Keilhoff Gerburg, Lucas Benjamin, Pinkernelle Josephine, Steiner Michael, Fansa Hisham
Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany.
Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany.
Exp Cell Res. 2014 Oct 1;327(2):234-55. doi: 10.1016/j.yexcr.2014.06.020. Epub 2014 Jul 2.
Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries.
尽管外周神经系统具有再生能力，但这种能力是有限的。作为增强神经再生的一种潜在手段，脑蛋白水解物（CL）——一种蛋白水解肽组分——的作用在体外对运动神经元样NSC-34细胞系和脊髓器官型培养物进行了测试。因此，通过更换培养基使NSC-34细胞受到机械应激，并通过氧糖剥夺使其受到代谢应激。之后，使用MTT和BrdU标记（流式细胞术）评估细胞存活/增殖情况，并使用ImageJ分析评估神经突萌发情况。通过蛋白质印迹法分析钙蛋白酶-1、Src和α-血影蛋白的蛋白表达。在器官型培养物中，通过免疫组织化学测试CL对运动神经元存活和神经突萌发的影响。CL对氧糖剥夺应激的NSC-34细胞具有暂时的抗增殖但最初具有神经保护作用。高剂量或反复应用CL对细胞存活有害。CL在有限程度上增强神经突重建，影响钙蛋白酶-1蛋白表达，并根据Src表达影响钙蛋白酶介导的血影蛋白裂解。在脊髓器官型切片培养物中，CL无法支持运动神经元存活/神经突萌发。此外，它还阻碍了星形胶质细胞和小胶质细胞的活性。数据表明，CL可能仅对受损的脊髓运动神经元有孤立的积极作用。高剂量或累积的CL在脊髓损伤治疗中似乎有不利影响。需要进一步的实验来优化CL在脊髓损伤中安全临床应用的条件。
