CAJ | 학술논문

目的观察基质细胞衍生因子-1(SDF-1)对兔膝关节软骨细胞体外增殖的影响,探讨SDF-1在软骨细胞凋亡过程中的作用.方法体外分离培养兔膝关节软骨细胞并随机分为4组:对照组、50 μg/L SDF-1组、50 μg/L SDF-1+5 mg/L CXC趋化因子受体-4特异性拮抗剂(AMD3100)作用组、5 mg/L AMD3100处理组,采用噻唑蓝(MTT)比色法和测定细胞周期观察各组软骨细胞增殖.同时将各组置于250 μmol/L过氧化氢条件下作用2h,锥虫蓝染色计数软骨细胞存活率；测定各组中半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)的活力；流式细胞仪观察各组软骨细胞凋亡率的变化；原位末端转移酶标记(TUNEL)法检测凋亡的软骨细胞.结果增殖实验中SDF-1组吸光度(A)值(0.504±0.024)均高于其他各组,且该组S+G2/M期细胞比例高于其他各组,差异有统计学意义(P＜0.05).在凋亡方面,锥虫蓝拒染实验结果显示SDF-1组中细胞存活率高于其他3组(P＜0.01):而 SDF-1 组膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)标记的细胞比例[(4.00±0.92)％低于其他各组(P ＜0.05);SDF-1组Caspase-3活力(40.03±2.56) nmolpNA/(h·mg)蛋白也较其他3组低(P＜0.01):TUNEL实验中SDF-1组细胞核呈棕黄色的软骨细胞数低于其他各组(P＜0.05).但各实验中SDF-1+ AMD3100组和AMD3100处理组与对照组比较,差异无统计学意义(P＞005).结论 SDF-1可促进兔膝关节软骨细胞体外增殖,且在50 μg/L时软骨细胞增殖活性好,同时 SDF-1 在过氧化氢诱导的细胞凋亡条件下可保护软骨细胞免于凋亡.
목적 관찰기질세포연생인자-1(SDF-1)대토슬관절연골세포체외증식적영향,탐토SDF-1재연골세포조망과정중적작용.방법 체외분리배양토슬관절연골세포병수궤분위4조:대조조、50 μg/L SDF-1조、50 μg/L SDF-1+5 mg/L CXC추화인자수체-4특이성길항제(AMD3100)작용조、5 mg/L AMD3100처리조,채용새서람(MTT)비색법화측정세포주기관찰각조연골세포증식.동시장각조치우250 μmol/L과양화경조건하작용2h,추충람염색계수연골세포존활솔；측정각조중반광안선천동안산특이성단백매-3(Caspase-3)적활력；류식세포의관찰각조연골세포조망솔적변화；원위말단전이매표기(TUNEL)법검측조망적연골세포.결과 증식실험중SDF-1조흡광도(A)치(0.504±0.024)균고우기타각조,차해조S+G2/M기세포비례고우기타각조,차이유통계학의의(P＜0.05).재조망방면,추충람거염실험결과현시SDF-1조중세포존활솔고우기타3조(P＜0.01):이 SDF-1 조막련단백V-이류청산형광소(Annexin V-FITC)표기적세포비례[(4.00±0.92)％ 저 우기타각조(P ＜0.05);SDF-1조Caspase-3활력(40.03±2.56) nmolpNA/(h·mg)단백야교기타3조저(P＜0.01):TUNEL실험중SDF-1조세포핵정종황색적연골세포수저우기타각조(P＜0.05).단각실험중SDF-1+ AMD3100조화AMD3100처리조여대조조비교,차이무통계학의의(P＞005).결론 SDF-1가촉진토슬관절연골세포체외증식,차재50 μg/L시연골세포증식활성호,동시 SDF-1 재과양화경유도적세포조망조건하가보호연골세포면우조망.
Objective To observe the effect of stromal cell-derived factor-1 (SDF-1) on the proliferation of articular chondrocytes in rabbits and discuss the role of SDF-1 in chondrocyte apoptosis.Methods The chondrocytes were isolated and cutured,then divided into 4 groups:control group,50 μg/L SDF-1 group,50 μg/L SDF-1 + 5 mg/L CXC chemokine receptor-4 specific antagonist (AMD3100) group,and 5 mg/L AMD3100 group.The MTT assay and methods for cell cycle were used to observe the proliferation of chondrocytes.In addition,each group was exposured to the condition of 250 μmol/L hydrogen peroxide.Then cell viability was detected by using trypan blue exclusion assay,apoptosis rate of chondrocytes was detected by using flow cytometry,and the dynamic changes of Caspase-3 were measured in each group.Furthermore,the apoptosis of chondrocytes was evaluated by TdT-mediated dUTP nick end labeling (TUNEL) assay.Results The absorbance value of SDF-1 set (0.504 ±0.024) was higher than other groups,and the S +G2/M cell ratio in SDF-1 group was higher than other groups with the differnce bing statistically significant (all P ＜0.05).In terms of apoptosis,the viable rate in SDF-1 group was higher than other groups (P ＜ 0.01),while the Annexin V-FITC (+) proportion of cartilage cells in the SDF-1 group (4.00 ± 0.92) ％ was lower than other groups (P ＜ 0.05).Caspase-3 activity in SDF-1 group [(40.03 ± 2.56) nmolpNA/(h·mg) protein] was lower than other groups (P ＜0.01),and in addition,the number of apoptotic cells in SDF-1 group was less than other groups (P ＜ 0.05),but there was no significant difference among control group,SDF-1 + AMD3100 group and AMD3100 group in all experiments.Conclusion SDF-1 could contribute to the proliferation of rabbit chondrocytes in vitro,and in 50 μg/L group the Proliferation activity is reasonable,meanwhile SDF-1 can protect the chondrocytes from apoptosis induced by hydrogen peroxide.