| Objective :To construct and identify the lentivirus vector of over-expression the Glycogen synthase kinase-3β(GSK3β),and investigate the effect of over-expression GSK3βin mouse hippocampal on the change of depression behavior. To observe the level of GSK3βprotein after injection over-expression GSK3βlentivirus vector, and the changes of the behavior in different tests after chronic mild stress (CMS).Our study initial explored the role of GSK3βin depression.Methods:1.Constructed and assembled the lentivirus vector of over-expression the GSK3βand Green fluorescent protein (GFP).After infection 293T cells 72h, we observed the number and morphous of GFP-positive cells.2.We divided C57BL/6J mice into normal group, lentivirus vector group and GSK3βover-expression group, and injected saline, lentivirus vector and over-expression GSK3βlentivirus vector in different group mice's hippocampus bilaterally. After sacrifice in 7th day, co-expression of GSK3βand GFP were observed by immunofluorescence.3.Healthy adult male C57BL/6J mice were randomly divided into 3 groups: normal group, lentivirus vector group and GSK3βover-expression group. Each group also have CMS group corresponding. We injected saline, lentivirus vector and over-expression GSK3βlentivirus vector in different group mice hippocampus, then tested the behavior changes in different tests after 21 days CMS. The level of GSK3βprotein and mRNA were detected by Western Blot and RT-PCR.Results:1.We successfully constructed a T-cloning vector GSK3β. 2.After infection 293T cells in 72h, GSK3βover-expression lentivirus infection 293T cells had more GFP-positive cells. 3.Compared with control group, both protein and mRNA levels were increased significantly in GSK3βover-expression group hippocampus(P=0.001), after injection the lentivirus vector in 7 day. After injection the lentivirus vector 7 day, GSK3βover-expression group mice had more darkly stained GSK3β-positive cells in hippocampal, while other two group mice had few lightly stained GSK3β-positive cells in hippocampal. 4. consumption of sucrose ANOVA show that it exist obvious differences between all groups (F = 11.5). T test between LVX - GSK3βgroup and LVX - GFP group, LVX - GSK3βgroup show obviously lower than LVX - GFP groups ( P= 0.001), t-test between LVX - GSK3βgroup and normal controls , displays LVX - GSK3βgroup obviously lower than normal control group (P= 0.001), but LVX - GFP group and controls were not found differences. T test between LVX-GSK3β+ CMS group and LVX-GFP+CMS group , LVX - GSK3β+CMS group show significantly lower preferences than LVX-GFP+CMS group(P=0.023);T test between LVX -GSK3β+CMS group and controls+CMS group , LVX-GSK3β+CMS displays obviously lower than controls+CMS group (P = 0.025). But it is not found differences between LVX-GFP+CMS and controls+CMS groups. T test between LVX-GSK3β+CMS and LVX-GSK3βwas found no difference , but there is a trend (P =0.88). 5. Forced swimming ANOVA show that it exist obvious differences between all groups (F = 35.35). T test between LVX - GSK3βgroup and LVX - GFP group, LVX-GSK3βgroup show Despaired time obviously higher than LVX - GFP groups ( P= 0.001), t-test between LVX - GSK3βgroup and normal controls , displays LVX-GSK3βgroup Despaired time obviously higher than normal control group (P= 0.001), but LVX-GFP group and controls were not found differences. T test between LVX-GSK3β+CMS group and LVX-GFP+CMS group, LVX-GSK3β+CMS group show Despaired time is significantly higher preferences than LVX-GFP+CMS group(P=0.003);T test between LVX -GSK3β+CMS group and controls+CMS group, LVX-GSK3β+CMS displays obviously higher than controls+CMS group (P= 0.035). But it is not found differences between LVX-GFP+CMS and controls+CMS groups. the results showed that LVX-GSK3β+CMS is significantly higher than LVX-GFP+CMS group from T test between two groups(P = 0.001). 6. tail suspension test ANOVA show that it exist obvious differences between all groups (F = 8.17). T test between LVX - GSK3βgroup and LVX - GFP group, LVX-GSK3βgroup show Despaired time obviously higher than LVX - GFP groups (P= 0.036), t-test between LVX - GSK3βgroup and normal controls , displays LVX-GSK3βgroup Despaired time obviously higher than normal control group (P= 0.026), but LVX-GFP group and controls were not found differences. T test between LVX-GSK3β+CMS group and LVX-GFP+CMS group, LVX-GSK3β+CMS group show Despaired time is significantly higher preferences than LVX-GFP+CMS group(P=0.03);T test between LVX -GSK3β+CMS group and controls+CMS group, LVX-GSK3β+CMS displays obviously higher than controls+CMS group (P= 0.046). But it is not found differences between LVX-GFP+CMS and controls+CMS groups. the results showed that LVX-GSK3β+CMS is significantly higher than LVX-GFP+CMS group from T test between two groups(P= 0.005).Conclusion:1. Successfully constructed and cloned over-expression GSK3βlentivirus vector.2.Over-expression GSK3βlentivirus vector successfully transfected to C57BL/6J mice.3.Over-expression GSK3βC57BL/6J mice were significantly different in consumption of sucrose test, forced swimming test and tail suspension test compared with the normal group in the absence of CMS. This result indicated that increased GSK3βexpression in hippocampus might produce depression-like behavior. 4.Compared with the LVX-GFP+CMS and controls+CMS groups, the results of forced swimming test and tail suspension test were decreased in LVX-GSK3β+CMS C57BL/6J mice, suggesting that high expression of GSK3βin mice may be more sensitive to stress, but needed further study to confirmed the conclusion. |
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