尿毒症患者血清对血管平滑肌细胞钙化及Ⅰ型胶原表达的影响

Effect of uremic serum on calcification and the expression of typeⅠcollagen in vascular smooth muscle cells

目的通过不同浓度尿毒症患者血清(以下简称尿毒症血清)刺激血管平滑肌细胞(VSMCs),评估其钙化情况及I型胶原(ColⅠ)表达,探讨尿毒症血清诱导VSMCs钙化的机制。方法原代培养VSMCs,随机分为健康血清组和尿毒症血清组,依据血清在培养基中所占比例每组各设5%、10%、15%(低、中、高)三个亚组,刺激6 d后对细胞进行钙化染色及钙含量测定,同时RT-PCR检测细胞细胞内核心结合因子(Cbfα1)及ColⅠmRNA表达,Western-Blot检测细胞及上清液ColⅠ蛋白表达,并分析相关性。结果与健康血清组比较,不同浓度尿毒症血清刺激VSMCs后钙结节增加,钙含量升高,均呈浓度依赖性(P<0.05);Cbfα1 mRNA表达、ColⅠmRNA及上清液中蛋白表达均升高,且呈浓度依赖性(P<0.05)。结论不同浓度尿毒症血清刺激可使VSMCs钙化,呈现浓度依赖性,其作用机制之一可能是其促进VSMCs表型转化,进而分泌ColⅠ蛋白增加。

Objective To explore the effects of different concentrations of uremic serum on the calcification of vascular smooth muscle cells（VSMCs） and the expression of type Ⅰ collagen（Col Ⅰ） and to discuss the mechanism of VSMCs calcification induced by uremic serum.Methods Primary VSMCs were obtained,then they were randomly divided into healthy serum group and uremic serum group,and each group was divided into three subgroups based on the proportion of serum in the culture medium,namely 5%,10% and 15%.Calcium content and the calcium deposition of VSMCs were measured after stimulating 6 d.The expression of Cbfα1 mRNA and Col Ⅰ mRNA was detected by RT-PCR,the expression of Col I protein in VSMCs and supernatant fluid was detected by Western blot method;and then the correlation was analyzed.Results Compared with the healthy serum group,the calcium content and calcium deposition in VSMCs were increased,and in a dose-dependent manner after stimulating with different concentrations of uremic serum（P〈0.05）.The core-binding factor α1（Cbfα1） mRNA,Col Ⅰ mRNA and protein in uremic group were all increased in a dose-dependent manner（P〈0.05）.Conclusion Different concentrations of uremic serum can induce VSMCs calcification and in a dose-dependent manner.One of the mechanisms may be the promotion of VSMCs phenotypic transformation and thereby it achieves the above result by increasing the secretion of Col I protein.