摘要
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—NP0．1mg／ml±H202组，CsA或CsA—NP1．0mg／ml±H202组，CsA或CsA—NP5．0mg／ml±H202组，CsA或CsA—NP10．0mg／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一8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一3（caspase-3）活性，蛋白质免疫印迹法（Westernblot）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~mol／L）能诱导ASC细胞凋亡，但添加CsA或CsA—NP（0．1～10．0mg／m1）预处理后细胞凋亡率均明显下降（P〈0．01），其中又以5mg／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1mg／ml（8．4％±3．2％比12．1％4-3．2％，P〈0．05）和5mg／ml（6．2％±2．6％比10．6％4-2．8％，P〈0．05）细胞凋亡率较低；添加CsA或CsA—NP（0．1～10．0mg／m1）预处理后细胞存活率均明显高于单纯H，0，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H。0：组比较，CsA或CsA—NP（0．1～10．0mg／m1）预处理后可下调easpase-3活性；与CSA组比较，CSA-NP组在5mg／ml浓度下调作用更为明显（1．42％±0．12％比1．68％±0．18％，P〈0．05）。Westernblot结果显示添加CSA或CSA-NP（5mg／m1）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
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 ： controul group, single H2O2 group, CsA or CsA-NP 0. 1 mg/ml ± H2O3 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 ± H2O3 group. ASC apoptosis was induced by hydrogen peroxide （H2 02100 p.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 cytomeu 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 （O. 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.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2013年第6期501-506,共6页
Chinese Journal of Cardiology
基金
国家863计划项目（2006AA02A105）
