| Alzheimer’s disease(AD) is a neural degenerative disease with progressive learning and memory loss, cognitive dysfunction, and change in personality and behavior, it is also known as dementia. AD is closely related with age and the present study reconfirmed that aging is one of the important influence factors of AD. With the aging of population, the incidence of AD increased year by year, so researching the pathogenesis of AD and seeking the ideal treatments are still the medical problems for the world. In recent years, stem cell therapy provides a new research strategy for the treatment of central nervous system disease. Human umbilical cord derived mesenchymal stem cells(h UC-MSCs) have the self-renewal and differentiation potentials, and because of easy obtainment and isolation, low immunogenicity, ease of isolation and culture in vitro, h UC- MSCs are deemed as seed cells for tissue engineering and cell therapy. Recent studies showed the clinical effectiveness for various diseases by stem cells therapy. However, stem cells, like other cells in the body, become senescence with increased passage, not only in terms of morphology characteristics, but also weakened proliferation and differentiation or out of control, thus can not maintain normal tissue renewal, leading to aging and aging related diseases. Therefore, searching for the key factors that regulate stem cell proliferation is essential for improving the activity of stem cell biology and therapeutic effects.Marine phytoplankton(marine phytoplankton, MPPT) is a natural and non-toxic original product cultivated in the marine environment, containing more than 200 kinds of algae categories, and is rich in unsaturated fatty acids, organic elements, trace elements and vitamins, etc. So, MPPT has many biological effects such as anti-virus, anti-oxidation, anti-allergic, anti-tumor, enhancing immunity and promoting the body’s metabolism. However, the anti-aging effects of MPPT on h UC-MSCs. Therefore, this research aimed to study the anti-aging effect of MPPT on h UC-MSCs and AD mice in vitro and in vivo.ObjectivesIn this study, we examined the anti-aging effect of MPPT on hUC-MSCs in vitro, analyzed the treatments by MPPT simply or in combination with h UC-MSCs transplantation into AD models and explored the mechanismsin vitro and in vivo, which will provide scientific bases and strategy for improving the effect of stem cell therapy and seeking new AD prevention strategies.Methods1. Explore the anti-aging effects of MPPT on h UC-MSCs in vitroh UC–MSCs were isolated from umbilical cords of mature healthy newborns delivery by caesarean section, and then cultured and passaged. The morphology was observed with inverted microscope and the cell surface markers such as CD45, CD90, CD44, CD34 and CD29 were detected in passage 3, 10, 15 h UC-MSCs by ?ow cytometry analysis(FCM). The m RNA expression of p16, p21, p53, p CNA, sirt1 and sirt2 in different passages of h UC–MSCs were detected by q RT-PCR. CCK-8 assay was performed to detect the effect of MPPT on cell proliferation and flow cytometry was used to detect the cell cycle and apoptosis. β-galactosidase staining was used to detect the effect of 1g/L MPPT on cell senescence. The effect of 1g/L MPPT on the expression of p16, p21, p53, p CNA, sirt2 and sirt1 in RNA and protein level were detected by q RT-PCR and Western Blot, respectively.2. Study the anti- aging effects of MPPT alone or combined with h UC-MSCs treatment on AD models3-month-old AD mice(all males) were randomly divided into six groups: APP + group, MPPT group(200 mg/kg·d dose), P3 h UC-MSCs transplantation group(P3 h UC-MSCs group, transplanted 5x105 P3 h UC-MSCs), the MPPT joint P3 h UC-MSCs transplantation group(MPPT+P3h UC MSCs group) and P15 h UC-MSCs transplantation group(P15 h UC-MSCs group, transplanted 5x105 P15 h UC-MSCs), the MPPT joint P15 h UC-MSCs transplantation group(MPPT + P15 h UC MSCs group), normal control group(APP-group) with the same genetic background ofgenetically modified mice, 10 mice in eachgroup. The MPPT was diluted with the same volume for each mouse, and the APP- group and APP + group were given the same volume of saline, the MPPT group and joint group were lavaged daily for a month until 4- month- old. The state of mind, activity and the change of body weight of the mice were regularly observed. Morris water maze was used to detect the learning and memory of mice after transplantation,immunohistochemical to detect the survival of h UC- MSCs.The MDA content and SOD activity in serum were analyzed by TBA method and Hydroxylamine method. The RNA and protein expression of p16, p21, p53, p CNA, sirt2 and sirt1 in the brain were detected by q RT-PCR and Western Blot, respectively.Results1. The cell morphology of P3 hUC-MSCs is fusiform and easily adherent, the morphology of P10 h UC-MSCs is different with P3 h UC-MSCs’ S, however, the P15 h UC–MSCs were flat and poorly adherent. Flow cytometry analysis showed that the h UC-MSCs in different passages candetect the marker of CD44,CD90 and CD29, while did not show the marker of CD45 and CD34. The m RNA expression of p CNA, sirt2 and sirt1 reduced gradually with passage, while p16, p21 and p53 increased..Compared to the P15 h UC-MSCs group, 1 g/L MPPT could promote the proliferation of h UC-MSCs, induced cells into S phase, decrease the positive rate of β-galactosidase staining, and increase the m RNA and protein expression of p CNA, sirt2 and sirt1, while decrease p16, p21 and p53expression(P<0.05).2. During the treatment, the mice were in good condition with shiny fur, no abnormal behavior, such as drowsiness, anxiety, etc; Compared to APP + group, h UC-MSCs combined with MPPT could significantly improve the learning and memory ability of AD mice, increase the survive rate of h UC-MSCs, increase SOD and decrease MDA content in the serum(P <0.05). The expression of p16, p21, p53 in the brain tissue were significantly decreased in all treatment groups at the m RNA and protein levels, and the m RNA and protein expression of p CNA, sirt2, sirt1 were increased; and combination of MPPT and h UC-MSCs achieve better effect thansingle treatment in all the aspects discussed above(P<0.05).Conclusion1.MPPT has anti-aging effects on h UC-MSCs by regulating the expression of p16, p21, p53, p CNA, sirt2, sirt1 to enhance cell activity;2.MPPT promote h UC-MSCs survive and regulation the age-related gene expression enhance the h UC-MSCs anti-aging effect of transplantation for AD mice, and the MPPT combination with h UC-MSCs anti-aging effect is obviously better than the single treatment effect. |
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