瘦素诱导体外培养大鼠脂肪间充质干细胞凋亡  

Leptin Induced Apoptosis in Rat Adipose-derived Stem Cells Cultured in vitro

作　　者：姜玉玲[1] 乔虹[1] 刘梦雪[1] 李强[1] 孙玉倩[1] 张巾超[1] 

机构地区：[1]哈尔滨医科大学附属第二医院内分泌科,哈尔滨150086

出　　处：《生物工程学报》2008年第7期1216-1220,共5页Chinese Journal of Biotechnology

基　　金：黑龙江省自然科学基金项目(No.D200646);黑龙江省研究生创新科研项目(No.YJSCX2007-0198HLJ);黑龙江省卫生厅医学科研课题(No.2004-228);哈尔滨医科大学研究生创新基金(No.HCXS2007005)资助~~

摘　　要：为观察瘦素诱导体外培养大鼠脂肪间充质干细胞凋亡的作用,采用胶原酶消化法分离培养大鼠附睾脂肪垫间充质干细胞,第3代细胞用于实验。细胞免疫荧光化学方法鉴定CD105、Vimentin表达阳性率约80%以上,10-6mol/L的瘦素作用细胞48h、72h后激光共聚焦显微镜观察分别可见早期及中晚期特征表现;0mol/L、10-8mol/L、10-7mol/L、10-6mol/L瘦素分别作用于细胞48h后,应用AnnexinⅤ/PI双染色法流式细胞仪检测早期凋亡率分别为2.50%±0.72%、6.78%±1.99%、11.99%±1.58%、17.93%±4.82%(P<0.05);随着瘦素浓度的增加和作用时间的延长,Caspase-3的活性逐渐增高,至48h时达到高峰。说明瘦素可以直接诱导脂肪间充质干细胞凋亡,从数量上减少脂肪组织的含量。To determine the direct effect of leptin on adipose tissue apoptosis in vitro using rat adipose-derived stem cells（ADSCs）, we isolated the ADSCs of rat epididymis adipose tissue by collagenase digestion, filtration, and subsequent centrifugation. Cell cultures with or without leptin （10^-9 mol/L, 10^-8 mol/L, 10^-7 mol/L and 10^-6 mol/L） were incubated for different time. We examined the cell surface phenotype by immunofluorescence and detected the apoptosis morphological changes of ADSCs by laser scanning confocal microscope（LCSM）. The number of apoptotic cells was determined by flow cytometry assay after annexin V binding and PI staining. Caspase-3 activity was measured by spectrofluorometry. The present study demonstrates that leptin treatment causes a marked increase in adipose-derived stem cell apoptosis. With the LCSM, after being treated with leptin, ADSCs showed the typical characteristic of apoptosis. Leptin in used concentrations （0 mol/L, 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L） caused a marked increase in cell apoptosis after 48 h incubation time （for 2.50%±0.72%, 6.78%±1.99%, 11.99%±1.58% and 17.93%±4.82%, respectively, P〈0.05）. Caspase-3 activity increased and reached a maximal level after 48 h in a linear fashion. The effect of leptin was dose-dependent and time-dependent. Leptin has been demonstrated to induce preadipocyte and adipocyte apoptosis, and today we demonstrate that leptin can induce ADSCs apoptosis, which can contribute to the decrease of adiposity. To our knowledge, this is the first study demonstrating the direct peripheral effect of leptin on ADSCs.

关 键 词：瘦素 脂肪间充质干细胞 凋亡 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]