧组牦牛PASMCs培养上清液中LDH蛋白浓度在72 h极显著上调(P<0.01);SOD蛋白浓度在72、96 h均极显著或显著上调(P<0.01;P<0.05);CK蛋白浓度在48、72 h均显著或极显著上调(P<0.05;P<0.01);MDA蛋白浓度在72 h显著上调、96 h显著下调(P<0.05)。在低氧条件下,各时间点上清液中LDH、SOD蛋白浓度差异不显著(P>0.05),CK、MDA蛋白浓度在24、48和72 h均显著高于96 h (P<0.05)。在常氧条件下,上清液中LDH蛋白浓度在48 h显著高于24、72 h (P<0.05);SOD蛋白浓度在24、48 h均显著高于72、96 h (P<0.05);CK蛋白浓度在24 h显著高于72、96 h (P<0.05);MDA蛋白浓度在48 h显著高于72 h (P<0.05)。【结论】长时间低氧能够增强牦牛PASMCs氧化应激反应和细胞损伤,抗氧化作用减弱,细胞增殖效率显著降低,LDH、SOD、CK和MDA 4种蛋白在低氧条件下牦牛PASMCs损伤与抗损伤以及抗氧化中发挥重要调节作用,这可能与牦牛肺脏血管的低氧适应性有关。

关键词: 牦牛; 肺动脉平滑肌细胞(PASMCs); 低氧; 氧化应激

Abstract: 【Objective】 This experiment was aimed to study the effects of hypoxia on the proliferation and oxidative stress injury of pulmonary artery smooth muscle cells (PASMCs) in yaks(Bos grunniens), and preliminarily explore the adaptive characteristics of pulmonary artery smooth muscle in yaks to hypoxia.【Method】 PASMCs of yaks were isolated and purified using α-smooth muscle actin (α-SMA).The test was divided into normoxia and hypoxia groups, and the proliferation efficiency of PASMCs in yaks was measured by CCK-8 method at 0, 2, 4, 6, 12, 24, 48, 72, 96 and 120 h under hypoxia and normoxia conditions.The protein concentrations of lactate dehydrogenase (LDH), superoxide dismutase (SOD), creatine kinase (CK) and malondialdehyde (MDA) in the cell culture supernatants of normoxia and hypoxia groups at 24, 48, 72 and 96 h of culture were measured by ELISA method.【Result】 The result of cell identification showed that more than 95% of cells could express α-SMA, which indicated that the isolated and purified cells were PASMCs in yaks.CCK-8 results showed that the cell proliferation folds in hypoxia group at 72, 96 and 120 h were extremely significantly lower than that in normoxia group (P<0.01), and the cell proliferation folds in both hypoxia and normoxia groups at 72 h were extremely significantly higher than 48 h (P<0.01), which indicated that the proliferation peak of PASMCs in yaks occurred at 72 h, and the prolonged hypoxia environment could reduce the proliferation efficiency of PASMCs in yaks.ELISA assay results were showed that compared with normoxia group, the LDH protein concentration of PASMCs culture supernatant of yaks in hypoxia group at 72 h was extremely significantly upregulated (P<0.01), the SOD protein concentration at 72 and 96 h were extremely significantly or significantly upregulated (P<0.01 or P<0.05), the CK protein concentration at 48 and 72 h were significantly or extremely significantly upregulated (P<0.05 or P<0.01), and the MDA protein concentrations at 72 h was significantly upregulated and at 96 h was significantly downregulated (P<0.05).Under hypoxia conditions, there was no significant difference of LDH and SOD protein concentrations in supernatant at different time (P>0.05), the CK and MDA protein concentrations at 24, 48 and 72 h were significantly higher than 96 h (P<0.05).Under normoxia conditions, the LDH protein concentration in supernatant at 48 h was significantly higher than 24 and 72 h (P<0.05), the SOD protein concentration at 24 and 48 h were significantly higher than 72 and 96 h (P<0.05), the CK protein concentration at 24 h was significantly higher than 72 and 96 h (P<0.05), the MDA protein concentration at 48 h was significantly higher than 72 h (P<0.05).【Conclusion】 Prolonged hypoxia could enhance oxidative stress and cell damage of PASMCs in yaks, weaken antioxidant effects, and significantly reduced cell proliferation efficiency.Four proteins of LDH, SOD, CK and MDA were played important regulatory roles in damage and anti-damage as well as antioxidant effects of PASMCs in yaks under hypoxia conditions, which might be related to the hypoxia adaptation of pulmonary vasculature in yaks.

Key words: yaks; pulmonary arterial smooth muscle cells (PASMCs); hypoxia; oxidative stress

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