CAJ | 학술논문

背景:大多学者使用5-氮胞苷作为诱导剂诱导骨髓间充质干细胞向心肌细胞定向分化.目的:观察联合应用黄芪甲苷及5-氮胞苷诱导骨髓间充质干细胞向心肌细胞定向分化中心肌细胞相关受体的表达.方法:选用生长良好的第3 代骨髓间充质干细胞,分为4 组.Ⅰ组:仅更换L-DMEM 培养液;Ⅱ组:100 mg/L 黄芪甲苷+5 μmol/L 5-氮胞苷诱导24h 后,更换L-DMEM 培养液.Ⅲ组:10 μmol/L 5-氮胞苷孵育24h 后,更换L-DMEM 培养液;Ⅳ组:5 μmol/L 5-氮胞苷孵育24 h 后,更换L-DMEM 培养液.各组均每3 d 换液1 次,诱导30 d 后对分化细胞进行鉴定.结果与结论:①Ⅲ组、Ⅳ组及Ⅱ组诱导后细胞心肌细胞特异性蛋白Nkx2.5 、cTnT 及Desmin 的表达均为阳性,与Ⅰ组比较,差异有非常显著性意义(P < 0.01).Ⅱ组及Ⅲ组诱导后2 周,镜下见cTnT 、Desmin 表达数量高于Ⅳ组(P < 0.01).Nkx2.5 在两组表达亦高于Ⅳ组,其中Ⅱ组与Ⅳ组比较,差异有极显著性意义(P < 0.01),Ⅲ组与Ⅳ组比较,差异有显著性意义(P < 0.05).Ⅰ组无上述蛋白的阳性表达.②诱导后2 周,镜下可见Ⅱ组、Ⅲ组细胞出现心肌细胞样的节律性跳动,证明部分细胞在诱导因素的作用下,已向心肌细胞分化.结果表明用100 mg/L 黄芪甲苷+5 μmol/L 5-氮胞苷联合诱导可产生与10 μmol/L 5-氮胞苷相似的诱导效果,这可能与黄芪甲苷对细胞具有保护作用,促血管内皮细胞增殖作用,增强细胞对5-氮胞苷细胞毒性的耐受,上调心肌特异性蛋白的表达有关.
배경:대다학자사용5-담포감작위유도제유도골수간충질간세포향심기세포정향분화.목적:관찰연합응용황기갑감급5-담포감유도골수간충질간세포향심기세포정향분화중심기세포상관수체적표체.방법:선용생장량호적제3 대골수간충질간세포,분위4 조.Ⅰ조:부경환L-DMEM 배양액;Ⅱ조:100 mg/L 황기갑감+5 μmol/L 5-담포감유도24h 후,경환L-DMEM 배양액.Ⅲ조:10 μmol/L 5-담포감부육24h 후,경환L-DMEM 배양액;Ⅳ조:5 μmol/L 5-담포감부육24 h 후,경환L-DMEM 배양액.각조균매3 d 환액1 차,유도30 d 후대분화세포진행감정.결과여결론:①Ⅲ조、Ⅳ조급Ⅱ조유도후세포심기세포특이성단백Nkx2.5 、cTnT 급Desmin 적표체균위양성,여Ⅰ조비교,차이유비상현저성의의(P < 0.01).Ⅱ조급Ⅲ조유도후2 주,경하견cTnT 、Desmin 표체수량고우Ⅳ조(P < 0.01).Nkx2.5 재량조표체역고우Ⅳ조,기중Ⅱ조여Ⅳ조비교,차이유겁현저성의의(P < 0.01),Ⅲ조여Ⅳ조비교,차이유현저성의의(P < 0.05).Ⅰ조무상술단백적양성표체.②유도후2 주,경하가견Ⅱ조、Ⅲ조세포출현심기세포양적절률성도동,증명부분세포재유도인소적작용하,이향심기세포분화.결과표명용100 mg/L 황기갑감+5 μmol/L 5-담포감연합유도가산생여10 μmol/L 5-담포감상사적유도효과,저가능여황기갑감대세포구유보호작용,촉혈관내피세포증식작용,증강세포대5-담포감세포독성적내수,상조심기특이성단백적표체유관.
BACKGROUND: 5-azacytidine (5-Aza) has been frequently used to induce bone marrow mesenchymal stem cells (BMSCs)differentiation into cardiomyocyte.OBJECTIVE: To observe expression of cardiomyocyte-related receptors in cardiomyogenic differentiation of rat BMSCs.METHODS: BMSCs of passage three were assigned to four groups: group Ⅰ: L-DMEM solution alone was replaced; Ⅱ:L-DMEM solution was replaced after induction of 100 mg/L AST+5 μmol/L 5-Aza for 24 hours; group Ⅲ: L-DMEM solution wasreplaced after induction of 10 μmol/L 5-Aza for 24 hours; and group Ⅳ: L-DMEM solution was replaced after induction of 5 μmol/L5-Aza for 24 hours. Culture medium was replaced every 3 days in each group. Differentiated cells were identified after 30 days ofinduction.RESULTS AND CONCLUSION: Expression of cardiomyocyte specific proteins Nkx2.5, cTnT and Desmin was detected in groupsⅢ, Ⅳ and Ⅱ after induction compared with group Ⅰ , with significant differences (P < 0.01). The amount of cTnT and Desminexpression expression was significantly higher in groups Ⅱ and Ⅲ compared with group Ⅳ (P < 0.01). The level of Nkx2.5expression was significantly higher in groups Ⅱ (P < 0.01) and Ⅲ (P < 0.05) compared with group Ⅳ. No Nkx2.5, cTnT andDesmin espression was detected in group Ⅰ. After induction for 2 weeks, cells with spontaneous contractility were observed ingroups Ⅱ and Ⅲ, indicating differentiation towards cardiomyocyte after induction. Results demonstrated that induction effectswere similar between 100 mg/L AST+5 μmol/L 5-Aza and 10 μmol/L 5-Aza. This may contribute to cytoprotective effects of AST,which can promote vascular endothelial cell proliferation, enhance celss tolerance to 5-Aza-induced cytotoxicity and upregulatecardiac-specific protein expression.