摘要
目的 评价自噬在低氧预处理骨髓间充质干细胞（BMSC）在大鼠脊髓缺血再灌注损伤组织中存活的作用.方法 离体实验 大鼠原代BMSC,以1＆#215;106个/ml密度接种于12孔板（1ml/孔）,采用随机数字表法,将其分为6组（n=30）：对照组（C组）、常氧培养组（N组）、低氧预处理组（H组）、低氧预处理＋腺苷酸活化蛋白激酶（AMPK）抑制剂复合物C组（HC组）、低氧预处理＋自噬抑制剂3-甲基腺嘌呤组（HM组）和低氧预处理＋哺乳动物雷帕霉素靶蛋白抑制剂雷帕霉素组（HR组）.低氧预处理前3h时HC组、HM组和HR组分别加入10 mmol/L复合物C、5 mmol/L 3-甲基腺嘌呤和10 nmol/L雷帕霉素.每组取12个培养孔,测定磷酸化AMPK（p-AMPK）、磷酸化mTOR（p-mTOR）、微管相关蛋白1轻链3Ⅰ （LC3Ⅰ）和微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）表达.每组取18个培养孔,加入500μmmol/L H2O2孵育24 h,检测细胞存活率、凋亡率、caspase-9和caspase-3的活性.在体实验成年雄性SD大鼠,体重300～350 g,3月龄,脊髓缺血再灌注损伤模型制备成功的大鼠192只,采用随机数字表法,将其分为6组（n=32）C组、N组、H组、HC组、HM组和MR组,于再灌注30 min时,于L1-5脊髓节段分别注射离体实验中相应各组处理的1＆#215; 106个/ml BMSC悬液.分别于再灌注4、12、24和48 h时进行神经行为学评分,取脊髓组织,测定凋亡BMSC情况.结果 离体实验与N组比较,H组p-AMPK表达上调,p-mTOR表达下调,LC3Ⅱ/LC3 Ⅰ和存活率升高,凋亡率、caspase-9和caspase-3的活性降低（P＜0.05）;与H组比较,HC组p-AMPK表达下调,p-mTOR表达上调,LC3Ⅱ/LC3 Ⅰ和存活率降低,凋亡率、caspase-9和caspase-3的活性升高,HM组LC3Ⅱ/LC3 Ⅰ和存活率降低,凋亡率、caspase-9和caspase-3的活性升高,HR组p-mTOR表达下调,LC3Ⅱ/LC3 Ⅰ和存活率升高,凋亡率、caspase-9和caspase-3的活性降低（P＜0.05）.在体实验 与N组比较,H组神经行为学评分
Objective To evaluate the role of autophagy in survival of hypoxia-preconditioned bone marrow mesenchymal stem cells （BMSCs） in damaged tissues resulting from spinal cord ischemiareperfusion （I/R） injury in rats.Methods In vitro experiment Rat BMSCs were seeded in 12-well plates at a density of 1＆#215; 106 cells/ml （1 ml/well） , and randomly divided into 6 groups （n =30 wells each） ： control group （group C） , normoxia-incubated group （group N） , hypoxic preconditioning （HP） group （group H）, HP ＋ AMP-activated protein kinase （AMPK） inhibitor Compound C group （group HC）, HP ＋ autophagy inhibitor 3-methyladenine group （group HM） and HP ＋ mammalian target of rapamycin （mTOR） inhibitor rapamycin group （group HR）.In HC, HM and HR groups, 10 mmol/L Compound C, 5 mmol/L 3-methyladenine and 10 nmol/L rapamycin were added to the culture medium, respectively, at 3 h before HP.Twelve wells in each group were selected, and the expression of phosphorylated AMPK （p-AMPK） , phosphorylated mTOR （p-mTOR）, microtubule-associated protein 1 light chain 3 Ⅰ （LC3 Ⅰ）, and LC3 Ⅱ in BMSCs was determined.Eighteen wells in each group were selected, and BMSCs were co-cultured with 500 μ mmol/L H2O2 for 24 h, the survival and apoptotic rate of BMSCs were measured, and the activities of caspase-9 and caspase-3 were detected.In vivo experiment Adult male Sprague-Dawley rats, weighing 300-350 g, aged 3 months, underwent spinal cord I/R.A total of 192 rats with spinal cord I/R injury were randomly divided into C, N, H, HC, HM and HR groups （n =32 each） using a random number table.At 30 min of reperfusion, BMSC suspension 5 μ l （1＆#215;106 cells/ml） processed in N, H, HC, HM and HR groups of in vitro experiment was implanted into the lumbar segment （L1-5） of the spinal cord in N, H, HC, HM and HR groups, respectively.Neurological function was scored at 4, 12, 24 and 48 h of reperfusion.The lumbar segment of spinal cord was removed for detection of apopt
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2015年第11期1370-1376,共7页
Chinese Journal of Anesthesiology
基金
国家自然科学基金面上项目（81272065,81471845）
全军医学科技青年培育项目（14QNP036）
福建省自然科学基金面上项目（2015J01410）
南京军区福州总医院医学科技研究计划（201127,2014J01）
关键词
自噬
细胞低氧
缺血预处理
间质干细胞移植
脊髓
再灌注损伤
Autophagy
Cell hypoxia
Ischemic preconditioning
Mesenchymal stem cell transplantation
Spinal cord
Reperfusion injury
