CAJ | 학술논문

探讨在缺氧/复氧所诱导乳鼠心肌细胞凋亡中，钙敏感受体（calcium - sensing receptor，CaSR）对Fas/Fas配体（Fas L）途径的影响。方法对8只2日龄Wistar大鼠心肌细胞进行原代培养，后随机分为三组：(1)对照组：细胞不经缺氧/复氧处理，在其他组复氧开始时培养基内加入与用药组等体积的生理盐水；(2)缺氧/复氧组：心室肌细胞缺氧2h/复氧24h，细胞培养基内于复氧开始时加入与用药组等体积的生理盐水；(3) CaSR激动剂氯化钆(GdCl3)组：细胞培养基内于复氧开始时加入300μmol/L CaSR激动剂GdCl3。流式细胞仪分析细胞凋亡率，透射电镜观察细胞超微结构，蛋白印迹法检测CaSR、Fas和FasL蛋白的表达。结果流式细胞仪检测显示,缺氧/复氧组心肌细胞凋亡为12.18％±1.54％,与对照组相比，心肌细胞凋亡明显增加(P＜0.01)。CaSR激动剂GdCl3使心肌细胞凋亡进一步增加至20.25％±2.87％ (P＜0.01)。透射电镜下可见线粒体絮状变、线粒体嵴溶解、空泡样变；CaSR激动剂使CaSR、Fas和FasL表达进一步上调，明显高于缺氧/复氧组(P＜0.05)。结论CaSR激活参与了缺氧/复氧所诱导的乳鼠心肌细胞凋亡，CaSR可通过激活Fas/FasL死亡受体途径导致乳鼠心肌细胞凋亡。
탐토재결양/복양소유도유서심기세포조망중，개민감수체（calcium - sensing receptor，CaSR）대Fas/Fas배체（Fas L）도경적영향。방법대8지2일령Wistar대서심기세포진행원대배양，후수궤분위삼조：(1)대조조：세포불경결양/복양처리，재기타조복양개시시배양기내가입여용약조등체적적생리염수；(2)결양/복양조：심실기세포결양2h/복양24h，세포배양기내우복양개시시가입여용약조등체적적생리염수；(3) CaSR격동제록화구(GdCl3)조：세포배양기내우복양개시시가입300μmol/L CaSR격동제GdCl3。류식세포의분석세포조망솔，투사전경관찰세포초미결구，단백인적법검측CaSR、Fas화FasL단백적표체。결과류식세포의검측현시,결양/복양조심기세포조망위12.18％±1.54％,여대조조상비，심기세포조망명현증가(P＜0.01)。CaSR격동제GdCl3사심기세포조망진일보증가지20.25％±2.87％ (P＜0.01)。투사전경하가견선립체서상변、선립체척용해、공포양변；CaSR격동제사CaSR、Fas화FasL표체진일보상조，명현고우결양/복양조(P＜0.05)。결론CaSR격활삼여료결양/복양소유도적유서심기세포조망，CaSR가통과격활Fas/FasL사망수체도경도치유서심기세포조망。
Objective To observe the influence of calcium - sensing receptor (CaSR) on Fas/Fas ligand (Fas L) pathway during anoxia/reoxygenation (A/R) - induced cardiomyocytes apoptosis in neonatal rat. Methods Single cells were dissociated from minced hearts of 2 - day - old Wistar rats with a 0. 25％ solution of crude trypsin and then cultured as monolayers at a density of 5 x 104cells/cm2 in DMEM medium equilibrated with humidified air containing 5％ CO2 at 37C. Three days after the cells were seeded, the cultured cardiomyocytes were randomly divided into three groups. ( 1 ) control group: cardiomyocytes were continuously cultured for 26 hours in DMEM medium. (2) A/R group: cardiomyocytes underwent anoxia for 2 hours and reoxygenation for 24 hours. (3) GdCl3 group: 300μmol/L GdCl3 was added to the culture medium at the beginning of reoxygenation. Apoptosis of cardiomyocytes was assessed by flow cytometer and morphological alterations were observed with transmission electron microscope. The expression of CaSR, Fas and Fas L were analyzed by Western blot.Results The result of flow cytometer showed that cardiomyocytes apoptosis was 12. 18％ ± 1.54％ in A/R group,and was higher than that in the control group (P ＜0. 01 ). At the same time, mitochondrial cristae dissolution and disappearance could be detected. Compared with A/R group, GdCl3, a specific activator of CaSR, further enhanced cardiomyocytes apoptosis to 20. 25％ + 2. 87％ ( P ＜ 0. 01 ), along with an increment in CaSR, Fas and Fas L expressions ( P ＜ 0. 05 ). Conclusion CaSR is closely involved in cardiomyocytes apoptosia during anoxia/reoxygenation. CaSR could induce apoptosis of neonatal rat cardiomyocytes through Fas/Fas L receptor pathway.