脂联素转染的内皮祖细胞对糖尿病大鼠脑缺血再灌注损伤的保护作用

Protective effect of adiponectin-transfected endothelial progenitor cells on diabetic rats with cerebral ischemic reperfusion injury

目的探讨脂联素(adiponectin,APN)基因转染的内皮祖细胞(endothelial progenitor cells,EPCs)移植对2型糖尿病大鼠(type 2 diabetes mellitus,T2DM)脑缺血再灌注损伤(ischemic-reperfusion injury,I/R)的保护作用及其可能的作用机制。方法 4周龄SD雄性大鼠骨髓分离培养EPCs,随机分组并干预:未干预组(EPCs组)、转染APN基因的EPCs(LV-APN-EPCs组)和空白病毒EPCs(LV-EPCs组);5周龄SD雄性大鼠70只,随机分为假手术组(sham组)、对照组(PBS组)、LV-APN-EPCs治疗组、LV-EPCs治疗组和EPCs治疗组,高脂结合链脲佐菌素诱导T2DM模型,线栓法造I/R模型,I/R损伤后1 h分别静脉注射1 mL PBS、PBS、LV-APN-EPCs、LV-EPCs和EPCs,I/R前和I/R后的第1、7、14 d,各组进行mNss评分;细胞治疗后第14 d,各组行脑组织HE染色、CD31+免疫荧光染色,ELISA法检测炎症因子IL-β和TNF-α的水平。结果 I/R后第7、14 d LV-APN-EPCs组的mNss评分明显小于LV-EPCs组、EPCs组和PBS组(P<0.05);细胞治疗后第14 d,LV-APN-EPCs组神经元破坏较LV-EPCs组、EPCs组和PBS组明显减轻;CD31+免疫荧光染色显示LV-APN-EPCs治疗显著增加了I/R损伤区皮质的血管密度(P<0.05);ELISA显示LV-APNEPCs组脑组织的炎症因子IL-β和TNF-α的水平明显较LV-EPCs组、EPCs组和PBS组低(P<0.05)。结论 LV-APN-EPCs治疗对T2DM大鼠脑I/R损伤发挥保护作用,其可能的作用机制是促进脑I/R损伤区血管再生和抑制炎症因子的表达。

Objective To investigate the neuroproteetive effects and the underlying mechanisms of treat- ment with adiponectin-transfected endothelial progenitor cells （LV-APN-EPCs） in T2DM stroke rats. Methods EPCs were isolated from the bone marrow of 4-week-old male SD rats and cultured, one-third EPCs contin- ued to be cultured （EPCs） and two-thirds EPCs were transfected with LV-GFP-APN and LV-GFP respectively （LV-APN-EPCs and LV-EPCs）. Seventy rats were divided into five groups at random： 1） sham group （sham- operated rats, n = 14）. 2） PBS group （phosphate-buffered saline control group, n = 14）. 3） LV-APN-EPCs group （rats treated with LV-APN-EPCs, n = 14）. 4） LV-EPCs group （rats treated with LV-EPCs, n = 14）. 5） EPCs group （ rats treated with EPCs, n = 14）. T2DM was induced using a combination of 2-week high-fat diet, followed by a low dose （35 mg/kg） of streptozotocin （STZ） intraperitoneal injection. T2DM rats were then subjected to transient （2 hours） left middle cerebral artery occlusion （MCAO） via intraluminal vascular occlusion. At 1 hour after reperfusion, LV-APN-EPCs, LV-EPCs and EPCs resuspended in PBS were injected into the tail vein of rats （1 x 106/mL per rat, LV-APN-EPCs group, LV-EPCs group and EPCs group）, whereas lmL PBS was IV-administered to the rats （PBS group and sham group）. The modified neurological severity score （mNSS） was evaluated before MCAO and on days 1, 7, 14 after MCAO. HS〈E staining was done to view the pathological changes in rat brain tissues on day 14 after MCAO. lmmunofluorescent staining was carried out for CD3I on day 14. The levels of tumour necrosis factor alpha （TNF-α） and interleukin-1β （IL-1β） in the ischemic brain were quantified by using the ELISA kits. Results Compared with PBS, LV- EPCs and EPCs groups, better neurological function were significantly found in the LV-APN-EPCs group （P〈0. 05）. LV-APN-EPCs alleviated the morphological damage in the ischemic brain. LV-APN-EPCs treatment significantly promoted capillary regeneration as indicated by increased cluster of differentiated （CD）31 （ ＋ ） mi- crovessels in the ischemic border zone. LV APN-EPCs significantly inhibited the expression of tumour necrosis factor alpha and interleukin-1β in the ischemic brain. Concision LV-APN-EPCs treatment initiated 14 days after stroke significantly improved neurological functional deficits and alleviated morphological damage, which had neuroprotective effects on T2DM stroke rats. Decreasing the neuroinflammatory factors and increasing an giogenesis effects might be the underlying mechanisms of treatment with LV-APN-EPCs.