| It is widely known that neural stem cells (NSCs) have the capacity of reproduction and multi-directional differentiation in the developing and adult central nervous system. NSCs can self-renew through division and have the ability to differentiate into neurons, astrocytes and oligodendrocytes. The proliferation and multipotent characteristics make NSCs become the promising choice for the treatment of various neurodegenerative diseases. NSCs transplantation as a strategy of treatment for CNS diseases has been adopted in neurodegenerative disorders researches such as Parkinson’s disease. Transplanted NSCs can supply exogenous cells and then recover the tissue function through proliferation, multi-directional differentiation and migration. However, the behaviors of transplanted NSCs in the host tissue are influenced by many factors. To understand the mechanisms of survival and differentiation of NSCs are very important for NSCs therapeutic application in nervous system diseases. Ghrelin, as an endogenous ligand for the growth hormone secretagogue receptor la (GHS-Rla), plays neurobiological roles in neuroprotection and neurogenesis. It is reported that ghrelin can promote the survival of dopaminergic (DAergic) neurons and regulate their electrophysiological activity. However, there are no evidences about the effect of ghrelin on the proliferation and differentiation of midbrain NSCs (miNSCs) which is the source of DAergic neurons in the development. In the present study, we used primary cell culture method to form miNSCs. Flow cytometry was used to detect the percentage of SPF (S-phase fraction, indicating the activity of cell proliferation) and PI (proliferation index, indicating the activity of cell proliferation) in cell cycle to observe the effect of ghrelin on the proliferation of miNSCs. Cell Counting Kit-8 (CCK-8) was used to detect OD values and observe the effect of ghrelin on cell viability; Immunofluorescence and Western blots were used to detect the expression of β-tubulin Ⅲ, TH, ChAT, GAD67, GFAP and NG2 for assessment of the effect of ghrelin on the differentiation of miNSCs.The results were as follows:1. Midbrain NSCs formed neurospheres with the diameter of 50-70 μm after cultured for 3 d, and they increased up to 200-300 μm after cultured for 7 d. Immunohistochemical staining experiments showed positive stainings both in Nestin and in Ki67. As the culture time prolonged, the OD values detected by CCK-8 kit increased, and they reached platform on the 5th day. After cultured in differentiate medium for 7 d, P-tubulin Ⅲ-positive neurons, GFAP-positive astrocytes and NG2-positive oligodendrocytes were observed.2. GHS-Rla, the receptor of ghrelin, expressed robustly on the membrane of miNSCs.3. MiNSCs were treated by different concentrations of ghrelin (10-9,10-8,10-7and 10-6 mol/L) for 24 hrs. The results of CCK-8 experiments indicated that 10-9～10-6mol/L ghrelin could increase the cell viability (0.121±0.004,0.109+0.017,0.111±0.015, 0.107±0.019) compared with controls (0.089±0.01). The effect of ghrelin on increasing cell viability was fully blocked when miNSCs were pretreated by GHS-Rla blocker [D-Lys3]-GHRP-6 (10-4 mol/L) for 30 min (P<0.05).4. MiNSCs were cultured for 24 hrs and then treated with ghrelin for 2 days. We found that the diameter of neurospheres increased in 10-8～10-6 mol/L ghrelin treatment groups (87.82±5.74 μm,89.25±4.26 μm,87.44±7.43 μm) rather than control group (74.02±4.45 μm), which indicated that the high proliferation ability induced by ghrelin (P<0.05). However, no significant changes in the diameter of neurospheres were observed in 10-10～10-9 mol/L ghrelin treatment groups (75.97±5.5μm,72.34±5.53 μm). Furthermore, these cells were collected to examine cell cycle phase using flow cytometer. Compared with control group (16.68±2.03%), PI significantly increased in 10-8～10-6 mol/L ghrelin treatment groups (23.95±2.49%,24.14±2.33%,26.28±2.50%), whereas it did not significantly change in 10-10～10-9 mol/L groups (17.37±2.03%,19.15±2.08%). Besides, SPF was also upregulated in 10-7～10-6 mol/L groups (16.8±2.06%,19.85-1.67%), while no change was observed in 10-10～10-8 mol/L groups (14±1.63%,14.43±1.52%, 15.15±1.56%) and control group (13±1.23%),. Meanwhile, the diameter of neurospheres, PI and SPF were all higher in 10-7～10-6 mol/L groups than in 10"10-10"9 mol/L groups (P<0.05).5. MiNSCs were incubated with 10-10～10-6 mol/L ghrelin for 7 d. As immunofluorescence and western blot results shown, the percentage of β-tubulin Ⅲ-positive cells as well as β-tubulin III protein level significantly increased in 7 days of 10-8～10-6 mol/L ghrelin treatment group (21.09±.46%,23.05±1.87%,28.8±3.05%) compared with control group (13.6±3.37%),10-10 mol/L group (13.04±2.86%) and 10"9 mol/L group (15.09±2.44%)respectively (P<0.05).10-8～10-6mol/L ghrelin could increase the number of TH-positive cells and promote the expression of TH.10-10 and 10-9 mol/L ghrelin had no effect on the expression of TH (P<0.05). Furthmore,10-8～10-6 mol/L ghrelin could reduce the expression of ChAT (P<0.05). Ghrelin had no effect on the expression of GAD67 (P>0.05). We also found that 10-7 and 10-6 mol/L ghrelin could reduce the number of GFAP-positive cells and inhibit the expression of GFAP (P<0.05) However, this effect was not found in the cells treated by 10-10～10-8 mol/L ghrelin. 10-8～10-6 mol/L ghrelin could increase the number of NG2-positive cells and promote NG2 expression (P<0.05). However, this effect was not observed in the cells treated by 10-10 and 10-9 mol/L ghrelin.6.The ratio of GFAP-positive cells to GFAP protein levels significantly decreased in 7 days of ghrelin post-treated groups, with 49.96±5.85% for 10-1 mol/L group and 44.4±6.69% for 10-6 mol/L group separately compared with both control (73.4±10.86%) and 10-10～10-8 mol/L groups (68.24±7.57%,70.65±10.24%,65.13±8.51%). However, no changes were observed in 10-10～10-8 mol/L groups compared to control. We also found that the number of NG2-positive cells and NG2 protein levels were higher in 10-1 mol/L (7.02±0.81%) and 10"6 mol/L (9.09±1.1%) ghrelin treatment groups than that of in control (1.94±0.36%) and lower concentration groups. Likewise, these parameters did not change in 10-10～10-8 mol/L groups (2.44±0.37%,2.71±0.5%,5.35±0.55%) compared to control.The above results suggest that miNSCs have potential abilities of self-proliferation and multi-directional differentiation and can both form neurospheres and differentiate to neurons, astrocytes and oligodendrocytes. In this study, we confirmed that ghrelin receptor GHS-Rla expressed on the surface of miNSCs and its antagonist [D-Lys3]-GHRP-6 could fully block the effect of ghrelin on the enhancement of miNSCs viability, which indicated that this effect might be through ligand-receptor pathway. In addition, the diameter of neurospheres and flow cytometry experiments suggested that certain concentrations ghrelin could promote the proliferation of miNSCs, which might be through adjusting the S and G2/M phase cells implementation. Our study confirmed that ghrelin could promote midbrain NSCs differentiation to neurons. As to different subtypes of neurons, ghrelin could promote the differentiation of miNSCs to dopaminergic neurons and inhibit the differentiation to acetylcholinesterase cholinergic neurons. But no significant effect was observed in GABA neurons. We also found that the effects of ghrelin on the differentiation of miNSCs to astrocytes and oligodendrocytes are different. Our study firstly demonstrate above results in primarily cultured miNSCs in vitro, which provides solid experimental evidences and theoretical supplements for the view that ghrelin can be used as a protective reagent in nerve system to therapy the neurodegenerative disorders. |
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