CAJ | 학술논문

目的建立人心脏瓣膜间质细胞体外钙化模型,并观察人骨形态发生蛋白2(BMP-2)在心脏瓣膜间质细胞钙化中的作用.方法体外培养人心脏瓣膜间质细胞后,将其分为2组:对照组在常规培养基础上,加用重组型人BMP-2蛋白;实验组在常规培养基础上加用钙化诱导剂,包括重组型人BMP-2蛋白、β甘油磷酸、L抗坏血酸和地塞米松.两组细胞均培养14 d,分别进行Von Kossa染色,并观察细胞钙化情况,建立心脏瓣膜间质细胞体外钙化模型.采用Western blot法检测两组细胞中细胞间黏附分子1、白细胞介素8、BMP-2和BMP-4的表达;采用ELISA法测定两组细胞分泌的BMP-2蛋白含量.结果对照组细胞结构清晰,呈梭形放射状生长,Von Kossa染色阴性;实验组细胞核颜色变深,可见颗粒状沉积物分布在细胞周围,Von Kossa染色阳性,可见钙化的细胞形成结节.实验组细胞中的细胞间黏附分子1、白细胞介素8、BMP-2和BMP-4表达均高于对照组(P均＜0.05).细胞培养液上清中的BMP-2水平实验组为(92.5±4.9)pg/ml,对照组为(22.2±1.9) pg/ml,差异有统计学意义(P＜0.05).结论采用重组型人BMP-2、β甘油磷酸、L抗坏血酸和地塞米松可诱导体外培养的瓣膜间质细胞钙化,并发生炎症反应,其BMP-2的表达以及分泌均增加.
목적 건립인심장판막간질세포체외개화모형,병관찰인골형태발생단백2(BMP-2)재심장판막간질세포개화중적작용.방법 체외배양인심장판막간질세포후,장기분위2조:대조조재상규배양기출상,가용중조형인BMP-2단백;실험조재상규배양기출상가용개화유도제,포괄중조형인BMP-2단백、β감유린산、L항배혈산화지새미송.량조세포균배양14 d,분별진행Von Kossa염색,병관찰세포개화정황,건립심장판막간질세포체외개화모형.채용Western blot법검측량조세포중세포간점부분자1、백세포개소8、BMP-2화BMP-4적표체;채용ELISA법측정량조세포분비적BMP-2단백함량.결과 대조조세포결구청석,정사형방사상생장,Von Kossa염색음성;실험조세포핵안색변심,가견과립상침적물분포재세포주위,Von Kossa염색양성,가견개화적세포형성결절.실험조세포중적세포간점부분자1、백세포개소8、BMP-2화BMP-4표체균고우대조조(P균＜0.05).세포배양액상청중적BMP-2수평실험조위(92.5±4.9)pg/ml,대조조위(22.2±1.9) pg/ml,차이유통계학의의(P＜0.05).결론 채용중조형인BMP-2、β감유린산、L항배혈산화지새미송가유도체외배양적판막간질세포개화,병발생염증반응,기BMP-2적표체이급분비균증가.
Objective To establish human heart valve interstitial cells calcification culture model in vitro,and observe the effect of bone morphogenetic protein-2 (BMP-2) on calcification of human heart valve interstitial cells.Methods Human heart valve interstitial cells were cultured in vitro,and divided into control group:cells were cultured in conventional media plus recombinant human BMP-2 treatmnent and experimental group:besides above treaments,calcification inducers (recombinant hunan BMP-2,β-glycerophosphate,L-ascorbic acid,dexamethasone) were added to the culture media.The two group of cells were cultured for 14 days and were stained by Von Kossa,then the cell calcification was observed in this valvular interstitial cells calcification culture model in vitro.Protein expression of intercellular adhesion molecule 1 (ICAM-1),interleukin 8,BMP-2 and BMP-4 was determined by Western blot and BMP-2 secretion was measured by ELISA.Results In the control group,the structure of human heart valve interstitial cells was clear,and the spindle and radial growth shaped cellular morphology was visible,and Von Kossa staining was negative.In the experimental group,the nuclei become darker in color,and granular sediment distribution was seen surrounding cells,and Von Kossa staining was positive,the cells were forming nodules of calcification.The protein expression of ICAM-1,interleukin 8,BMP-2 and BMP-4 in the experimental was significantly higher than that of the control group (all P ＜ 0.05).The expression of BMP-2 in the experimental group was also significantly higher than that in control group ((92.5 ±4.9) pg/ml vs.(22.2 ± 1.9) pg/ml,P ＜ 0.05).Conclusion Human BMP-2,β-glycerophosphate,L-ascorbic acid,and dexamethasone can induce human heart valve interstitial cells calcification and enhance inflammation in vitro by stimulating the secretion of BMP-2.