载脂蛋白D在常染色体显性多囊肾病中的表达及对囊肿衬里上皮细胞增殖的影响

Expression of apolipoprotein D in autosomal dominant polycystic kidney disease and its influence on proliferation of cyst-lining epithelial cells

目的探讨载脂蛋白D(apolipoprotein D,ApoD)在常染色体显性多囊肾病(autosomal dominant polycystic kidneydisease,ADPKD)发病中的作用。方法采用免疫组织化学及蛋白质印迹方法分析ApoD在ADPKD患者肾脏囊肿组织中的表达;蛋白质印迹法和实时荧光定量PCR法分别检测Han:SPRD大鼠肾组织ApoD蛋白和基因表达的变化。应用MTT法检测人重组ApoD蛋白对ADPKD囊肿衬里上皮细胞(WT9-12)增殖的影响;流式细胞术检测经人重组ApoD蛋白作用后WT9-12细胞的凋亡情况及细胞周期改变。结果 ADPKD患者及Han:SPRD杂合型大鼠(cy/+)肾组织中ApoD表达量分别低于正常人及Han:SPRD正常大鼠(+/+)肾组织(P<0.05,P<0.01)。8、12、16、24周龄Han:SPRD杂合型大鼠(cy/+)肾脏中Ap-oD基因表达量均低于同周龄Han:SPRD正常大鼠(+/+)(P<0.05)。人重组ApoD蛋白以100、200、400 ng/ml作用于WT9-12细胞,作用48 h细胞增殖抑制率分别为8.21%、7.59%、8.07%(P<0.05),作用72 h细胞增殖抑制率分别为8.62%、6.43%、9.42%(P<0.05)。人重组表达ApoD蛋白以400 ng/ml浓度刺激细胞72 h,对细胞凋亡无明显影响,但可使G1期细胞增加8.26%,S期细胞减少8.09%(P<0.05)。结论 ApoD蛋白可能通过调节细胞周期减弱WT9-12细胞增殖,其表达减少可能在ADPKD的发病过程中具有一定作用。

Objective To investigate the role of apolipoprotein D（ApoD） in the pathogenesis of autosomal dominant polycystic kidney disease（ADPKD）.Methods The expression of ApoD protein in renal cystic tissues of ADPKD patients was examined by immunofluorescence and Western blotting analysis.Western blotting analysis and real-time PCR were used to detect the expression of ApoD protein and mRNA in the renal tissues of Han：SPRD rats.The cystic lining immortalized epithelial cells（WT9-12 cells） of ADPKD were stimulated with human recombinant ApoD protein and their proliferation was examined by MTT assay;flow cytometry was used to determine the cell apoptosis and cell cycle distribution.Results Immunohistochemisty and Western blotting analysis showed that ApoD expression was significantly lower in the renal tissues of ADPKD patients than that in healthy controls,and that in the Han：SPRD cy/＋ rats was significantly lower than that in the Han：SPRD ＋/＋ rats（P0.05,P0.01）.ApoD mRNA expression in the kidneys of Han： SPRD cy/＋ rats of 8,12,16,and 24 weeks old was significantly lower than that of Han： SPRD ＋/＋ rats of same ages（P0.05）.Recombinant ApoD protein at 100,200,and 400 ng/ml inhibited the proliferation of WT9-12 cells by 8.21%,7.59% and 8.07% after 48 h stimulation（P0.05）,and by 8.62%,6.43% and 9.42% after 72 h stimulation,respectively（P0.05）.Treatment with 400 ng/ml human recombinant ApoD protein for 72 h increased G1 phase WT9-12 cells by 8.26% and decreased S phase cells by 8.09%（P0.05）,but it had no significant effect on cell apoptosis.Conclusion ApoD may inhibit WT9-12 cell proliferation by regulating cell cycle,and decreased ApoD expression may participate in the pathogenesis of ADPKD.