Oligodendrocyte progenitor cell transplantation in combination with miconazole for myelin repair in mice with leukodystrophy

doi:10.3969/j.issn.2095-4344.0961

Abstract

Abstract:

BACKGROUND: Oligodendrocyte progenitor cells are precursor cells of oligodendrocytes that are myelinating cells in the central nervous system. These cells are closely related to central demyelinating diseases such as leukodystrophy. Recent studies have shown that miconazole can promote oligodendrocyte progenitor cell differentiation and myelin regeneration. The combination of miconazole and oligodendrocyte progenitor cells is likely to be used for the treatment of leukodystrophy.
OBJECTIVE: To investigate the effect of oligodendrocyte progenitor cell transplantation combined with miconazole on myelin reparation in mice with leukodystrophy.
METHODS: Myelin basic protein (MBP) knockout was performed to establish animal models of leukodystrophy in shiverer mice. Twenty-six newborn shi-/- mice were randomly assigned to negative control group and treatment group (n=13 per group). In addition, 13 newborn shi+/+ mice were selected as positive control group. In the treatment group, oligodendrocyte progenitor cells were transplanted within 24 hours after birth, and in the control groups, oligodendrocyte progenitor cells were replaced by PBS. At the same time, in the treatment group miconazole was injected intraperitoneally at 1-5 days after birth, and in the control groups, miconazole was replaced by normal saline. At the age of 8 weeks, MBP immunofluorescence staining and western blot were used to detect MBP protein expression. The myelin sheath structure of the corpus callosum was observed by transmission electron microscopy.
RESULTS AND CONCLUSION: Compared with the negative control group, the MBP expression in the treatment group was significantly enhanced and the average fluorescence intensity was significantly increased (P < 0.05). But when compared with the positive control group, the MBP expression in the treatment group was significantly weakened and the average fluorescence intensity significantly decreased (P < 0.05). Under the transmission electron microscope, the myelin sheath structure in the corpus callosum region was significantly diverse among the groups. In the negative control group, the myelin sheath was rare and irregular, and the lamellar arrangement was loose and disordered. In the positive control group, the myelin was significantly increased in number and thickened. With the normal morphology, the lamellar layer was closely arranged and the boundary was clear. In the treatment group, the number of myelin was significantly higher than that of the negative control group, and some myelin sheaths were normal in shape, but most myelin sheaths were still slender. These findings indicate that oligodendrocyte progenitor cells combined with miconazole helps to improve demyelination in mice with leukodystrophy, but cannot completely reverse the pathological status of damaged myelin.

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: Leukoencephalopathies, Myelin Sheath, Oligodendroglia, Miconazole, Tissue Engineering

CLC Number:

R394.2

Wu Cheng-jun, Su Xue-wen, Wang Zhao-yan, Yang Yin-xiang, Luan Zuo. Oligodendrocyte progenitor cell transplantation in combination with miconazole for myelin repair in mice with leukodystrophy[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4041-4046.

Figures/Tables 1

2.1 实验动物大体观察结果 OPCs、PBS的立体定位移植进展顺利，进针、细胞注射均无阻滞，期间小鼠反应平稳，拔针后观察针孔附近无出血、无液体渗漏等情况发生，术后小鼠无不良反应，苏醒后活动如常。咪康唑、生理盐水的腹腔注射过程顺利，注射完毕留针2 min，拔针后观察小鼠腹部表面未见出血、液体渗漏等情况发生。观察期内小鼠正常长大，无提前死亡、无不良疾病等现象发生。 2.2 免疫荧光染色结果 MBP免疫荧光染色观察结果见图1。阴性对照组MBP荧光黯淡，几乎没有荧光表达，相比较之下，治疗组MBP荧光表达明显增强，但与阳性对照组比较，治疗组和阴性对照组的荧光强度均明显偏弱。荧光定量分析结果见表1，与阴性对照组比较，治疗组的平均荧光强度值明显增高，差异有显著性意义(P < 0.05)；但与阳性对照组比较，治疗组的平均荧光强度值明显降低，差异有显著性意义(P < 0.05)。 2.3 Western blot检测MBP表达量取各组小鼠脑组织进行Western blot检测，结果见图2。MBP蛋白表达量(以目的条带和内参条带吸光度值比值表示)在3组间均有明显差异。治疗组的MBP蛋白表达量(0.28±0.03)比阴性对照组(0.14±0.01)高，且比阳性对照组(0.74±0.07)低，差异有显著性意义。结果显示OPCs治疗可增加shi-/-鼠的MBP蛋白表达量，提示其有助于改善脱髓鞘小鼠的脑白质髓鞘形成不良状况，但不足以完全修复髓鞘，与MBP免疫荧光染色结果相一致。 2.4 透射电镜观察结果透射电镜观察小鼠胼胝体区域的髓鞘结构，见图3。阴性对照组罕见髓鞘结构，轴突形态不规则，板层排列松散，紊乱，与轴索间隙宽大，髓鞘厚度明显降低；治疗组髓鞘数量较阴性对照组明显增多，轴突形态规则，部分髓鞘厚度增加且板层排列紧密，近似正常髓鞘，但多数髓鞘厚度仍明显减低；阳性对照组髓鞘明显增多，所有髓鞘均形态均匀一致，厚度增加，髓鞘边界清晰，板层排列紧密，无松散、紊乱现象。结果提示OPCs治疗可促进脱髓鞘小鼠的髓鞘再生，但尚不足以恢复正常髓鞘结构。"

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