慢性肾衰竭兔血清对主动脉平滑肌细胞增殖及核因子κB活化的作用

Effects of chronic renal failure rabbit serum on proliferation and nuclear factor kappa B activation of rabbit arterial smooth muscle cells

目的 研究慢性肾衰竭兔血清对其主动脉平滑肌细胞增殖和核因子κB（NF-κB）活化的影响,并探讨其作用的可能机制.方法 建立慢性肾衰竭兔模型,采集血清.采用原代培养的兔主动脉平滑肌细胞,用不同浓度的慢性肾衰竭兔血清刺激不同时间,四甲基偶氮噻唑盐（MTF）法检测细胞增殖;Hoechst33342染色观察细胞凋亡;Western印迹检测慢性肾衰竭血清对主动脉平滑肌细胞增殖细胞核抗原（PCNA）、NF-κB p65蛋白表达的影响;免疫荧光观察NF-κB p65核转位.结果 （1）在较低浓度（≤10%）时,慢性肾衰竭兔血清对平滑肌细胞的增殖有明显的促进作用,且呈浓度、时间依赖性;但血清浓度继续增加后,对平滑肌细胞的促增殖作用却明显减弱,与正常血清组差异有统计学意义（P＜0.05）.（2）Hoechst33342染色表明,慢性肾衰竭血清在低浓度时（≤10%）细胞凋亡率和正常血清组差异无统计学意义（P＞0.05）,但高浓度（＞10%）时,平滑肌细胞凋亡率增加,与正常血清组差异有统计学意义（P＜0.01）.（3）慢性肾衰竭血清刺激24 h后,低浓度促进PCNA、NF-κB p65蛋白表达,高浓度抑制PCNA、NF-κB p65表达,与正常血清组差异有统计学意义（P＜0.01）.（4）10%慢性肾衰竭血清与平滑肌细胞孵育24 h后免疫荧光显示,NF-κB p65发生了核转位.结论 不同浓度的慢性肾衰竭兔血清可导致平滑肌细胞增殖或凋亡,其促增殖作用可能与其活化了NF-κB有关.本研究为防治慢性肾衰竭加速性动脉粥样硬化提供理论依据.

Objective To investigate the effects of chronic renal failure rabbit serum on proliferation and nuclear factor kappa B （NF-κB） activation of rabbit arterial smooth muscle cells （ASMCs） and to explore the possible mechanism. Methods Rabbit model of chronic renal failure was established by the ligation of renal arterial branches. ASMCs were incubated in the media with various concentrations of chronic renal failure serum cultured in vitro. Cell proliferation was assessed by MTT. Cell apoptosis was detected by Hoechst33342 staining. NF-κB p65 nuclear translocation was analyzed by immunofluorescence. Expression of proliferating cell nuclear antigen （PCNA） and NF-κB p65 proteins in response to chronic renal failure serum in ASMCs was determined by Western blotting. Results Lower concentrations of chronic renal failure serum （≤ 10%） could significantly promot the proliferation of ASMCs in a dose- and time-dependent manner. Higher concentrations of chronic renal failure serum （〉10%） could significantly inhibit the proliferation and induce apoptosis of ASMCs compared to the normal control （P〈0.05）. Under the stimulation of lower concentrations of chronic renal failure serum, the expression of PCNA and NF-κB p65 increased significantly compared to the normal control （P〈0.01）, while decreased markedly under the stimulation of higher concentrations of chronic renal failure serum compared to the normal control （P〈0.01）. Under the stimulation of 10% chronic renal failure serum, nuclear translocation of NF-κB p65 in ASMCs was found. Conclusions Different concentrations of chronic renal failure rabbit serum can effectively induce ASMCs proliferation or apoptosis. The mechanism of promoting proliferation may be mediated by activating NF-κB, which will be useful for the treatment of accelerated atherosclerosis in chronic renal failure.