Human erythropoietin-modified human amniotic mesenchymal stem cells via subarachnoid transplantation promote neurologic recovery from brain injury

doi:10.3969/j.issn.2095-4344.2016.23.013

Abstract

Abstract:

BACKGROUND: Studies have shown that as a regulator of bone marrow function erythropoietin is a glycoprotein that controls the development of the central nervous system and has neurotrophic and neuroprotective potential. Therefore, transplantation of human amniotic mesenchymal stem cells genetically modified by human erythropoietin is a new choice for brain injury treatment.
OBJECTIVE: To observe the effect of transplantation of human amniotic mesenchymal stem cells genetically modified by human erythropoietin on the functional recovery from brain injury in rats.
METHODS: Eukaryotic expression plasmid pcDNA3.1 carrying erythropoietin was successfully constructed and transferred into amniotic mesenchymal stem cells cultured in vitro. Expression of erythropoietin was detected using western blot assay before and after transfection. Rat models of middle cerebral arterial occlusion was made and given transplantation of transfected amniotic mesenchymal stem cells via the tail vein (transfection group). Additionally, model and simple cell transplantation groups were set in a comparative study.
RESULTS AND CONCLUSION: Findings from western blot detection showed that transfected cells could express human erythropoietin. Compared with the other groups, modified neurologic severity scores, growth-associated protein 43 and aquaporin 9 at mRNA and protein levels were all decreased significantly in the transfection group. Furthermore, the number of cells positive for CM-Dil was highest in the transfection group, followed by simple cell transplantation group, and lowest in the model group (all P < 0.05). Overall findings from this study show that human erythropoietin-modified human amniotic mesenchymal stem cell transplantation promotes neurologic recovery from brain injury through eliciting a reduction in growth-associated protein 43 and aquaporin 9 at mRNA and protein levels as well as inhibiting cell apoptosis.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, Brain Injuries

CLC Number:

R394.2

Lv Gang, Zhang Hong-yuan. Human erythropoietin-modified human amniotic mesenchymal stem cells via subarachnoid transplantation promote neurologic recovery from brain injury[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(23): 3432-3438.

Figures/Tables 1

2.1 实验动物数量分析纳入大鼠67只，建模过程中死亡1只，最终66只进入最终结果分析。 2.2 人羊膜间充质干细胞形态最初培养的人羊膜间充质干细胞呈贴壁生长，形态多样，呈梭形、多边形、星形、纺锤形或多角形等(图1A)。传至第2代后人羊膜间充质干细胞贴壁速度加快，培养8 h几乎完全贴壁，细胞呈现均匀一致的成纤维细胞样，细胞多呈单层放射形状、长梭形或旋窝状生长，倒置显微镜下观察到，人羊膜间充质干细胞平均直径约为15 μm，胞浆内含有丰富的中间丝，含有中等量的高尔基复合体、线粒体和致密体，胞浆周围有自噬空泡和胞饮小泡，细胞或细胞突起之间可见到细胞连接结构(图1B)。 2.3 Western blot检测人促红细胞生成素蛋白表达结果重组腺相关病毒作为载体介导的促红细胞生成素基因在转染48 h后，人促红细胞生成素基因转染组的人羊膜间充质干细胞检测到人促红细胞生成素蛋白表达明显，而模型组和空载病毒组人促红细胞生成素蛋白未见表达，说明了人促红细胞生成素基因已稳定整合入人促红细胞生成素转染组人羊膜间充质干细胞中，且可以行目的蛋白的稳定表达，见图2。 2.4 大鼠脑织生长相关蛋白43及水通道蛋白9基因和蛋白表达的变化 RT-PCR检测显示，移植后7 d，脑损伤组脑损伤周围组织水通道蛋白9、生长相关蛋白43 mRNA和蛋白的表达高于人羊膜间充质干细胞移植组(P < 0.05)。人羊膜间充质干细胞移植组高于联合治疗组(P < 0.05)，见图3。 2.5 神经学缺损评分各组大鼠改良神经功能缺损评分，在损伤后24 h，联合治疗组与人羊膜间充质干细胞移植组、脑损伤组比较差异无显著性意义；在损伤后3 d开始，联合治疗组与人羊膜间充质干细胞移植组、脑损伤组比较，联合治疗组明显低于人羊膜间充质干细胞移植组及脑损伤组(P < 0.05)，见表1。 2.6 荧光显微镜观察结果移植2周后，荧光显微镜观察结果显示，大鼠梗死灶脑组织中的CM-Dil阳性细胞数：脑损伤组为(0±0.00)个/高倍视野，人羊膜间充质干细胞移植组为(21.64±4.57)个/高倍视野，联合治疗组为(32.94±8.46)个/高倍视野，见图4。CM-Dil的阳性细胞数联合治疗组最多，人羊膜间充质干细胞移植组次之，脑损伤组最少(P < 0.05)。"

References

[1] 黄俊海.颅脑外伤的CT影像诊断解析[J].医药前沿,2015, 5(32):80-81.
[2] 张安庆.外伤性脑梗塞28例临床诊治分析[J].淮海医药, 2014,3(4):238-238,239.
[3] 曹莹莹,王晴,陈冰,等.用荧光原位杂交技术检测hEPO转基因山羊染色体上外源基因的整合状况[J].中国比较医学杂志,2003,13(3):132-134.
[4] 冯士军.NGF联合骨髓间充质干细胞移植治疗大鼠脑损伤[J].中国现代医学杂志,2011,21(8):924-928.
[5] Komblum HI. Introduction to neural stem cells. Stroke. 2007;38(2 Suppl):810-816.
[6] 卑贵光,李松柏,马虹,等.脑梗死非溶栓治疗出血性转化的MR分型与预后相关性研究[J].中国临床医学影像杂志, 2011,22(8):533-536.
[7] Bi B, Guo J, Marlier A, et al. Erythropoietin expands a stromal cell population that can mediate renoprotection. Am J Physiol Renal Physiol. 2008; 295(4):1017-1022.
[8] Shiozawa Y, Jung Y, Ziegler AM, et al. Erythropoietin couples hematopoiesis with bone formation. PLoS One. 2010;5(5):10853.
[9] 薛政民,胡萌,张长海,等.不同浓度重组人促红细胞生成素对神经干细胞体外培养增殖的影响[J].中国组织工程研究与临床康复,2011,15(23):4194-4198.
[10] 李宗义,李鹏,高芙蓉,等.EPO修饰增强Müller细胞对RCS大鼠视网膜变性的干预效果[J].中华细胞与干细胞杂志(电子版),2014,4(1):52-59.
[11] 杨智,程春凤,黄昕艳,等.Trizol法提取新生小鼠脑组织总RNA优化比较[J].中风与神经疾病杂志,2012,29(2): 155-156.
[12] 吴洪磊,李洪斌.中西医结合治疗重症颅脑损伤并发肺部感染的临床分析[J].中医临床研究,2015,25(5):120-121.
[13] 韩大明,冯伟.儿童重型颅脑损伤56例临床分析[J].中国实用医刊,2015,42(1):55-57.
[14] 赵建华,张杰文,李玮,等.中枢神经系统结核继发急性脑梗死的临床和MRI/MRA[J].中风与神经疾病杂志,2011, 28(4):364-365.
[15] 刘勤,张红宇,王璐.灯盏花药理作用研究进展[J].云南中医中药杂志,2013,34(2):61-64.
[16] Wu H, Yang SF, Dai J, et al. Combination of early and delayed ischemic postconditioning enhances brain-derived neurotrophic factor production by upregulating the ERK-CREB pathway in rats with focal ischemia. Mol Med Rep. 2015;12(5):6427-6434.
[17] 袁丽丽,杜红梅,管英俊,等.促红细胞生成素体外培养鼠胚脑皮质神经干细胞的分化[J].中国组织工程研究与临床康复,2011,15(49):9174-9177.
[18] 徐颖,凌伟华,陆士奇,等.尾静脉移植羊膜间充质干细胞治疗大鼠脑缺血再灌注损伤[J].中国组织工程研究与临床康复,2011,15(49):9198-9201.
[19] Zhang X, Arnott JA, Rehman S, et al. Src is a major signaling component for CTGF induction by TGF-beta1 in osteoblasts. J Cell Physiol. 2010;224(3):691-701.
[20] Wu MF,Zhang SQ,Gu R,et al.Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord. Neural Regen Res. 2015;10(9):1483-1490.
[21] 张建军,史焕昌,杨卫山,等.亚低温条件下脊髓损伤模型大鼠神经再生微环境及运动功能的变化[J].中国组织工程研究,2015,19(27):4316-4321.
[22] Wang Y, Gang XK, Liu Q, et al. Transplantation of X-box-binding protein-1 gene-modified neural stem cells in the lateral ventricle of brain ischemia rats. Neural Regen Res. 2011;6(1):6-11.
[23] 张志涛,陈彬,白哗,等.缺血缺氧性脑病模型幼鼠脑损伤区神经元电生理特性分析[J].中国医药,2015,10(4): 496-500.
[24] 胡伟,彭亚文,蒋帅,等.自噬在缺血性脑损伤中的作用研究进展[J].中华神经医学杂志,2015,14(5):529-531.
[25] 孙晓全,张瑜,徐清,等.基层医院颅脑损伤的治疗现状与对策[J].临床神经外科杂志,2013,10(5):301-302.
[26] Chang YC, Huang CC. Perinatal brain injury and regulation of transcription.Curr Opin Neurol. 2006;19: 141-147.
[27] 曹健锋,晋鑫,史载祥,等.亚低温治疗创伤性脑损伤的研究进展[J].中华神经医学杂志,2015,14(4):444-448.
[28] Song J, Sun H, Xu F, et al. Recombinant human erythropoietin improves neurological outcomes in very preterm infants. Ann Neurol. 2016.
[29] Barton SK, Tolcos M, Miller SL, et al. Ventilation-induced brain injury in preterm neonates: a review of potential therapies. Neonatology. 2016; 110(2): 155-162.
[30] Lan KM, Tien LT, Cai Z, et al. Erythropoietin Ameliorates Neonatal Hypoxia-Ischemia-Induced Neurobehavioral Deficits, Neuroinflammation, and Hippocampal Injury in the Juvenile Rat. Int J Mol Sci. 2016;17(3):E289.
[31] Kumarasinghe G, Gao L, Hicks M, et al. Improved heart function from older donors using pharmacologic conditioning strategies. J Heart Lung Transplant. 2016.
[32] Robinson S, Winer JL, Berkner J, et al. Imaging and serum biomarkers reflecting the functional efficacy of extended erythropoietin treatment in rats following infantile traumatic brain injury. J Neurosurg Pediatr. 2016.
[33] Skitek M, Jerin A. Proenkefalin A and protachykinin in ischemic neurological complications after cardiac surgery. Med Glas (Zenica). 2016;13(1):8-13.
[34] Hassouna I, Ott C, Wüstefeld L, et al. Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus. Mol Psychiatry. 2016.
[35] Kochanek PM, Clark RS. Traumatic brain injury research highlights in 2015. Lancet Neurol. 2016;15(1): 13-15.
[36] Kochanek PM, Bramlett HM, Shear DA, et al. Synthesis of findings, current investigations, and future directions: operation brain trauma therapy. J Neurotrauma. 2016;33(6):606-614.
[37] Millet A, Bouzat P, Trouve-Buisson T, et al. Erythropoietin and Its Derivates Modulate Mitochondrial Dysfunction after Diffuse Traumatic Brain Injury. J Neurotrauma. 2016.
[38] Lv W,Li WY,Xu XY,et al.Bone marrow mesenchymal stem cells transplantation promotes the release of endogenous erythropoietin after ischemic stroke. Neural Regen Res. 2015; 10 (8): 1265-1270.
[39] Yamal JM, Benoit JS, Doshi P, et al. Association of transfusion red blood cell storage age and blood oxygenation, long-term neurologic outcome, and mortality in traumatic brain injury. J Trauma Acute Care Surg. 2015;79(5):843-849.
[40] Gatto R, Chauhan M, Chauhan N. Anti-edema effects of rhEpo in experimental traumatic brain injury. Restor Neurol Neurosci. 2015;33(6):927-941.