骨髓间充质干细胞移植对蛛网膜下腔出血大鼠早期脑损伤的治疗作用及机制

Therapeutic effect and mechanism of BMSC transplantation on early brain injury in rats with subarachnoid hemorrhage

目的探讨骨髓间充质干细胞(BMSC)移植对蛛网膜下腔出血(SAH)大鼠早期脑损伤(EBI)的治疗作用及机制。方法将健康雄性SD大鼠随机分为假手术组、蛛网膜下腔出血(SAH)组、BMSC组、VitE组,每组10只,采用血管内穿刺法构建SAH模型。分离、培养、鉴定BMSC。造模后24 h,将第4代BMSC 3×10^(6)个重悬于0.6 mL PBS中,经股静脉注入BMSC组;假手术组、SAH组、VitE组经股静脉注入等量PBS。VitE组给予维生素E(100 mg/kg)灌胃,其他组给予等量橄榄油灌胃。造模后48 h,用改良Garcia评分评估大鼠的神经功能,计算脑组织水含量,用HE染色法观察脑组织病理变化,统计坏死的神经细胞数量,普鲁士蓝染色(铁染色)法检测脑组织铁沉积情况,吸光光度法检测脑组织铁含量、MDA、GSH,Western blotting法检测脑组织NOX2蛋白,ROS探针法检测脑组织ROS。结果与假手术组比较,SAH组改良Garcia评分低,脑组织含水量高,SAH组神经细胞坏死数量多,脑组织中铁沉积量、铁含量、MDA含量高,GSH含量低,脑组织中NOX2蛋白表达、ROS水平高(P均<0.05);与SAH组比较,BMSC组、VitE组改良Garcia评分高,脑组织含水量低,神经细胞坏死数量少,脑组织中铁沉积量、铁含量、MDA含量低,GSH含量高,脑组织中NOX2蛋白表达、ROS水平低(P均<0.05)。除改良Garcia评分外,BMSC组、VitE组以上其他指标比较差异有统计学意义(P均<0.05)。假手术组神经细胞形态规则,均匀;SAH组神经细胞排列紊乱,细胞核破碎;与SAH组比较,BMSC组神经细胞损伤情况明显改善;VitE组神经细胞损伤修复情况比BMSC组弱。结论BMSC移植可有效治疗SAH大鼠EBI,其作用机制可能通过NOX2/ROS通路调控铁死亡。

Objective To investigate the therapeutic effect and mechanism of bone marrow mesenchymal stem cell(BMSC)transplantation on early brain injury(EBI)in rats with subarachnoid hemorrhage(SAH).Methods Forty-eight healthy male SD rats were randomly divided into the sham-operated group,SAH group,BMSC group and VitE group,with 10 rats in each group,and SAH models were constructed by intravascular puncture method.BMSCs were isolated,cultured,and identified.At 24 h after modeling,3×10^(6) BMSCs of the 4th generation were suspended in 0.6 mL PBS and were injected into the BMSC group via the femoral vein;equal volume of PBS was injected into the sham-operated,SAH and VitE groups via the femoral vein.Vitamin E(100 mg/kg)was administered to the VitE group by gavage,and equal volume of olive oil was administered to the other groups by gavage.At 48 h after modeling,the neurological function of rats was assessed by modified Garcia score;the water content of brain tissues was calculated;the pathological changes of brain tissues were observed by HE staining;the number of necrotic neuronal cells was counted;the iron deposition in the brain tissues was detected by Prussian blue staining(iron staining);the iron content,MDA,and GSH in the brain tissues were detected by absorption photometry;the NOX2 protein in the brain tissues was detected by Western blotting,and ROS was detected by ROS probe method.Results Compared with the sham-operated group,the SAH group had lower modified Garcia score,higher brain tissue water content,lager number of necrotic nerve cells,higher brain tissue iron deposition,iron content,MDA content,lower GSH content,and higher NOX2 protein expression and higher ROS level in the brain tissues(all P<0.05).Compared with the SAH group,the modified Garcia score was higher,the water content of brain tissue was lower,the necrotic nerve cells was less,the iron deposition,iron content and MDA content in the brain tissue were lower,the GSH content was higher,the expression of NOX2 protein and the level of ROS in brain tissues were lower in the BMSC group and VitE group(all P<0.05).Except for modified Garcia score,there were significant differences in the above indexes between BMSC group and VitE group(all P<0.05).The morphology of nerve cells in the sham-operated group was regular and uniform.In the SAH group,the nerve cells were arranged disorderly and the nucleus was broken.Compared with the SAH group,the injury of nerve cells in the BMSC group was significantly improved.The repair of nerve cell injury was better in the in BMSC group than in the VitE group.Conclusion BMSC transplantation can effectively treat EBI in SAH rats,and its mechanism of action may be the regulation of iron death through NOX2/ROS pathway.