有机磷酸盐-与无机磷酸盐-钙化诱导培养基诱导主动脉瓣膜间质细胞钙化效果的对比研究

Comparative Study for Effect on Aortic Valve Interstitial Cells Calcification between Organic Phosphate-and Inorganic Phosphate-Osteogenic Induction Medium

目的比较有机磷酸盐(OP)-与无机磷酸盐(IP)-钙化诱导培养基(OIM)诱导主动脉瓣膜间质细胞(AVICs)钙化的效果。方法主动脉瓣膜来源于2017年1—10月在第二军医大学附属长海医院行心脏移植的患者,取外观正常的主动脉瓣膜备用,采用改良的二次胶原酶消化法分离AVICs并随机分为A组和B组,A组采用普通培养基+OP-OIM进行培养,B组采用普通培养基+IP-OIM进行培养。采用茜素红染色检测钙化结节,采用邻甲酚酞络合酮比色法检测钙含量,采用实时荧光定量聚合酶链反应检测Runt相关转录因子2(RUNΧ2)、碱性磷酸酶(ALP)mRNA相对表达量,采用蛋白质免疫印迹法检测RUNΧ2蛋白相对表达量,采用对硝基苯酚比色法检测ALP活性。结果 (1)细胞免疫荧光检测结果显示,分离得到的细胞Vimentin阳性率接近100%。流式细胞术检测结果显示,分离得到的细胞CD31阳性率为1.17%。(2)A组细胞培养21 d可见大量橘红色钙化结节,B组细胞培养7 d可见大量橘红色钙化结节。A组细胞培养21 d和B组细胞培养7 d钙含量比较,差异无统计学意义(P>0.05)。(3)以细胞培养0 d作为对照,A组细胞培养21 d与B组细胞培养7 d RUNΧ2 mRNA相对表达量比较,差异无统计学意义(P>0.05);但A组细胞培养21 d与B组细胞培养7 d ALP mRNA相对表达量比较,差异有统计学意义(P<0.05)。(4)两组细胞培养0 d RUNΧ2蛋白相对表达量比较,差异无统计学意义(P>0.05);A组细胞培养21 d与B组细胞培养7 d RUNΧ2蛋白相对表达量比较,差异无统计学意义(P>0.05)。(5)两组细胞培养0 d ALP活性比较,差异无统计学意义(P>0.05);A组细胞培养21 d与B组细胞培养7 d ALP活性比较,差异无统计学意义(P>0.05)。结论IP-OIM培养7 d与OP-OIM培养21 d诱导的AVICs钙化效果相当,IP-OIM诱导AVICs钙化较OP-OIM缩短14 d,可替代OP-OIM快速有效地建立体外AVICs钙化模型。

Objective To compare effect on aortic valve interstitial cells（AVICs）calcification between organic phosphate（OP）-and inorganic phosphate（IP）-osteogenic induction medium（OIM）. Methods Normal-looking aortic valves were collected from patients undergoing heart transplantation in Changhai Hospital Affiliated to the Second Military Medical University from January to October 2017,and AVICs were separated by modified secondary collagenase digestion method;then the AVICs were divided into A group and B group,thereinto AVICs in A group were cultivated in ordinary medium and OP-OIM,while AVICs in B group were cultivated in ordinary medium and IP-OIM. Alizarin red staining method was used to detect the calcified nodules,o-cresolphthalein-complexone colorimetric method was used to detect the calcium content,real-time fluorescence quantitative PCR was used to detect the relative expression of RUNΧ2 mRNA and ALP mRNA,Western blotting method was used to detect the relative expression of RUNΧ2 protein,and p-nitrophenol colorimetric method was used to detect the activity of ALP. Results（1）Cell immunofluorescence detection results showed that,positive rate of Vimentin approach to 100%;Flow cytometry test results showed that,positive rate of CD31 was 1.17%.（2）A large number of orange calcified nodules were found in A group 21 days after cultivation and in B group 7 days after cultivation. No statistically significant difference of calcium content was found between A group 21 days after cultivation and B group 7 days after cultivation（P〉0.05）.（3）Taking 0 day after cultivation as control,no statistically significant difference of relative expression of RUNΧ2 mRNA was found between A group 21 days after cultivation and B group 7 days after cultivation（P〉0.05）,but there was statistically significant difference of relative expression of ALP mRNA between A group 21 days after cultivation and B group 7 days after cultivation（P〈0.05）.（4）No statistically significant difference of relative expression of RUNΧ2 protein was found between the two groups 0 day after cultivation,nor was relative expression of RUNΧ2 protein between A group 21 days after cultivation and B group 7 days after cultivation（P〉0.05）.（5）No statistically significant difference of activity of ALP was found between the two groups,nor was activity of ALP between A group 21 days after cultivation and B group 7 days after cultivation（P〉0.05）. Conclusion IP-OIM culture for 7 days has similar induced effect on AVICs calcification with OP-OIM culture for 21 days,which shortens for 14 days and may replace OP-OIM to quickly and effectively establish AVICs calcification model in vitro.