中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2013年
6期
501-506
,共6页
尹巧香%裴志勇%王恒%赵玉生
尹巧香%裴誌勇%王恆%趙玉生
윤교향%배지용%왕항%조옥생
干细胞%环孢菌素%细胞凋亡%纳米粒子
榦細胞%環孢菌素%細胞凋亡%納米粒子
간세포%배포균소%세포조망%납미입자
Stem cells%Cyclosporine%Apoptosis%Nanoparticles
目的 通过与环孢菌素A(CsA)比较,评估环孢菌素A-纳米乳(CsA-NP)预处理保护猪脂肪组织来源干细胞(ASC)抗凋亡作用及可能机制.方法 采用过氧化氢(H2O2)体外诱导ASC凋亡模型,将ASC细胞分为6组:对照组(无特殊干预),单纯H2O2组,CsA或CsA-NP 0.1 mg/ml+H2O2组,CsA或CsA-NP 1.0 mg/ml+H2O2组,CsA或CsA-NP 5.0 mg/ml+H2O2组,CsA或CsA-NP10.0 mg/ml+H2O2组.观察凋亡细胞形态并计算凋亡率,用流式细胞仪检测ASC细胞凋亡,用细胞计数CCK-8试剂检测细胞活性,分光光度法检测半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性,蛋白质免疫印迹法(Western blot)检测细胞色素C表达.结果 细胞形态和生化检测结果显示,采用H2O2(100 μmol/L)能诱导ASC细胞凋亡,但添加CsA或CsA-NP(0.1 ～ 10.0 mg/ml)预处理后细胞凋亡率均明显下降(P＜0.01),其中又以5 mg/ml浓度组下降最多(与单纯H2O2比较,CsA:10.6％±2.8％比25.2％±3.8％；CsA-NP:6.2％ ±2.6％比25.2％±3.6％；P均＜0.01).与CsA相同浓度组比较,CsA-NP组在1 mg/ml(8.4％±3.2％比12.1％±3.2％,P＜0.05)和5mg/ml (6.2％±2.6％比10.6％±2.8％,P＜0.05)细胞凋亡率较低；添加CsA或CsA-NP (0.1 ～10.0 mg/ml)预处理后细胞存活率均明显高于单纯H2O2组(P＜0.01).同时检测CsA或CsA-NP保护细胞抗凋亡的可能机制,与单纯H2O2组比较,CsA或CsA-NP (0.1 ～10.0 mg/ml)预处理后可下调caspase-3活性；与CsA组比较,CsA-NP组在5 mg/ml浓度下调作用更为明显(1.42％±0.12％比1.68％±0.18％,P＜0.05).Western blot结果显示添加CsA或CsA-NP(5mg/ml)可完全抑制H2O2诱导的细胞色素C释放.结论 CsA-NP预处理能保护ASC抗凋亡,可能机制是减轻细胞凋亡率,提高细胞存活率,下调caspase-3活性和抑制细胞色素C释放.
目的 通過與環孢菌素A(CsA)比較,評估環孢菌素A-納米乳(CsA-NP)預處理保護豬脂肪組織來源榦細胞(ASC)抗凋亡作用及可能機製.方法 採用過氧化氫(H2O2)體外誘導ASC凋亡模型,將ASC細胞分為6組:對照組(無特殊榦預),單純H2O2組,CsA或CsA-NP 0.1 mg/ml+H2O2組,CsA或CsA-NP 1.0 mg/ml+H2O2組,CsA或CsA-NP 5.0 mg/ml+H2O2組,CsA或CsA-NP10.0 mg/ml+H2O2組.觀察凋亡細胞形態併計算凋亡率,用流式細胞儀檢測ASC細胞凋亡,用細胞計數CCK-8試劑檢測細胞活性,分光光度法檢測半胱氨痠天鼕氨痠蛋白酶-3(caspase-3)活性,蛋白質免疫印跡法(Western blot)檢測細胞色素C錶達.結果 細胞形態和生化檢測結果顯示,採用H2O2(100 μmol/L)能誘導ASC細胞凋亡,但添加CsA或CsA-NP(0.1 ～ 10.0 mg/ml)預處理後細胞凋亡率均明顯下降(P＜0.01),其中又以5 mg/ml濃度組下降最多(與單純H2O2比較,CsA:10.6％±2.8％比25.2％±3.8％；CsA-NP:6.2％ ±2.6％比25.2％±3.6％；P均＜0.01).與CsA相同濃度組比較,CsA-NP組在1 mg/ml(8.4％±3.2％比12.1％±3.2％,P＜0.05)和5mg/ml (6.2％±2.6％比10.6％±2.8％,P＜0.05)細胞凋亡率較低；添加CsA或CsA-NP (0.1 ～10.0 mg/ml)預處理後細胞存活率均明顯高于單純H2O2組(P＜0.01).同時檢測CsA或CsA-NP保護細胞抗凋亡的可能機製,與單純H2O2組比較,CsA或CsA-NP (0.1 ～10.0 mg/ml)預處理後可下調caspase-3活性；與CsA組比較,CsA-NP組在5 mg/ml濃度下調作用更為明顯(1.42％±0.12％比1.68％±0.18％,P＜0.05).Western blot結果顯示添加CsA或CsA-NP(5mg/ml)可完全抑製H2O2誘導的細胞色素C釋放.結論 CsA-NP預處理能保護ASC抗凋亡,可能機製是減輕細胞凋亡率,提高細胞存活率,下調caspase-3活性和抑製細胞色素C釋放.
목적 통과여배포균소A(CsA)비교,평고배포균소A-납미유(CsA-NP)예처리보호저지방조직래원간세포(ASC)항조망작용급가능궤제.방법 채용과양화경(H2O2)체외유도ASC조망모형,장ASC세포분위6조:대조조(무특수간예),단순H2O2조,CsA혹CsA-NP 0.1 mg/ml+H2O2조,CsA혹CsA-NP 1.0 mg/ml+H2O2조,CsA혹CsA-NP 5.0 mg/ml+H2O2조,CsA혹CsA-NP10.0 mg/ml+H2O2조.관찰조망세포형태병계산조망솔,용류식세포의검측ASC세포조망,용세포계수CCK-8시제검측세포활성,분광광도법검측반광안산천동안산단백매-3(caspase-3)활성,단백질면역인적법(Western blot)검측세포색소C표체.결과 세포형태화생화검측결과현시,채용H2O2(100 μmol/L)능유도ASC세포조망,단첨가CsA혹CsA-NP(0.1 ～ 10.0 mg/ml)예처리후세포조망솔균명현하강(P＜0.01),기중우이5 mg/ml농도조하강최다(여단순H2O2비교,CsA:10.6％±2.8％비25.2％±3.8％；CsA-NP:6.2％ ±2.6％비25.2％±3.6％；P균＜0.01).여CsA상동농도조비교,CsA-NP조재1 mg/ml(8.4％±3.2％비12.1％±3.2％,P＜0.05)화5mg/ml (6.2％±2.6％비10.6％±2.8％,P＜0.05)세포조망솔교저；첨가CsA혹CsA-NP (0.1 ～10.0 mg/ml)예처리후세포존활솔균명현고우단순H2O2조(P＜0.01).동시검측CsA혹CsA-NP보호세포항조망적가능궤제,여단순H2O2조비교,CsA혹CsA-NP (0.1 ～10.0 mg/ml)예처리후가하조caspase-3활성；여CsA조비교,CsA-NP조재5 mg/ml농도하조작용경위명현(1.42％±0.12％비1.68％±0.18％,P＜0.05).Western blot결과현시첨가CsA혹CsA-NP(5mg/ml)가완전억제H2O2유도적세포색소C석방.결론 CsA-NP예처리능보호ASC항조망,가능궤제시감경세포조망솔,제고세포존활솔,하조caspase-3활성화억제세포색소C석방.
Objective To investigate the effect of cyclosporine A-nanopaticals emulsion (CsA-NP) on protecting apoptosis of swine adipose tissue-derived stem cells (ASC) and related mechanisms.Methods ASC were randomized to six groups:contronl group,single H2O2 group,CsA or CsA-NP 0.1 mg/ml + H2O2 group,CsA or CsA-NP 1.0 mg/ml + H2O2 group,CsA or CsA-NP 5.0 mg/ml + H2O2 group,CsA or CsA-NP 10.0 mg/ml + H2O2 group.ASC apoptosis was induced by hydrogen peroxide (H2O2 100 μ mol/L) in vitro.The morphology of apoptotic cells was observed and the number of apoptotic cells was measured.Apoptosis of ASC was detected by flow cytometry using an apoptosis kit.Cell activity was determined by CCK-8 assay.Caspase-3 activity was detected by applying a caspase-3 assay kit.Expression of cytochrome C was investigated by Western blot.Results H2O2 induced ASC apoptosis was evidenced by morphological and biochemical changes,which could be significantly reduced by pre-treatment with CsA or CsA-NP at concentration of 0.1-10.0 mg/ml,and the best effect was observed at concentration of 5 mg/ml (apoptosis rate:CsA:10.6％ ±2.8％ vs.25.2％ ±3.8％; CsA-NP:6.2％ ±2.6％ vs.25.2％ ±3.6％ in control group,all P ＜ 0.01).The cell activity was significantly higher in CsA or CsA-NP pre-treated ASC at concentration of 0.1-10.0 mg/ml than in H2O2 group (P ＜0.01).Pre-treatment with CsA or CsA-NP (0.1-10.0 mg/ml) significantly down-regulated caspase-3 activity.Furthermore,CsA or CsA-NP (5 mg/ ml) completely inhibited the H2O2-induced release of cytochrome C.Conclusions These results suggest that CsA-NP and CsA could protect the oxidative stress-induced ASC apoptosis through decreasing the activation of caspase-3 and inhibiting the release of cytochrome C.
