Rac1在草酸所致的肾小管上皮细胞氧化损伤中的作用
发布时间：2018-12-31 16:31
【摘要】：目的：通过抑制暴露在高浓度草酸中MDCK细胞中Ras相关的C3肉毒素底物1(Rac1)的表达,观察细胞还原型烟酰胺腺嘌呤二核苷酸(NADPH)氧化酶活性、活性氧物质含量、细胞氧化损伤程度的改变以及细胞表面对草酸钙结晶粘附情况的变化,探讨Racl在草酸所致的NADPH氧化酶介导的肾小管上皮细胞氧化损伤过程中的作用,以验证我们关于Rac1表达在草酸钙肾结石形成和复发中扮演重要角色的猜测,为草酸钙肾结石形成及复发机制研究提供新思路。 方法：体外培养犬肾小管上皮(MDCK)细胞制成爬片后,随机分为A组(空白对照组)、B组(Rac1抑制组)、C组(草酸组)、D组(Rac1抑制+草酸组)。B组和D组分别暴露在含Rac1抑制剂NSC23766(100umol/L)培养液中,A组和C组加入含磷酸盐缓冲液(PBS)的培养液。30min后C组及D组更换为含草酸(5mmol/L)的培养液,余对照更换为含PBS培养液。4h后使用化学发光法分别测定各组培养基中过氧化氢(H202)含量、乳酸脱氢酶(LDH)活性和MDCK细胞NADPH氧化酶活性；免疫细胞化学染色观察细胞Rac1表达强度；向培养基中加入草酸钙(0.75mmol/L)15min后在倒置显微镜下观察细胞对草酸钙结晶的粘附情况。 结果：A组(空白对照组)H202浓度、LDH活性及细胞NADPH氧化酶活性分别为(24.23±2.44)mmol/L、(0.62±0.06)U/mL.(1042.83±61.00) RLU/min/mg;C组(草酸组)分别为(67.84±4.25)mmol/L、(1.71±0.08)U/mL、(2852.50±105.71)RLU/min/mg; D组(Rac1抑制+草酸组)分别为(34.79±3.07)mmol/L、(0.96±0.07)U/mL、(1118.83±57.56)RLU/min/mg。与A组相比,C组细胞Rac1的表达增强(p0.05),NADPH氧化酶活性、H202含量、LDH活性也明显增强(p均0.05),MDCK细胞对草酸钙结晶的粘附数量增多(p0.05)；而与C组相比,D组细胞Rac1表达减弱(p0.05),NADPH氧化酶活性、H202含量、LDH活性均显著降低(p均0.05),粘附在细胞表面的草酸钙结晶数量也明显减少(p0.05)。 结论：草酸通过Rac1调节的NADPH氧化酶致肾小管上皮细胞氧化损伤,促进细胞对草酸钙结晶的粘附作用,抑制Rac1可显著减少活性氧物质的产生及细胞氧化损伤,减少结晶在细胞表面的粘附。因此,Rac1在草酸所致的肾小管上皮细胞氧化损伤中发挥了重要作用,在促进草酸钙肾结石形成过程中扮演了重要角色。
[Abstract]:Aim: to observe the activity of reduced nicotinamide adenine dinucleotide (NADPH) oxidase (NADPH) oxidase) and the content of reactive oxygen species (Ros) in MDCK cells exposed to high concentrations of oxalic acid by inhibiting the expression of Ras related C 3 botulinum toxin substrate 1 (Rac1). To explore the role of Racl in the oxidative injury of renal tubular epithelial cells mediated by oxalic acid-induced NADPH oxidase, and the changes of cell surface adhesion to calcium oxalate. In order to verify our hypothesis that the expression of Rac1 plays an important role in the formation and recurrence of calcium oxalate nephrolithiasis, it provides a new idea for the study of the formation and recurrence mechanism of calcium oxalate renal calculi. Methods: canine renal tubular epithelial (MDCK) cells were cultured in vitro, and then were randomly divided into group A (blank control group,), B group) (Rac1 inhibitory group,), C group, oxalic acid group). Group D (Rac1 inhibited oxalic acid group). B group and D group were exposed to NSC23766 (100umol/L) medium containing Rac1 inhibitor, respectively. Group A and group C were added with culture medium containing phosphate buffer (PBS). After 30min, group C and group D were replaced with culture medium containing oxalic acid (5mmol/L). After 4 hours, the content of hydrogen peroxide (H202), the activity of lactate dehydrogenase (LDH) and the activity of NADPH oxidase in MDCK cells were measured by chemiluminescence method. The intensity of Rac1 expression was observed by immunocytochemical staining, and the adhesion of cells to calcium oxalate crystals was observed under inverted microscope after the addition of calcium oxalate (0.75mmol/L) 15min to the culture medium. Results: the concentration of H202, the activity of LDH and the activity of NADPH oxidase in group A (blank control group) were (24.23 卤2.44) mmol/L, (0.62 卤0.06) U / mL. (1042.83 卤61.00) RLU/min/mg;, respectively. Group C (oxalic acid group) was (67.84 卤4.25) mmol/L, (1.71 卤0.08) U / mL, (2852.50 卤105.71) RLU/min/mg;, respectively. Group D (Rac1 inhibited oxalic acid) was (34.79 卤3.07) mmol/L, (0.96 卤0.07) U / mL, (1118.83 卤57.56) RLU/min/mg., respectively. Compared with group A, the expression of Rac1 in group C was increased (p0.05), NADPH oxidase activity, H202 content and LDH activity were also significantly increased (p0.05). Compared with group C, the expression of Rac1 in group D was significantly decreased (p0.05), NADPH oxidase activity, H202 content, LDH activity were significantly decreased (p0.05), and the number of calcium oxalate crystals adhered to the cell surface was also significantly decreased (p0.05). Conclusion: oxalic acid induced oxidative damage of renal tubular epithelial cells by NADPH oxidase regulated by Rac1, promoted the adhesion of cells to calcium oxalate crystals, and inhibited the production of reactive oxygen species and oxidative damage of cells by inhibiting Rac1. Reduce the adhesion of crystal on cell surface. Therefore, Rac1 plays an important role in oxalic acid-induced oxidative injury of renal tubular epithelial cells and plays an important role in the formation of calcium oxalate nephrolithiasis.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.4
本文编号：2396837
[Abstract]:Aim: to observe the activity of reduced nicotinamide adenine dinucleotide (NADPH) oxidase (NADPH) oxidase) and the content of reactive oxygen species (Ros) in MDCK cells exposed to high concentrations of oxalic acid by inhibiting the expression of Ras related C 3 botulinum toxin substrate 1 (Rac1). To explore the role of Racl in the oxidative injury of renal tubular epithelial cells mediated by oxalic acid-induced NADPH oxidase, and the changes of cell surface adhesion to calcium oxalate. In order to verify our hypothesis that the expression of Rac1 plays an important role in the formation and recurrence of calcium oxalate nephrolithiasis, it provides a new idea for the study of the formation and recurrence mechanism of calcium oxalate renal calculi. Methods: canine renal tubular epithelial (MDCK) cells were cultured in vitro, and then were randomly divided into group A (blank control group,), B group) (Rac1 inhibitory group,), C group, oxalic acid group). Group D (Rac1 inhibited oxalic acid group). B group and D group were exposed to NSC23766 (100umol/L) medium containing Rac1 inhibitor, respectively. Group A and group C were added with culture medium containing phosphate buffer (PBS). After 30min, group C and group D were replaced with culture medium containing oxalic acid (5mmol/L). After 4 hours, the content of hydrogen peroxide (H202), the activity of lactate dehydrogenase (LDH) and the activity of NADPH oxidase in MDCK cells were measured by chemiluminescence method. The intensity of Rac1 expression was observed by immunocytochemical staining, and the adhesion of cells to calcium oxalate crystals was observed under inverted microscope after the addition of calcium oxalate (0.75mmol/L) 15min to the culture medium. Results: the concentration of H202, the activity of LDH and the activity of NADPH oxidase in group A (blank control group) were (24.23 卤2.44) mmol/L, (0.62 卤0.06) U / mL. (1042.83 卤61.00) RLU/min/mg;, respectively. Group C (oxalic acid group) was (67.84 卤4.25) mmol/L, (1.71 卤0.08) U / mL, (2852.50 卤105.71) RLU/min/mg;, respectively. Group D (Rac1 inhibited oxalic acid) was (34.79 卤3.07) mmol/L, (0.96 卤0.07) U / mL, (1118.83 卤57.56) RLU/min/mg., respectively. Compared with group A, the expression of Rac1 in group C was increased (p0.05), NADPH oxidase activity, H202 content and LDH activity were also significantly increased (p0.05). Compared with group C, the expression of Rac1 in group D was significantly decreased (p0.05), NADPH oxidase activity, H202 content, LDH activity were significantly decreased (p0.05), and the number of calcium oxalate crystals adhered to the cell surface was also significantly decreased (p0.05). Conclusion: oxalic acid induced oxidative damage of renal tubular epithelial cells by NADPH oxidase regulated by Rac1, promoted the adhesion of cells to calcium oxalate crystals, and inhibited the production of reactive oxygen species and oxidative damage of cells by inhibiting Rac1. Reduce the adhesion of crystal on cell surface. Therefore, Rac1 plays an important role in oxalic acid-induced oxidative injury of renal tubular epithelial cells and plays an important role in the formation of calcium oxalate nephrolithiasis.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.4
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