| Alzheimer's disease(AD) is a neurodegenerative disease, which is related to aging. The etiology and molecular mechanism of the disease is still uncertain. Current pharmacological treatments for AD can relieve AD patients' symptoms, but they cannot delay or halt the degeneration and the loss of neurons in the brains. The discovery of neural stem cells supports a new method to cure AD. There are two strategies in stem cell treatment of AD:activating the endogenous neural stem cells effectively and using exogenous stem cells to repair or replace the impaired neurons. However, stem cells can become aging in vivo and in vitro, resulting in aging related disease and limiting the use of stem cells. Wnt/?-catenin signaling pathway is essential for the development of embryo. Studies show that the activation of Wnt/?-catenin signaling pathway can promote stem cells differentiation into neurons, and regulate the neurogenesis in hippocampus. Engrafted mesenchymal stem cells facilitates hippocampal neurogenesis and neural differentiation in mouse model of AD by activating Wnt/?-catenin signaling pathway. Lithium chloride(Li Cl), which has been used as a drug in clinical for over a century, has been proved to be safety and efficiency. Li Cl, as a direct inhibitor of glycogensynthase kinase-3?(GSK-3?) which plays a key role in the Wnt/?-catenin signaling pathway, leads to an increase in intracellular ?-catenin and to the consequent activation of the Wnt/?-catenin signaling pathway. Li Cl can alleviate spatial memory impairment and neurodegeneration in brains of mouse models of AD. However, Li Cl combined with stem cells transplantation has not been reported to cure AD up to now.Objectives Therefore, we use Li Cl as the target and human umbilical cord derived mesenchymal stem cells(h UC-MSCs) as the object to observe the influence of Li Cl on the proliferation, aging, apoptosis and neural differentiation of h UC-MSCs in vitro. Then we evaluate the nerve repairment and intervention effect of Li Cl combined with h UC-MSCs transplantation on AD in vivo. The aim of this research is to discover the possible molecular mechanism of AD and observe the effect of stem cells on AD, in order to provide a basal theory for the treatment of AD.Method 1. Explore the influence of Li Cl on the proliferation, aging, apoptosis and neural differentiation of h UC-MSCs in vitro Experiments were allocated into two groups: control group(MSCs group) and Li Cl treatment group(MSCs+Li Cl group). CCK-8 assay and Ed U staining were used to detect the influence of Li Cl on h UC-MSCs' proliferation; flow cytometry was performed to detect the cell cycle and apoptosis; ?-galactosidase staining, q RT-PCR and Western Blot were used to detect the senescence. After neural differentiation induction, the expression of NSE, DCX, ?-III Tubulin of h UC-MSCs were detected by immunocytochemistry; the expression of Ngn1, Ngn2, Mash1 were analyzed by q RT-PCR. 2. The intervention effect of Li Cl combined with h UC-MSCs transplantation on AD mouse models A total of 60 AD mouse models(APP695v717 transgenic mouse) were allocatedinto 6 groups randomly: APP- group, APP+ group, Li Cl group, Li Cl pretreatment group, and Li Cl combined with h UC-MSCs group. The learning and memory ability was studied by Morris maze test, the survival and migration of h UC-MSCs and the expression of neuronal markers ?-III Tubulin in hippocampe were tested by immunohistochemistry; the expression of m RNAs and proteins associated with Wnt/?-catenin pathway in the hippocampus were detected by q RT-PCR and Western Blot, and the expression of neurotrophic factors in the hippocampus were studied by q RT-PCR.Results 1. Compared with MSCs group, Li Cl facilitated the proliferation of h UC-MSCs, increased the proportion of cells in S phase, decreased the rate of ?-galactosidase positive cells and apoptosis cells(P<0.05). Li Cl also decreased the expression of GSK-3?, p16, p21 and p53 in h UC-MSCs, increased the expression of c-Myc, p CNA, sirt1 and sirt2, and increased the expression of ?-catenin only at the level of protein(P<0.05). After neural differentiation induction, the cells expressed NSE, DCX, ?-III Tubulin, Ngn2 and Mash1 at higher level in contrast with MSCs group(P<0.05). Compared with the merely inducted group, the cells added Li Cl before inducting expressed these markers higher(P<0.05). 2. Compared with APP+ group, the transplantation of h UC-MSCs and the intraperitoneal injection of Li Cl both could improve the learning and memory ability of AD mice, and the effect of Li Cl combined with h UC-MSCs was proved to be more significant(P<0.05). The h UC-MSCs which survived in Li Cl+ MSCs group were higher than which in MSCs group(P<0.05). The expression of p16, p21, p53 were decreased significantly, and the expression of p CNA, sirt1, sirt2 were significantly increased in the hippocampus of APP+ mice which were treated. The expression of sirt1 in the hippocampus of Li Cl combined with h UC-MSCs treated mice increased higher than single treated mice(P<0.05). The expression of GSK-3? decreased, ?-catenin and c-Myc increased in the hippocampus of APP+ mice which were treated, and the expression of c-Myc in the hippocampus of Li Cl combined with h UC-MSCstreated mice increased higher than single treated mice(P<0.05). The expression of ?-catenin, BDNF, NGF and NT3 increased significantly in the hippocampus of APP+ mice which were treated, and the mice in Li Cl combined with h UC-MSCs increased more significantly(P<0.05).Conclusion 1. Li Cl could promote the proliferation, inhibit the apoptosis and senescence, accelerate the neural differentiation of h UC-MSCs, most likely by activing the Wnt/?-catenin signaling pathway and regulating the senescence associated gene, and then enhance the activity of h UC-MSCs. 2. Li Cl could improve the learning and memory ability of APP+ mice, and improve the expression of neurotrophic factors in hippocampus by increasing the migration and survival, facilitating the differentiation of stem cells in the injury area, and then delay the aging of AD mice. The therapy effect of Li Cl combined with h UC-MSCs transplantation were much better than single treated on APP+ mice. |
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