缺氧预处理骨髓间充质干细胞对大鼠脊髓缺血再灌注时血红素氧合酶-1表达的影响

Effect of hypoxia-preconditioned BMSCs on expression of HO-1 during spinal ischemia-reperfusion in rats

目的 评价缺氧预处理骨髓间充质干细胞（BMSC）对大鼠脊髓缺血再灌注时血红素氧合酶-1（HO-1）表达的影响.方法 健康清洁级SD雄性大鼠30只,6～8周龄,体重200～ 250 g,采用随机数字表法,将其分为5组（n=6）：假手术组（S组）、缺血再灌注组（I/R组）、PBS组、BMSC移植组（BMSC组）和缺氧预处理组（HP）.HP组BMSC置于3％O2-5％ CO2-92％N2的培养箱中培养24 h,进行缺氧预处理.PBS组、BMSC组和HP组分别鞘内注射PBS、BMSC和缺氧预处理的BMSC,60 min后采用主动脉阻断法建立大鼠脊髓缺血再灌注损伤模型.分别于再灌注6、12、24、48 h和7d时行神经功能评分.于再灌注7d时,处死大鼠,取脊髓组织,采用Western blot法测定HO-1蛋白表达水平,采用RT-PCR法测定HO-1 mRNA表达水平.结果 与S组比较,其余各组再灌注各时点神经功能评分降低,脊髓组织HO-1 mRNA及其蛋白表达上调（P＜0.05）;与I/R组比较,BMSC组和HP组再灌注各时点神经功能评分升高,脊髓组织HO-1 mRNA及其蛋白表达上调（P＜0.05）,PBS组再灌注各时点神经功能评分、脊髓组织HO-1 mRNA及其蛋白表达差异无统计学意义（P＞0.05）;与BMSC组比较,HP组再灌注各时点神经功能评分升高,脊髓组织HO-1 mRNA及其蛋白表达上调（P＜0.05）.结论 缺氧预处理增强BMSC移植减轻大鼠脊髓缺血再灌注损伤作用的机制可能与HO-1表达进一步上调有关.

Objective To evaluate the effect of hypoxia-preconditioned bone marrow mesenchymal stem cells （BMSCs） on the expression of HO-1 during spinal ischemia-reperfusion （I/R） in rats.Methods Thirty healthy adult Sprague-Dawley rats,weighing 200-250 g,were randomly divided into 5 groups with 6 animals in each group using a random number table：sham operation group （group S）,I/R group,PBS group,BMSC transplantation group （BMSC group） and hypoxic preconditioning group （group HP）.In group HP,BMSCs were exposed to 3％O2-5％CO2-92％N2 for 24 h for hypoxia preconditioning.In PBS,BMSC and HP groups,PBS,BMSCs and BMSCs for hypoxic preconditioning were injected intrathecally,respectively,and 60 min later spinal I/R was induced by clamping the aorta in the rats anesthetized with chloral hydrate.At 6,12,24 and 48 h and 7 days of reperfusion,the neurological function was evaluated and scored.At day 7 of reperfusion,the rats were sacrificed,and spinal cord tissues were obtained for determination of HO-1 expression （by Western blot） and HO-1 mRNA expression （using real-time PCR）.Results Compared with group S,the neurological function score was significantly decreased at each time point of reperfusion,and the expression of HO-1 mRNA and protein in spinal cord tissues was up-regulated in the other groups.Compared with group I/R,the neurological function score was significantly increased at each time point of reperfusion,and the expression of HO-1 mRNA and protein in spinal cord tissues was upregulated in BMSC and HP groups,and no significant change was found in group PBS in the parameters mentioned above.Compared with group BMSC,the neurological function score was significantly increased at each time point of reperfusion,and the expression of HO-1 mRNA and protein in spinal cord tissues was up-regulated in group HP.Conclusion The mechanism by which hypoxic preconditioning enhances protection of spinal cord by BMSC transplantation may be related to up-regulation of HO-1 expression in rats.