【摘要】：目的：通過抑制暴露在高濃度草酸中MDCK細(xì)胞中Ras相關(guān)的C3肉毒素底物1(Rac1)的表達(dá),觀察細(xì)胞還原型煙酰胺腺嘌呤二核苷酸(NADPH)氧化酶活性、活性氧物質(zhì)含量、細(xì)胞氧化損傷程度的改變以及細(xì)胞表面對(duì)草酸鈣結(jié)晶粘附情況的變化,探討Racl在草酸所致的NADPH氧化酶介導(dǎo)的腎小管上皮細(xì)胞氧化損傷過程中的作用,以驗(yàn)證我們關(guān)于Rac1表達(dá)在草酸鈣腎結(jié)石形成和復(fù)發(fā)中扮演重要角色的猜測(cè),為草酸鈣腎結(jié)石形成及復(fù)發(fā)機(jī)制研究提供新思路。 方法：體外培養(yǎng)犬腎小管上皮(MDCK)細(xì)胞制成爬片后,隨機(jī)分為A組(空白對(duì)照組)、B組(Rac1抑制組)、C組(草酸組)、D組(Rac1抑制+草酸組)。B組和D組分別暴露在含Rac1抑制劑NSC23766(100umol/L)培養(yǎng)液中,A組和C組加入含磷酸鹽緩沖液(PBS)的培養(yǎng)液。30min后C組及D組更換為含草酸(5mmol/L)的培養(yǎng)液,余對(duì)照更換為含PBS培養(yǎng)液。4h后使用化學(xué)發(fā)光法分別測(cè)定各組培養(yǎng)基中過氧化氫(H202)含量、乳酸脫氫酶(LDH)活性和MDCK細(xì)胞NADPH氧化酶活性；免疫細(xì)胞化學(xué)染色觀察細(xì)胞Rac1表達(dá)強(qiáng)度；向培養(yǎng)基中加入草酸鈣(0.75mmol/L)15min后在倒置顯微鏡下觀察細(xì)胞對(duì)草酸鈣結(jié)晶的粘附情況。 結(jié)果：A組(空白對(duì)照組)H202濃度、LDH活性及細(xì)胞NADPH氧化酶活性分別為(24.23±2.44)mmol/L、(0.62±0.06)U/mL.(1042.83±61.00) RLU/min/mg;C組(草酸組)分別為(67.84±4.25)mmol/L、(1.71±0.08)U/mL、(2852.50±105.71)RLU/min/mg; D組(Rac1抑制+草酸組)分別為(34.79±3.07)mmol/L、(0.96±0.07)U/mL、(1118.83±57.56)RLU/min/mg。與A組相比,C組細(xì)胞Rac1的表達(dá)增強(qiáng)(p0.05),NADPH氧化酶活性、H202含量、LDH活性也明顯增強(qiáng)(p均0.05),MDCK細(xì)胞對(duì)草酸鈣結(jié)晶的粘附數(shù)量增多(p0.05)；而與C組相比,D組細(xì)胞Rac1表達(dá)減弱(p0.05),NADPH氧化酶活性、H202含量、LDH活性均顯著降低(p均0.05),粘附在細(xì)胞表面的草酸鈣結(jié)晶數(shù)量也明顯減少(p0.05)。 結(jié)論：草酸通過Rac1調(diào)節(jié)的NADPH氧化酶致腎小管上皮細(xì)胞氧化損傷,促進(jìn)細(xì)胞對(duì)草酸鈣結(jié)晶的粘附作用,抑制Rac1可顯著減少活性氧物質(zhì)的產(chǎn)生及細(xì)胞氧化損傷,減少結(jié)晶在細(xì)胞表面的粘附。因此,Rac1在草酸所致的腎小管上皮細(xì)胞氧化損傷中發(fā)揮了重要作用,在促進(jìn)草酸鈣腎結(jié)石形成過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 鹵2.44) mmol/L, (0.62 鹵0.06) U / mL. (1042.83 鹵61.00) RLU/min/mg;, respectively. Group C (oxalic acid group) was (67.84 鹵4.25) mmol/L, (1.71 鹵0.08) U / mL, (2852.50 鹵105.71) RLU/min/mg;, respectively. Group D (Rac1 inhibited oxalic acid) was (34.79 鹵3.07) mmol/L, (0.96 鹵0.07) U / mL, (1118.83 鹵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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.4

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