Effect Of Transfection With Recombinant Adenovirus Cardiotrophin-1 On The Neural Stem Cells

Objective:To observe the protective role of transfection with recombinant adenovirus cardiotrophin-1 (Adv-CT-1) on the neural stem cells (NSCs) and its effect on differentiating into GABAergic neurons. Methods:1. NSCs were isolated, cultured and identified from hippocampus of neonatal Wistar rats. NSCs were subcultured by using different methods.2. NSCs were transfected with Adv-CT-1 or recombinant adenovirus enhanced green fluorescence protein (Adv-EGFP). NSCs were transfected with Adv-CT-1 or Adv-EGFP at various multiplies of infection (MOI) ranging from 25,50,100 to 200. The transfection efficiency was determined by fluorescence microscope for Adv-EGFP and the expression rate of CT-1 was detected by immunofluorescent staining after transfecting for 24,48,72 and 96 h.3. To study the protective role of transfection with Adv-CT-1 on the neural stem cells. NSCs were randomly divided into glutamate, CT-1 (transfected with Adv-CT-1 at MOI of 200) and control group. NSCs were treated with 50μmol/L glutamate for 30 minutes in glutamate and CT-1 group, and NSCs were untreated in control group. At 12,24,48 and 72 h after treatment with glutamate, the states of survival and proliferation with NSCs were estimated by MTT method, and the apoptosis of NSCs was observed by TUNEL.4. To investigate the effect of transfection with Adv-CT-1 on the differentiation of NSCs. NSCs were randomly divided into CT-1 (transfected with Adv-CT-1 at MOI of 200), serum (added 10% fetal bovine serum to medium) and control (untreated) group. At 1, 3,5 and 7 d, the ratio of differentiation into GABAergic neurons was assayed by immunofluorescent staining. Result:1. NSCs isolated from hippocampus of neonatal rats were positive for Nestin expression, with the potential for further proliferation and capable of differentiation into neurons and astroglias. Using NeuroCult Chemical Dissociation Kit could decrease death of cells and benefit proliferation of NSCs during subculturing.2. After transfection with Adv-CT-1 for 24,48,72 and 96 h, the expression rates of CT-1 were 27.09%±4.25%,32.21%±4.96%,36.49%±2.31% and 34.24%±4.02% at MOI of 25, and 37.73%±3.50%,47.49%±2.87%,55.19%±4.54% and 53.16%±4.87% at MOI of 50, and 54.82%±3.00%,67.19%±3.54%,75.10%±3.97% and 72.81%±2.94% at MOI of 100, and 65.40%±3.74%,80.36%±4.35%,83.55%±3.07% and 81.20%±3.13% at MOI of 200, respectivly. The expression rates of CT-1 increased with increasing in MOI of Adv-CT-1 in a dose-dependent manner. Transfected with Adv-CT-1 at different MOI, the expression rates of CT-1 all reached to the peak at 72 h. After transfection with Adv-CT-1 for 24,48, 72 and 96 h, the transfection efficiency of EGFP were 53.95%±5.19%,63.35%±2.43%, 65.25%±2.29% and 67.41%±4.16% at MOI of 25, and 63.80%±6.43%,72.03%±4.34%, 75.52%±2.51% and 77.52%±4.29% at MOI of 50, and 71.50%±3.80%,84.96%±3.49%, 86.71%±4.00% and 88.05%±3.59% at MOI of 100, and 73.30%±2.50%,87.19%±3.50%, 90.06%±2.87% and 91.50%±3.40% at MOI of 200, respectivly. The transfection efficiency of EGFP increased with increasing in MOI of Adv-EGFP in a dose-dependent manner. Transfected with Adv-EGFP at different MOI, the transfection efficiency of EGFP all reached the peak at 96 h.3. At 12,24,48 and 72 h after treatment with glutamate, the values of optical density (OD) were 0.317±0.035,0.262±0.034,0.332±0.050 and 0.386±0.030 in glutamate group, and 0.375±0.033,0.323±0.031,0.412±0.027 and 0.480±0.055 in CT-1 group, which declined minimum value at 24 h in both groups.At 12, 24,48 and 72 h, the values of OD in control group were 0.445±0.043,0.534±0.039, 0.646±0.065 and 0.766±0.058.At all time points, the values of OD in control group were significant higher than those in glutamate group and CT-1 group(P<0.05), which in CT-1 group were significant higher than those in glutamate group(P<0.05). At 12,24,48 and 72 h after treatment with glutamate, the apoptosis rates of cells were 30.45%±5.91%, 41.65%±4.35%,30.15%±2.96% and 21.13%±3.91% in glutamate group, and 20.47%±2.30%,26.63%±3.31%,20.77%±2.61% and 10.70%±3.30% in CT-1 group, which reached the peak at 24 h in both groups. At 12,24,48 and 72 h, the apoptosis rates of cells in control group were 2.48%±0.65%,2.94%±0.68%,2.80%±0.62% and 3.08%±0.51%. At all time points, the apoptosis rates of cells in control group were significant lower than those in glutamate group and CT-1 group(P<0.05), which in CT-1 group were lower than those in glutamate group (P<0.05).4. At 1,3,5 and 7 d, the ratios of differentiation into GABAergic neurons were 8.49%±2.05%,22.65%±2.60%,36.70%± 3.05% and 46.02%±5.77% in Adv-CT-1 group, and 3.19%±0.79%,8.92%±1.58%, 17.79%±3.33% and 19.73%±3.83% in serum group, and 0.34%±0.27%,0.92%±0.55%, 1.24%±0.45% and 1.46%±0.52% in control group, respectivly. The ratios of differentiation into GABAergic neurons reached the peak at 7 d in all groups. At all time points, the ratios of differentiation into GABAergic neurons in Adv-CT-1 group were significant higher than those in serum group and in control group (P<0.05), which in serum group were significant higher than those in control group (P<0.05). No significiant difference was observed in the nummber of differentiation into GABAergic neurons of CT-1 group between 7 d and 14 d (t=2.445, P>0.05), while significiant difference was observed in serum group (t=5.96, P<0.01). Conclusion:1. Neural stem cells were successfully isolated and cultured from hippocampus of neonatal rats, and could be subcultured efficiently by using NeuroCult Chemical Dissociation Kit.2. Both Adv-CT-1 and Adv-EGFP can transfect NSCs efficiently, and furthermore, CT-1 and EGFP can be expressed highly in the NSCs. The expression of CT-1 could be reflected by EGFP indirectly. EGFP could effectively trace CT-1 as a reporter gene.3. Adv-CT-1 possessed the protective effects on NSCs injured by glutamate, which can inhibit NSCs apoptosis and improve cells survival.4. Adv-CT-1 can promote NSCs to differentiate into GABAergic neurons and promot survival of GABAergic neurons.