Effect of Intrauterine Transplantation of Bone Marrow Mesenchymal Stem Cells on the Expression of Bone Morphogenetic Protein 4 in Spinal Cord of Fetal Rats with Spina Bifida Aperta

doi:10.12114/j.issn.1007-9572.2019.00.481

Abstract

Abstract: Background　Bone morphogenetic proteins-4 （BMP4） plays an important role in forming dorsal ventral axis pattern in neural tube，cell apoptosis as well as differentiation and transformation of stem cells.If BMP4 is transferred into bone marrow mesenchymal stem cells（MSCs）by transgenic method，whether it can secrete a certain level of BMP4，so that stem cells can replace damaged nerve cells as well as reconstruct collateral circulation and neuroprotection，is an urgent problem to be solved.Objective　To observe the expression of BMP4 in spinal cord of fetal rats with spina bifida aperta before and after MSCs transplantation.Methods　From November 2017 to October 2018，a total of 50 Wistar rats （8 males and 42 females） were purchased from the Laboratory Animal Center of the First Clinical College，Harbin Medical University.Five Wistar rats aged 10 days after birth were retained as stem cell sources.Feeding under normal conditions，cage closing at midnight and mating were confirmed.The rat fetuses with spina bifida were established by a teratogenic drug named as retinoic acid.MSCs were transplanted into rat fetuses by microsurgical injection.The fetuses from the same pregnant rat were divided into five groups:retinoic acid-free malformation group （n=10），spina bifida malformation group （n=12），stem cell transplantation group with retinoic acid-free malformation （n=10），stem cell transplantation group with spina bifida malformation （n=10） and blank transplantation group with spina bifida malformation （n=8）.Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of BMP4 and antagonist Noggin in each group.Results　The expression of BMP4 mRNA in spina bifida malformation group was higher than that in retinoic acid-free malformation group，and the expression of Noggin mRNA was lower than that in retinoic acid-free malformation group（P<0.05）.The comparisons of BMP4 and Noggin mRNA expression in spina bifida malformation group，stem cell transplantation group and blank transplantation group with spina bifida malformation also showed significant statistical differences（P<0.05）.The expression of BMP4 mRNA in stem cell transplantation group with spina bifida malformation was higher than that in spina bifida malformation group and blank transplantation group with spina bifida malformation.The expression of Noggin mRNA was lower than that in spina bifida malformation group and blank transplantation group with spina bifida malformation（P<0.05）.However，The comparisons of BMP4 and Noggin mRNA between retinoic acid-free malformation group and stem cell transplantation group with retinoic acid-free malformation confirmed no statistical differences （P>0.05）.Conclusion　BMP4 is related to the pathogenesis of spina bifida aperta.Furthermore，MSCs transplantation can up-regulate BMP4 mRNA expression to promote the recovery of neurological function so as to achieve therapeutic efficiency of congenital spina bifida malformation.

Key words: Mesenchymal stem cell, Spina bifida aperta, Bone morphogeneric protein 4, Stem cell transplantation, Real-time polymerase chain reaction

摘要： 背景　骨形态发生蛋白4（BMP4）在神经管背腹轴模式的形成、细胞凋亡以及干细胞的分化和转化过程中均起重要作用，通过转基因的方法将BMP4基因转入骨髓间充质干细胞（MSCs）后，是否能分泌一定水平的BMP4，使干细胞既能替代受损神经元，又具有重建侧支循环和神经保护作用，一直是未解决的热点问题。目的　观察MSCs移植前后，显性脊柱裂胎鼠脊髓组织中BMP4表达情况。方法　于2017年11月—2018年10月，在哈尔滨医科大学附属第一医院实验动物中心购买Wistar实验大鼠50只（雄鼠8只，雌鼠42只），出生后10 d的Wistar大鼠5只留作干细胞来源。常规条件下饲养，午夜合笼，确认交配。利用致畸药物维甲酸建立显性脊柱裂胎鼠模型，利用显微外科注射技术对胎鼠进行MSCs移植治疗。将同一只孕鼠所孕的胎鼠分为维甲酸无畸形组（10只）、脊柱裂畸形组（12只）、维甲酸无畸形干细胞移植组（10只）、畸形干细胞移植组（10只）和畸形空白移植组（8只）。采用实时荧光定量聚合酶链反应（Real-time PCR）法检测各组BMP4和拮抗剂Noggin mRNA表达情况。结果　脊柱裂畸形组BMP4 mRNA表达水平高于维甲酸无畸形组，Noggin mRNA表达水平低于维甲酸无畸形组（P<0.05）。脊柱裂畸形组、畸形干细胞移植组和畸形空白移植组BMP4、Noggin mRNA表达水平比较，差异有统计学意义（P<0.05）；其中畸形干细胞移植组BMP4 mRNA表达水平高于脊柱裂畸形组和畸形空白移植组，Noggin mRNA表达水平低于脊柱裂畸形组和畸形空白移植组（P<0.05）。维甲酸无畸形组和维甲酸无畸形干细胞移植组BMP4、Noggin mRNA表达水平比较，差异均无统计学意义（P>0.05）。结论　BMP4与先天性显性脊柱裂畸形的发病机制有关；在MSCs移植治疗先天性脊柱裂畸形过程中可能通过上调BMP4 mRNA的表达而最终达到治疗的目的。

关键词: 骨髓间充质干细胞, 显性脊柱裂, 骨形态发生蛋白质4, 干细胞移植, 实时聚合酶链反应

JIANG Mingyu*,ZHANG Jianbai,REN Mingyong, et al. Effect of Intrauterine Transplantation of Bone Marrow Mesenchymal Stem Cells on the Expression of Bone Morphogenetic Protein 4 in Spinal Cord of Fetal Rats with Spina Bifida Aperta　[J]. Chinese General Practice, 2020, 23(2): 189-193. DOI: 10.12114/j.issn.1007-9572.2019.00.481.
姜明宇,张剑白,任明永等. 胚胎期宫内移植骨髓间充质干细胞对显性脊柱裂胎鼠脊髓骨形态发生蛋白4表达水平的影响研究[J]. 中国全科医学, 2020, 23(2): 189-193. DOI: 10.12114/j.issn.1007-9572.2019.00.481.

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