Composition for preventing or treating amyotrophic lateral sclerosis using two or more isoforms of hepatocyte growth factor
一种肝细胞生长因子、侧索硬化症的技术，应用在利用肝细胞生长因子的两种以上的异构体的肌萎缩性侧索硬化症预防或治疗用组合物领域，能够解决无肌萎缩性侧索硬化症预防或治疗剂等问题
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Embodiment 1
[0091] Example 1: Confirmation of the efficacy of pCK-hepatocyte growth factor X7 production in the maturation of embryonic neuronal cells (ENC)
[0092] Only the cerebral cortex was harvested from mouse embryos (embryo) and monocellularized, and 10 μM of cytarabine (Ara-C) was added to the culture medium to culture only neurons. To confirm the effect of pCK-HGFX7 production on the maturation of embryonic neurons, seed 2×10 4 cells, and the next day, the cells were treated with 1.25 ng / mL protein obtained from 293F cells transfected with pCK-HGF X7 (Lifetechnologies, USA) to confirm The degree of neurite outgrowth exhibited in the maturation of the cells. Regarding the degree of neurite outgrowth of cells, expression of TUJ-1, which is a neuron-specific tubulin, was confirmed by immunocytochemistry.
[0093] As a result, such as figure 1 As shown, it was confirmed that the pCK-hepatocyte growth factor X7-treated group more significantly increased the length of neurite compa...
Embodiment 2
[0094] Example 2: Confirmation of the effect of pCK-hepatocyte growth factor X7 on cell growth after maturation of embryonic nerve cells
[0095] The effect of pCK-hepatocyte growth factor X7 on cell growth after maturation of embryonic neural cells was confirmed. For this, seed 5×10 4 embryonic neurons and undergo a 6-day maturation process. Six days later, the protein obtained from 293F cells transfected with pCK-hepatocyte growth factor X7 was treated with 1.25 ng / mL to confirm the effect of pCK-hepatocyte growth factor X7 on the growth of the cells. After 3 days of pCK-hepatocyte growth factor X7 treatment, MTT assay was performed to measure cell growth.
[0096] As a result, such as figure 2 As shown, it was confirmed that cell growth increased by about 40% in the pCK-hepatocyte growth factor X7-treated group compared with the pCK-treated group as the control group.
Embodiment 3
[0097] Example 3: Confirmation of the effect of pCK-hepatocyte growth factor X7 on cell growth and apoptosis in mouse motor neuron cells (NSC-34)
[0098] 3-1. Cell lines and cell culture
[0099] The mouse motor nerve cells (Cellution Biosystem, Vancouver, CA) used in this experiment are motor nerve cells derived from mice. Mouse motor nerve cells are a cell line that is a mixture of motor nerve cells and neuroblastoma cells derived from mouse spinal nerves during the embryonic period, and is widely used in studies related to motor nerves. Incubate the above cells at 37°C and 5% CO 2 Cultivate under conditions, and culture in Dulbecco's modified Eagle including 10% bovine fetal serum (Gibco, USA) and antibiotics (Gibco, BRL, USA) culture medium (DMEM, Sigma). Cell culture media, reagents and sera were purchased from Gebico and Sigmaaldrich.
[0100] 3-2. Production and quantification of supernatant expressing hepatocyte growth factor protein
[0101] To produce supernata...
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