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目的探讨高糖高脂刺激下血小板活化因子(PAF)对系膜细胞分泌细胞外基质(ECM)的影响。方法人肾小球系膜细胞单独培养,实验分为六组:A组采用D-葡萄糖5.5mmol/L处理作为对照;B组采用PAF C-16 2×10~(-8) mol/L;C组采用D-葡萄糖30mmol/L+溶血卵磷脂20mg/L;D组采用D-葡萄糖30mmol/L+溶血卵磷脂20mg/L+PAF C-16 2×10~(-8) mol/L;E组采用PAF C-162×10~(-8) mol/L+转化生长因子β1(TGF-β1)中和性抗体5μg/ml;F组采用D-葡萄糖30mmol/L+溶血卵磷脂20mg/L+PAF C-16 2×10~(-8) mol/L+TGF-β1中和性抗体5μg/ml。采用qRT-PCR及Western blot法检测细胞TGF-β1的表达,ELISA法检测细胞上清液Ⅳ型胶原(ColⅣ)和纤维连接蛋白(Fn)的含量。结果与A组相比,B、C、D组系膜细胞的TGF-β1mRNA和蛋白表达水平均上调(P<0.05),而D组系膜细胞的TGF-β1mRNA表达水平亦高于B、C组(P<0.05)。与A组相比,B、C、D组系膜细胞上清液ColⅣ、Fn含量均升高(P<0.05)。与D组相比,F组细胞上清液中ColⅣ和Fn含量降低(P<0.05)。结论高糖高脂联合PAF可通过介导系膜细胞TGF-β1表达从而增加ECM分泌,是糖尿病肾病发生的潜在风险因素。
Objective To investigate the effect of platelet-activating factor (PAF) on the secretion of extracellular matrix (ECM) by mesangial cells under high glucose and lipid stimulation. Methods Human glomerular mesangial cells were cultured in vitro. The experiment was divided into six groups: group A was treated with 5.5 mmol / L D-glucose as control; group B was treated with PAF C-16 2 × 10 -8 mol / L; In group C, D-glucose 30 mmol / L and lysolecithin 20 mg / L were used in group C; D-glucose 30 mmol / L and lysolecithin 20 mg / L and PAF C-16 2 × 10 -8 mol / The mice in group F were treated with 30 mmol / L D-glucose and 20 mg / L lysophosphatidylcholine (PAF) and 10 mg / L neutralizing antibody against transforming growth factor β1 (TGF- -16 2 × 10 ~ (-8) mol / L + TGF-β1 neutralizing antibody 5μg / ml. The expression of TGF-β1 was detected by qRT-PCR and Western blot. The contents of collagen Ⅳ and fibronectin (Fn) were detected by ELISA. Results Compared with group A, the mRNA and protein expressions of TGF-β1 in mesangial cells of groups B, C and D were both increased (P <0.05), while the expression of TGF-β1 in mesangial cells of group D was also higher than that of group B and C Group (P <0.05). Compared with group A, the contents of ColⅣ and Fn in mesangial cells of group B, C and D were increased (P <0.05). Compared with group D, the content of ColⅣ and Fn in group F cell supernatant decreased (P <0.05). Conclusion High glucose and high fat combined with PAF can increase the secretion of ECM by mediating the expression of TGF-β1 in mesangial cells, which is a potential risk factor for diabetic nephropathy.