骨髓间充质干细胞移植对缺氧缺血性脑损伤大鼠脑内肿瘤坏死因子-α及白细胞介素-1β水平的影响

Effects of bone marrow mesenchymal stem cells transplantation on tumor necrosis factor-α and interleukin-1β in preterm rats with hypoxic-ischemic brain damage

目的探讨骨髓间充质干细胞移植对缺氧缺血性脑损伤（hypoxic—ischemic brain damage，HIBD）模型大鼠脑内肿瘤坏死因子-α（tumornecrosis factor—q，TNF—α）及白细胞介素-1β（interleukin-1β，IL-1β）水平的影响。方法3日龄清洁级Sprague—Dawley大鼠随机分为移植组、HIBD组和假手术组。移植组和HIBD组大鼠麻醉后结扎左侧颈总动脉，并吸入氧浓度为6％的氧氮混合气体2h，制作HIBD模型；假手术组大鼠麻醉后仅游离左侧颈总动脉，不做缺血缺氧处理。移植组造模后经左侧脑室定位注射增强型绿色荧光蛋白转基因大鼠骨髓间充质干细胞2μl（含细胞2×10^-5个），HIBD组造模后经左侧脑室定位注射2μl磷酸盐缓冲液，假手术组不予干预。采用HE染色法观察脑组织病理改变；免疫组织化学法检测各组大鼠脑中单核巨噬细胞抗原-1阳性细胞数；免疫荧光法检测增强型绿色荧光蛋白转基因大鼠骨髓间充质干细胞在脑部的定植；酶联免疫吸附试验法和实时荧光定量-聚合酶链反应技术检测各组大鼠脑组织中TNF—α和IL—1β蛋白及mRNA的表达水平。采用单因素方差分析和LSD检验进行统计学分析。结果干预7d后，HE染色结果显示移植组脑白质区未见明显细胞水肿、变性和坏死；HIBD组脑白质区见部分细胞变性、坏死；假手术组脑部未见明显异常。免疫组织化学检测结果显示HIBD组单核巨噬细胞抗原一1阳性细胞数为（33．0±4．0）个，明显多于移植组[（26．3±2．5）个]，假手术组[（2．3±0．6）个]明显少于移植组和HIBD组（LSD检验，P值均〈0．05）。免疫荧光显微镜观察到增强型绿色荧光蛋白转基因大鼠骨髓间充质干细胞存活并定植于脑组织。干预6、12、24、48h和7d后，HIBD组TNF-α、IL—1β蛋白水平均在24h达到峰值，随后呈下降趋势，至7d时仍未降至假手术组水平；HIBD组各时间点TNF—α、IL-1β蛋白表达水平[TNF—α（3．03±0．10）、（5．57±0．19）、（7．78±0．19）、（4．39±O．20）、（2．70±0．19）μg／L；IL-1β：（293．1±7．9）、（369．8±17．5）、（303．6±23．9）、（226．7±21．6）、（183．9±33．4）ng／L]分别显著高于移植组[TNF—α：（2．84±O．20）、（3．80±0．14）、（4．63±0．17）、（3．56±0．03）、（1．99±0．17）μg／L；IL-1β：（267．6±14．5）、（323．5±26．9）、（211．2±24．9）、（140．8±7．4）、（100．2±8．3）ng／L]，假手术组TNF—α和IL-1β蛋白表达水平最低[TNF—α：（1．03±0．02）、（1．13±0．03）、（1．05±0．02）、（1．09±O．02）、（1．07±0．02）μg／L；IL-1β：（63．6±13．0）、（64．0±11．3）、（60．8±10．0）、（67．9土13．5）、（66．2±11．7）ng／L1，差异均有统计学意义（LSD检验，P值均〈0．05）。干预24h后，HIBD组TNF- α和IL-1βmRNA表达水平分别为2．69±0．43和3．07±0．38，高于移植组（分别为1．61±0．29和1．08±0．11），假手术组表达水平（分别为0．94±0．16和1．08±0．11）明显低于移植组和HIBD组，差异均有统计学意义（LSD检验，P值均〈0．05）。结论骨髓间充质干细胞修复HIBD可能与其抑制小胶质细胞激活，下调炎症因子表达从而减轻炎症反应有关。

Objective To determine the changes in tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β ） expression after bone marrow mesenchyrnal stem cells （BMSCs） transplantation in a rat pup model of hypoxie-ischemic brain damage （HIBD） and discuss the anti-inflammatory mechanism of BMSCs transplantation in the repair of HIBD. Methods Three-day-old Sprague-Dawley rat pups were randomly divided into three groups： the transplantation group, in which a model of HIBD was established by ligation of left common carotid artery and hypoxia for two hours followed by the injection of 2 p. 1 BMSCs （2 × 10^5 cells） into the lateral ventricle; the HIBD model group, in which HIBD model was established and 2μl phosphate saline buffer was injected into the lateral ventricle; and the sham-operation group, in which no intervention was given. Histological changes in the brain were detected by HE staining and the number of cells positive for ectodermal dysplasia-1 （ED-1） staining, which is a specific marker for activated microglia, was detected by immunohistochemistry. The protein and mRNA levels of TNF-q and IL-1w were determined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. One-way analysis of variance and LSD test were applied for statistical analysis. Results HE staining showed that cellular edema and necrocytosis was not observed in cerebral white matter on the 7th post-transplantation day in the transplantation group, but observed in the HIBD model group. In the sham-operation group, cerebral white matter was normal. The number of ED-1 positive cells in the transplantation group （26.3 ± 2.5） was significantly lower than that in the HIBD model group （33.0±4.0）, but higher than that in the sham-operation group （2.3 ± 0.6） （LSD test, all P〈0.05）. The contents of TNF-α and IL-1β both in the transplantation group and the HIBD model group increased and peaked 24 b after transplantation, then gradually decreased, but did not reach normal levels （sham-operation group） on the 7th day. The contents of TNF- α and IL-1β in brain tissue in the HIBD model group [TNF- α： （3.03±0.10）, （5.57±0.19）, （7.78±0.19）, （4.39±0.20）, （2.70±019） μg/L; IL-1β： （293.1±7.9）, （369.8± 17.5）, （303.6±23.9）, （226.7±21.6）, （183.9±33.4） ng/L] were significantly higher than those in the transplantation group [TNF- α ： （2.84±0.20）, （3.80±0.14）, （4.63±0.17）, （3.56±0.03）, （1.99±0.17） μg/L; IL-1 [3： （267.6± 14.5）, （323.5±26.9）, （211.2±24.9）, （140.8±7.4）, （100.2±8.3） ng/L] at 6, 12, 24, 48 h and 7 days after BMSCs transplantation, respectively. The contents of TNF-α and IL-1β in the shamoperation group [TNF- α：（1.03 ±0.02）, （1.13±0.03）, （1.05±0.02）, （1.09±0.02）, （1.07±0.02） μ g/L; IL-1 β： （63.6 ±13.0）, （64.0 ±11.3）, （60.8 ±10.0）, （67.9 ±13.5）, （66.2 ±11.7） ng/L] were significantly lower than those in the transplantation group and HIBD model group （LSD test, all P〈0.05）. TNF- α and IL-1β mRNA at 24 h after transplantation in the HIBD model group （TNF- α ： 2.694-0.43; IL-1 β ： 3.07±0.38） were significantly higher than those in the transplantation group （TNF- α ： 1.61 4- 0.29; IL- 1 β ： 1.08 ± 0.11） and those in the sham- operation group （TNF-α： 0.94±0.16; IL-1β： 1.08±0.11） （LSD test, all P〈0.05）, Conclusions BMSCs may play an important role in the recovery of preterm HIBD and the mechanism may involve the inhibition of microglia activation and down-regulation of the expression of inflammatory factors.