人脐血间充质干细胞移植对新生大鼠缺氧缺血性脑损伤的神经保护
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摘要
背景:目前研究虽已证实脐血间充质干细胞移植可促进神经功能的恢复,但其具体作用机制尚不明确。目的:观察人脐血间充质干细胞移植对新生大鼠缺氧缺血性脑损伤的治疗作用,分析其可能的作用机制。方法:取10 d龄新生SD大鼠(由川北医学院实验动物中心提供),置于6 000 m海拔高原低压环境模拟仓生活和运动(运动方式为在舱内游泳槽内进行为期15 d的运动,60 min/d),建立高原缺血缺氧性脑损伤模型。造模成功24 h后,将60只大鼠随机分为移植组、模型组,移植组脑组织海马内注射DAPI标记的脐血间充质干细胞悬液,模型组以同样方法注射等量PBS;另取30只大鼠作为空白对照组,不建模不移植。细胞移植48 h后,TUNEL法检测神经细胞凋亡,Western blot法检测脑组织Caspase-3、p-Akt蛋白表达;细胞移植后21 d,Morris水迷宫实验测试大鼠学习记忆能力;细胞移植28 d后,ELISA法检测大鼠脑组织炎性因子表达,组织学观察脑组织病理变化。结果与结论:①TUNEL染色显示空白对照组仅见极少量凋亡细胞,模型组可见大量细胞凋亡,移植组细胞凋亡介于空白对照组与模型组之间,3组间细胞凋亡数量比较差异有显著性意义;②移植组脑组织Caspase-3蛋白表达明显低于模型组(P <0.05),p-Akt蛋白表达明显高于模型组(P <0.05);③Morris水迷宫实验测试结果显示,移植组大鼠逃避潜伏期与游泳距离均明显短于模型组(P <0.05);④与模型组比较,移植组肿瘤坏死因子α、白细胞介素1β、白细胞介素10质量浓度明显降低(P <0.05),白细胞介素8质量浓度明显升高(P <0.05);⑤模型组大鼠海马区神经元水肿,核染色质结构不清,有空泡形成,室管膜细胞重度水肿;移植组大鼠海马区神经元水肿程度轻于模型组,核染色质结构不清,未见空泡形成;⑥结果提示,人脐血间充质干细胞可通过调控炎性因子的表达、抑制神经细胞凋亡来改善新生大鼠缺氧缺血性脑损伤的学习记忆能力,发挥脑保护作用的,其中PI3K/Akt信号通路在抑制细胞凋亡中可能发挥了一定的作用。
BACKGROUND: Although it has been confirmed that human umbilical cord blood mesenchymal stem cells transplantation can promote the recovery of neural function, its mechanism is unclear.OBJECTIVE: To observe the therapeutic effect of human umbilical cord blood mesenchymal stem cells transplantation on hypoxic-ischemic brain injury in neonatal rats and to analyze its possible mechanism.METHODS: Sixty 10-day-old neonatal Sprague-Dawley rats(provided by the Experimental Animal Center of North Sichuan Medical College in China) were placed in a simulated hypobaric environment at 6 000-meter altitude to live and exercise. The animal model of ischemic-hypoxic brain injury at high altitude was established by exercising 60 min/d for 15 days in the swimming tank of the cabin. At 24 hours after modeling, 60 rats were randomly divided into transplantation group and model group. In the transplantation group, DAPI labeled human umbilical cord blood mesenchymal stem cells were injected into the rat hippocampus, while in the model group, the same volume of PBS was injected into the rat hippocampus. Another 30 rats were taken as blank control group, with no modeling and treatment. At 48 hours after cell transplantation, apoptosis of neurons was detected by TUNEL method, and western blot assay was used to detect the expression of Caspase-3 and p-Akt protein in brain tissue. At 21 days after cell transplantation, the learning and memory abilities of rats were measured by Morris water maze test. At 28 days after cell transplantation, the expression of inflammatory factors in rat brain tissue was detected by ELISA and pathological changes of the rat brain tissue were observed histologically.RESULTS AND CONCLUSION:(1) The results of TUNEL staining showed that only a small number of apoptotic cells were observed in the blank control group, and a large number of apoptotic cells were observed in the model group. The number of apoptotic cells in the transplantation group was higher than that in the blank control group but lower than that in the model group. There was a significant difference in the number of apoptotic cells among the three groups.(2) The expression of Caspase-3 protein in the rat brain tissue was significantly lower in the transplantation group than the model group(P < 0.05), while the expression of p-Akt protein in the transplantation group was significantly higher than that in the model group(P < 0.05).(3) In the Morris water maze test, the escape latency and swimming distance in the transplantation group were significantly shorter than those in the model group(P < 0.05).(4) Compared with the model group, the levels of tumor necrosis factor α, interleukin 1β and interleukin 10 were significantly reduced in the transplantation group(P < 0.05), while the level of interleukin 8 was significantly increased in the transplantation group(P < 0.05).(5) In the model group, the rats appeared to have neuronal edema in the hippocampus, with unclear chromatin structure and vacuoles, and severe ependymal cell edema. In the transplantation group,there was milder neuronal edema in the rat hippocampus compared with the model group, and the chromatin structure was still unclear but with no formation of vacuoles. To conclude, human umbilical cord blood mesenchymal stem cells can improve the learning and memory abilities of neonatal rats with hypoxic-ischemic brain injury by regulating the expression of inflammatory factors and inhibiting neuronal apoptosis. PI3K/Akt signaling pathway may play a role in inhibiting apoptosis.
BACKGROUND: Although it has been confirmed that human umbilical cord blood mesenchymal stem cells transplantation can promote the recovery of neural function, its mechanism is unclear.OBJECTIVE: To observe the therapeutic effect of human umbilical cord blood mesenchymal stem cells transplantation on hypoxic-ischemic brain injury in neonatal rats and to analyze its possible mechanism.METHODS: Sixty 10-day-old neonatal Sprague-Dawley rats(provided by the Experimental Animal Center of North Sichuan Medical College in China) were placed in a simulated hypobaric environment at 6 000-meter altitude to live and exercise. The animal model of ischemic-hypoxic brain injury at high altitude was established by exercising 60 min/d for 15 days in the swimming tank of the cabin. At 24 hours after modeling, 60 rats were randomly divided into transplantation group and model group. In the transplantation group, DAPI labeled human umbilical cord blood mesenchymal stem cells were injected into the rat hippocampus, while in the model group, the same volume of PBS was injected into the rat hippocampus. Another 30 rats were taken as blank control group, with no modeling and treatment. At 48 hours after cell transplantation, apoptosis of neurons was detected by TUNEL method, and western blot assay was used to detect the expression of Caspase-3 and p-Akt protein in brain tissue. At 21 days after cell transplantation, the learning and memory abilities of rats were measured by Morris water maze test. At 28 days after cell transplantation, the expression of inflammatory factors in rat brain tissue was detected by ELISA and pathological changes of the rat brain tissue were observed histologically.RESULTS AND CONCLUSION:(1) The results of TUNEL staining showed that only a small number of apoptotic cells were observed in the blank control group, and a large number of apoptotic cells were observed in the model group. The number of apoptotic cells in the transplantation group was higher than that in the blank control group but lower than that in the model group. There was a significant difference in the number of apoptotic cells among the three groups.(2) The expression of Caspase-3 protein in the rat brain tissue was significantly lower in the transplantation group than the model group(P < 0.05), while the expression of p-Akt protein in the transplantation group was significantly higher than that in the model group(P < 0.05).(3) In the Morris water maze test, the escape latency and swimming distance in the transplantation group were significantly shorter than those in the model group(P < 0.05).(4) Compared with the model group, the levels of tumor necrosis factor α, interleukin 1β and interleukin 10 were significantly reduced in the transplantation group(P < 0.05), while the level of interleukin 8 was significantly increased in the transplantation group(P < 0.05).(5) In the model group, the rats appeared to have neuronal edema in the hippocampus, with unclear chromatin structure and vacuoles, and severe ependymal cell edema. In the transplantation group,there was milder neuronal edema in the rat hippocampus compared with the model group, and the chromatin structure was still unclear but with no formation of vacuoles. To conclude, human umbilical cord blood mesenchymal stem cells can improve the learning and memory abilities of neonatal rats with hypoxic-ischemic brain injury by regulating the expression of inflammatory factors and inhibiting neuronal apoptosis. PI3K/Akt signaling pathway may play a role in inhibiting apoptosis.
引文
[1]李丽珍,陈成彩,梁玉美,等.新生儿缺氧缺血性脑病的发病机制及诊断进展[J].右江民族医学院学报,2017,39(5):397-399.
[2]Massaro AN,Govindan RB,Vezina G,et al.Impaired cerebral autoregulation and brain injury in newborns with hypoxic-ischemic encephalopathy treated with hypothermia.JNeurophysiol.2015;114(2):818-824.
[3]Tamplin OJ,Durand EM,Carr LA,et al.Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.Cell.2015;160(1-2):241-252.
[4]陈阳,宋良萍,察鹏飞,等.水动力辅助吸取成人脂肪来源干细胞的体外分离、培养和鉴定研究[J].中国美容医学,2018,27(1):77-80.
[5]王雪丽.骨母特异性因子2促进瘢痕疙瘩间充质干细胞体外增殖的研究[D].重庆:陆军军医大学,2017.
[6]朱向情,王强,蔡学敏,等.新生猕猴UCMSC的制备、鉴定及对老年猕猴自发DM的治疗作用[J].西南国防医药,2018,2(1):16-19.
[7]Li J,Guo W,Xiong M,et al.Erythropoietin facilitates the recruitment of bone marrow mesenchymal stem cells to sites of spinal cord injury.Exp Ther Med.2017;13(5):1806-1812.
[8]Feng Y,Ju Y,Cui J,et al.Bone marrow stromal cells promote neuromotor functional recovery,via upregulation of neurotrophic factors and synapse proteins following traumatic brain injury in rats.Mol Med Rep.2017;16(1):654-660.
[9]汪皖君,管雅琳,王雅静,等.骨髓间充质干细胞移植对大鼠脑缺血再灌注损伤的神经保护作用[J].中风与神经疾病杂志,2016,33(11):1017-1020.
[10]Ali EHA,Ahmed-Farid OA,Osman AAE.Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model.Neural Regen Res.2017;12(12):1990-1999.
[11]曹蕊,严笠,张辰,等.人小涎腺间充质干细胞与骨髓间充质干细胞的增殖和成脂比较[J].中国美容医学,2017,26(5):31-35.
[12]Guo L,Yin F,Meng HQ,et al.Differentiation of mesenchymal stem cells into dopaminergic neuron-like cells in vitro.Biomed Environ Sci.2005;18(1):36-42.
[13]王丹妮.人脐血间充质干细胞与大鼠骨髓间充质干细胞神经分化的实验研究[D].北京:首都医科大学,2008.
[14]Dong HJ,Shang CZ,Li G,et al.The distribution of transplanted umbilical cord mesenchymal stem cells in large blood vessel of experimental design with traumatic brain injury.J Craniofac Surg.2017;28(6):1615-1619.
[15]吴芳,杨佳勇,张敏,等.脐血间充质干细胞移植对脑性瘫痪儿童神经系统功能的影响:20例分析[J].中国组织工程研究与临床康复,2008,12(16):3198-3200.
[16]徐春香,张琴芬,黄颖兰,等.新生鼠缺血性脑损伤行人脐血间充质干细胞移植两种注射途径比较[J].护理学杂志,2014,29(3):43-45.
[17]庞炜.新生儿缺氧缺血脑损伤大鼠模型制备以及间充质干细胞对模型损伤疗效的研究[D].广州:南方医科大学,2016.
[18]杨静丽,乔丽丽,程秀永,等.脂多糖与缺氧缺血对新生大鼠脑损伤的协同作用[J].中国急救医学,2003,23(1):1-2.
[19]Kim M,Yu JH,Seo JH,et al.Neurobehavioral assessments in a mouse model of neonatal hypoxic-ischemic brain injury.JVis Exp.2017;(129).doi:10.3791/55838.
[20]Wu W,Wei W,Lu M,et al.Neuroprotective Effect of Chitosan Oligosaccharide on Hypoxic-Ischemic Brain Damage in Neonatal Rats.Neurochem Res.2017;42(11):3186-3198.
[21]宋文杰,沈瑞乐,武海波.两种新生大鼠缺氧缺血性脑损伤模型制备的比较[J].中国误诊学杂志,2005,5(17):3221-3223.
[22]周龙,林也容,陈慧,等.Mg SO4对急性胎兔宫内窘迫脑损伤NMDAr1表达影响的病理学研究[J].中国当代医药,2013,20(36):11-13.
[23]吴至凤,温恩懿,赵聪敏,等.针刺对发育期脑损伤大鼠脑组织细胞凋亡及Bcl-2、Bax蛋白表达的影响[J].重庆医学,2010,39(21):2898-2900,2903.
[24]王海伟,王自勤,苏丹,等.参茸益精片对急性高原缺氧脑损伤的保护作用研究[J].实用药物与临床,2017,20(10):1112-1115.
[25]Qiao L,Fu J,Xue X,et al.Neuronalinjury and roles of apoptosis and autophagy in a neonatal rat model of hypoxia-ischemia-induced periventricular leukomalacia.Mol Med Rep.2018;17(4):5940-5949.
[26]Cui D,Sun D,Wang X,et al.Impaired autophagosome clearance contributes to neuronal death in a piglet model of neonatal hypoxic-ischemic encephalopathy.Cell Death Dis.2017;8(7):e2919.
[27]Li D,Luo L,Xu M,et al.AMPK activates FOXO3a and promotes neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia.Brain Res Bull.2017;132:1-9.
[28]熊飞,詹7),唐于平,等.PI3K/Akt信号转导通路在非小细胞肺癌中的作用[J].中国药理学通报,2010,26(10):1264-1267.
[29]王维,张琍.P13K/Akt信号转导通路的研究进展[J].现代医药卫生,2010,26(7):1051-1052.
[30]姜恩平,王帅群,王卓,等.北五味子总木脂素对脑缺血模型大鼠神经细胞凋亡及p-AKT表达的影响[J].中国中药杂志,2014,39(9):1680-1684.
[31]Zhou ZQ,Li YL,Ao ZB,et al.Baicalin protects neonatal rat brains against hypoxic-ischemic injury by upregulating glutamate transporter 1 via the phosphoinositide 3-kinase/protein kinase B signaling pathway.Neural Regen Res.2017;12(10):1625-1631.
[32]Driscoll DJO',Felice VD,Kenny LC,et al.Mild prenatal hypoxia-ischemia leads to social deficits and central and peripheral inflammation in exposed offspring.Brain Behav Immun.2018;69:418-427.
[33]Huang J,Zhang L,Qu Y,et al.Histone acetylation of oligodendrocytes protects against white matter injury induced by inflammation and hypoxia-ischemia through activation of BDNF-TrkB signaling pathway in neonatal rats.Brain Res.2018;1688:33-46.
[34]刘慧娟,戴王娟,康树敏,等.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[J].中华围产医学杂志,2016,19(4):301-307.
[35]陈瑶,张馨月,陆玉霞,等.脐血间充质干细胞分化为神经样细胞的诱导因素的比较及优化[J].临床检验杂志,2016,3(9):669-673.
[36]赵迪诚,杜鹃,陈红,等.脐血间充质干细胞治疗大鼠脑缺血性损伤的实验研究[J].中华全科医学,2012,10(2):172-173,182.
[2]Massaro AN,Govindan RB,Vezina G,et al.Impaired cerebral autoregulation and brain injury in newborns with hypoxic-ischemic encephalopathy treated with hypothermia.JNeurophysiol.2015;114(2):818-824.
[3]Tamplin OJ,Durand EM,Carr LA,et al.Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.Cell.2015;160(1-2):241-252.
[4]陈阳,宋良萍,察鹏飞,等.水动力辅助吸取成人脂肪来源干细胞的体外分离、培养和鉴定研究[J].中国美容医学,2018,27(1):77-80.
[5]王雪丽.骨母特异性因子2促进瘢痕疙瘩间充质干细胞体外增殖的研究[D].重庆:陆军军医大学,2017.
[6]朱向情,王强,蔡学敏,等.新生猕猴UCMSC的制备、鉴定及对老年猕猴自发DM的治疗作用[J].西南国防医药,2018,2(1):16-19.
[7]Li J,Guo W,Xiong M,et al.Erythropoietin facilitates the recruitment of bone marrow mesenchymal stem cells to sites of spinal cord injury.Exp Ther Med.2017;13(5):1806-1812.
[8]Feng Y,Ju Y,Cui J,et al.Bone marrow stromal cells promote neuromotor functional recovery,via upregulation of neurotrophic factors and synapse proteins following traumatic brain injury in rats.Mol Med Rep.2017;16(1):654-660.
[9]汪皖君,管雅琳,王雅静,等.骨髓间充质干细胞移植对大鼠脑缺血再灌注损伤的神经保护作用[J].中风与神经疾病杂志,2016,33(11):1017-1020.
[10]Ali EHA,Ahmed-Farid OA,Osman AAE.Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model.Neural Regen Res.2017;12(12):1990-1999.
[11]曹蕊,严笠,张辰,等.人小涎腺间充质干细胞与骨髓间充质干细胞的增殖和成脂比较[J].中国美容医学,2017,26(5):31-35.
[12]Guo L,Yin F,Meng HQ,et al.Differentiation of mesenchymal stem cells into dopaminergic neuron-like cells in vitro.Biomed Environ Sci.2005;18(1):36-42.
[13]王丹妮.人脐血间充质干细胞与大鼠骨髓间充质干细胞神经分化的实验研究[D].北京:首都医科大学,2008.
[14]Dong HJ,Shang CZ,Li G,et al.The distribution of transplanted umbilical cord mesenchymal stem cells in large blood vessel of experimental design with traumatic brain injury.J Craniofac Surg.2017;28(6):1615-1619.
[15]吴芳,杨佳勇,张敏,等.脐血间充质干细胞移植对脑性瘫痪儿童神经系统功能的影响:20例分析[J].中国组织工程研究与临床康复,2008,12(16):3198-3200.
[16]徐春香,张琴芬,黄颖兰,等.新生鼠缺血性脑损伤行人脐血间充质干细胞移植两种注射途径比较[J].护理学杂志,2014,29(3):43-45.
[17]庞炜.新生儿缺氧缺血脑损伤大鼠模型制备以及间充质干细胞对模型损伤疗效的研究[D].广州:南方医科大学,2016.
[18]杨静丽,乔丽丽,程秀永,等.脂多糖与缺氧缺血对新生大鼠脑损伤的协同作用[J].中国急救医学,2003,23(1):1-2.
[19]Kim M,Yu JH,Seo JH,et al.Neurobehavioral assessments in a mouse model of neonatal hypoxic-ischemic brain injury.JVis Exp.2017;(129).doi:10.3791/55838.
[20]Wu W,Wei W,Lu M,et al.Neuroprotective Effect of Chitosan Oligosaccharide on Hypoxic-Ischemic Brain Damage in Neonatal Rats.Neurochem Res.2017;42(11):3186-3198.
[21]宋文杰,沈瑞乐,武海波.两种新生大鼠缺氧缺血性脑损伤模型制备的比较[J].中国误诊学杂志,2005,5(17):3221-3223.
[22]周龙,林也容,陈慧,等.Mg SO4对急性胎兔宫内窘迫脑损伤NMDAr1表达影响的病理学研究[J].中国当代医药,2013,20(36):11-13.
[23]吴至凤,温恩懿,赵聪敏,等.针刺对发育期脑损伤大鼠脑组织细胞凋亡及Bcl-2、Bax蛋白表达的影响[J].重庆医学,2010,39(21):2898-2900,2903.
[24]王海伟,王自勤,苏丹,等.参茸益精片对急性高原缺氧脑损伤的保护作用研究[J].实用药物与临床,2017,20(10):1112-1115.
[25]Qiao L,Fu J,Xue X,et al.Neuronalinjury and roles of apoptosis and autophagy in a neonatal rat model of hypoxia-ischemia-induced periventricular leukomalacia.Mol Med Rep.2018;17(4):5940-5949.
[26]Cui D,Sun D,Wang X,et al.Impaired autophagosome clearance contributes to neuronal death in a piglet model of neonatal hypoxic-ischemic encephalopathy.Cell Death Dis.2017;8(7):e2919.
[27]Li D,Luo L,Xu M,et al.AMPK activates FOXO3a and promotes neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia.Brain Res Bull.2017;132:1-9.
[28]熊飞,詹7),唐于平,等.PI3K/Akt信号转导通路在非小细胞肺癌中的作用[J].中国药理学通报,2010,26(10):1264-1267.
[29]王维,张琍.P13K/Akt信号转导通路的研究进展[J].现代医药卫生,2010,26(7):1051-1052.
[30]姜恩平,王帅群,王卓,等.北五味子总木脂素对脑缺血模型大鼠神经细胞凋亡及p-AKT表达的影响[J].中国中药杂志,2014,39(9):1680-1684.
[31]Zhou ZQ,Li YL,Ao ZB,et al.Baicalin protects neonatal rat brains against hypoxic-ischemic injury by upregulating glutamate transporter 1 via the phosphoinositide 3-kinase/protein kinase B signaling pathway.Neural Regen Res.2017;12(10):1625-1631.
[32]Driscoll DJO',Felice VD,Kenny LC,et al.Mild prenatal hypoxia-ischemia leads to social deficits and central and peripheral inflammation in exposed offspring.Brain Behav Immun.2018;69:418-427.
[33]Huang J,Zhang L,Qu Y,et al.Histone acetylation of oligodendrocytes protects against white matter injury induced by inflammation and hypoxia-ischemia through activation of BDNF-TrkB signaling pathway in neonatal rats.Brain Res.2018;1688:33-46.
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[35]陈瑶,张馨月,陆玉霞,等.脐血间充质干细胞分化为神经样细胞的诱导因素的比较及优化[J].临床检验杂志,2016,3(9):669-673.
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