高原鼠兔肝中Ldh-c基因的表达及其对无氧糖酵解水平的影响  

Expression of the Sperm-specific Lactate Dehydrogenase Gene(Ldh-c) in Plateau Pika(Ochotona curzoniae) Liver and Its Influence on the Anaerobic Glycolysis

作　　者：魏琳娜[1] 魏莲[1] 汪洋[1] 李筱[1] 许利娜[1] 魏登邦[1] 

机构地区：[1]青海大学农牧学院,西宁810016

出　　处：《动物学杂志》2015年第6期846-854,共9页Chinese Journal of Zoology

基　　金：国家自然科学基金项目(No.31260512);青海省自然科学基金项目(No.2012-Z-905)

摘　　要：高原鼠兔(Ochotona curzoniac)对高原低氧环境有很强的适应性。我们研究发现,精子特异性乳酸脱氢酶(LDH-C4)在高原鼠兔肝组织中表达。为了阐明LDH-C4对高原鼠兔肝组织无氧糖酵解水平的影响,将20只高原鼠兔随机分为抑制剂组和空白对照组,每组10只。抑制剂组注射1 mol/L LDH-C4特异性抑制剂N-异丙基草氨酸盐(N-isopropyl oxamate)于高原鼠兔股二头肌,双侧各注射500μl,空白对照组注射等剂量的0.9%生理盐水。应用荧光定量PCR和western blot方法,测定了精子特异性乳酸脱氢酶基因(Ldh-c)在高原鼠兔肝组织中的表达水平,应用高压液相色谱法测定了血液中抑制剂的浓度,用生物化学方法测定了抑制剂组和空白对照组高原鼠兔肝组织中乳酸脱氢酶(LDH)比活力以及乳酸(LD)和ATP含量。结果表明,在m RNA和蛋白水平,Ldh-c基因在高原鼠兔肝组织中均有表达,m RNA相对表达水平为0.22±0.04,蛋白相对表达水平为0.97±0.20;当肌肉注射1 mol/L的抑制剂30 min后,血液中抑制剂浓度为0.08 mmol/L,肝组织中乳酸脱氢酶比活力抑制剂组和空白对照组分别为(4 990±290)U/g和(7 360±420)U/g,抑制剂组和空白对照组乳酸含量分别为(0.38±0.05)mmol/g和(0.53±0.03)mmol/g,抑制剂组和空白对照组ATP含量分别为(5.84±0.83)μmol/g和(7.78±1.06)μmol/g,经单因素方差分析,抑制剂组的乳酸脱氢酶比活力和乳酸及ATP含量均显著下降(P<0.01);抑制剂对乳酸脱氢酶比活力和乳酸及ATP含量的抑制率分别为30.19%±3.90%、32.22%±8.70%和24.94%±7.80%。以上结果说明,Ldh-c在高原鼠兔肝组织中表达,LDH-C4通过催化无氧糖酵解过程,为其肝组织生命活动提供至少24%的ATP,这可能使高原鼠兔减小了在低氧环境中对氧气的依赖,增强了对低氧环境的适应力。The plateau pika（Ochotona curzoniae） has a strong adaptability to hypoxic plateau environment. We found that the sperm-specific lactate dehydrogenase（LDH-C4） gene was expressed in plateau pika liver. In order to shed light on the effect of LDH-C4 on the anaerobic glycolysis in plateau pika liver, 20 pikas were randomly divided into the inhibitor group and the control group, the sample size of each group was 10. The pikas of inhibitor group were injected 1ml 1mol N-isopropyl oxamate, a LDH-C4 specific inhibitor, in biceps femoris muscle of hind legs, each leg 500 μl, and the control individuals were injected with the same amount of 0.9% saline. The expression level of Ldh-c gene in plateau pika liver was determined by real-time PCR and western blot. The concentration of N-isopropyl oxamate in blood was determined by high pressure liquid performance（HPLP）, and the activities of lactate dehydrogenase（LDH） and the contents of lactate（LD） and ATP in liver were measured by biochemical methods. The results in Fig. 1 indicated that the expression levels of Ldh-c m RNA and protein were 0.22 ± 0.04 and 0.97 ± 0.20, respectively. After 30 min of injection of 1 ml N-isopropyl oxamate（1 mol/L） in biceps femoris muscle, the concentration of N-isopropyl oxamate in blood was 0.08 mmol/L. In the liver of inhibitor group and control group, the LDH activities were 4 990 ± 290 U/g and 7 360 ± 420 U/g, the contents of LD were 0.38 ± 0.05 mmol/g and 0.53 ± 0.03 mmol/g, and ATP centents were 5.84 ± 0.83 μmol/g and 7.78 ± 1.06 μmol/g, respectively, as shown in Fig. 2a, 2b and 2c. The data were analyzed statistically by one-way analysis of variance, and the results showed that the LDH activities, the contents of LD and ATP in liver of the inhibitor group were decreased significantly（P 0.01）, and the inhibition rate of N-isopropyl oxamate to LDH, LD and ATP were 30.19% ± 3.90%、32.22% ± 8.70% and 24.94% ± 7.80%, respectively, as shown in Fig. 2d. These results suggest that the sperm-specific lact

关 键 词：高原鼠兔 肝组织 Ldh-c基因 N-异丙基草氨酸盐 乳酸脱氢酶活力 

分 类 号：Q494[生物学—生理学] Q78