目的 探究细胞外酸碱环境对主动脉瓣膜间质细胞钙化的影响及相关机制.方法 主动脉瓣膜来源于2017年1—6月在第二军医大学附属长海医院心血管外科接受心移植手术的患者,采用二次胶原酶消化法分离主动脉瓣膜间质细胞,采用细胞免疫荧光法和流式细胞术鉴定主动脉瓣膜间质细胞.将传代的主动脉瓣膜间质细胞随机分为A、B、C、D 4组,A组采用普通培养基培养,pH值为7.4,B、C、D组均采用普通培养基+钙化培养基培养,pH值分别为7.1、7.4、7.7.采用实时荧光定量聚合酶链反应(PCR)检测4组细胞骨形态发生蛋白2(BMP-2)、Runt相关转录因子2(Runx2)和碱性磷酸酶(ALP)mRNA表达情况,采用Western Blot法检测4组细胞BMP-2、Runx2蛋白表达情况,采用比色法检测4组细胞ALP活性,采用茜素红染色观察4组细胞钙化结节情况.结果 (1)细胞免疫荧光法检测结果显示,波形蛋白(Vimentin)阳性率接近100%.流式细胞术检测结果显示,细胞CD31阳性率为1.17%.(2)将A组作为对照,C、D组细胞BMP-2、Runx2和ALP mRNA相对表达量高于B组,D组细胞BMP-2、Runx2和ALP mRNA相对表达量高于C组(P<0.05).(3)B、C、D组细胞BMP-2、Runx2蛋白相对表达量高于A组,C、D组细胞BMP-2、Runx2蛋白相对表达量高于B组,D组细胞BMP-2、Runx2蛋白相对表达量高于C组(P<0.05).(4) B、C、D组细胞ALP活性高于A组,C、D组细胞ALP活性高于B组,D组细胞ALP活性高于C组(P<0.05).(5)茜素红染色结果显示,A组细胞钙化结节较少,B、C、D组细胞钙化结节逐渐增多;与C组相比,B组钙化结节明显减少,D组钙化结节明显增多.结论 细胞外酸性环境可抑制主动脉瓣膜间质细胞钙化,碱性环境则可促进主动脉瓣膜间质细胞钙化,其机制可能与细胞外酸碱环境影响BMP-2信号通路有关.%Objective To investigate the impact of extracellular acidic or alkaline environment on aortic valve interstitial cells calcification and the underlying mechanism.Methods From January to June 2017,aortic valves were collected from patients underwent heart transplantation surgery in the Department of Cardiovascular Surgery Changhai Hospital Affiliated to the Second Military Medical University,secondary collagenase digestion method was used to isolate aortic valve interstitial cells,cell immunofluorescence method and flow cytometry were used to identify the aortic valve interstitial cells.Passaged aortic valve interstitial cells were randomly divided into A group,B group,C group and D group,thereinto cells in A group were cultivated in ordinary culture medium(pH was 7.4),cells in B group,C group and D group were cultivated in ordinary culture medium and calcification medium with different pH(7.1,7.4 and 7.7,respectively).Real-time fluorescence quantitative PCR was used to detect the mRNA expression of BMP-2,Runx2 and ALP,Western Blot method was used to detect the protein expression of BMP-2 and Runx2,colorimetric method was used to detect the activity of ALP,alizarin red staining method was used to observe the calcified nodules in cells.Results (1) Cell immunofluorescence test results showed that,positive rate of Vimentin was nearly 100%;flow cytometry results showed that,the positive rate of CD31 was 1.17%.(2) Taking A group as control,relative mRNA expression of BMP-2,Runx2 and ALP in C group and D group was statistically significantly higher than that in B group,respectively,meanwhile relative mRNA expression of BMP-2,Runx2 and ALP in D group was statistically significantly higher than that in C group,respectively(P<0.05).(3) Relative protein expression of BMP-2 and Runx2 in B group,C group and D group was statistically significantly higher than that in A group,respectively,relative protein expression of BMP-2 and Runx2 in C group and D group was statistically significantly higher than that in B group,respectively, meanwhile relative protein expression of BMP-2 and Runx2 in D group was statistically significantly higher than that in C group, respectively(P<0.05).(4) Activity of ALP in B group,C group and D group was statistically significantly higher than that in A group,respectively,activity of ALP in C group and D group was statistically significantly higher than that in B group, respectively,meanwhile activity of ALP in D group was statistically significantly higher than that in C group(P<0.05).(5) Alizarin red staining results showed that,calcified nodules in A group were little,while calcified nodules in B group,C group and D group gradually increased;compared with C group,calcified nodules in B group significantly reduced,while calcified nodules in D group significantly increased.Conclusion Extracellular acidic environment can inhibit the aortic valve interstitial cells calcification,while extracellular alkaline environment may promote the aortic valve interstitial cells calcification,the mechanism possibly correlated with influence on BMP-2 signaling pathway.
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