【作者】 姚宁；

【导师】 关方霞；

【作者基本信息】 郑州大学 ， 细胞生物学， 2015， 硕士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是一种渐进性的学习记忆功能丧失及空间认知功能障碍的中枢神经系统退行性疾病,同时伴随有人格和行为的改变,并且也是与年龄密切相关的老年性疾病,亦称老年痴呆。随着人口老龄化日益加重,AD的发病率逐年上升,研究结果表明衰老是AD发病的重要影响因素之一。研究AD的发病机制和寻求理想的治疗药物和手段仍是当今世界医学的难题。干细胞移植作为一种新的治疗方法为中枢神经系统疾病的治疗带来了希望。人脐带间充质干细胞具有自我更新和多向分化的潜能。因h UC-MSCs取材方便、来源广泛,低免疫源性,并且体外易于分离培养,因此h UC-MSCs成为组织工程及细胞替代治疗的种子细胞。近年来研究报道显示,国内外研究者通过干细胞治疗各种疾病的临床疗效观察其治疗疾病的效果取得了较好的进展。但是,研究发现干细胞在体外培养过程中与其他正常细胞一样,随着传代次数的增加,细胞在不仅形态上表现出衰老特征,而且增殖活性和分化能力减弱,细胞发生衰老。由于干细胞活力降低从而不能够维持机体正常的组织更新,引发机体的衰老甚至导致相关疾病的发生。因此,寻找调控干细胞增殖和抗衰老的关键因子对于提高干细胞生物学活性和治疗效果至关重要。海洋单细胞海藻(marine phytoplankton,MPPT)是一种无毒害,纯天然提取的海洋生物产品。MPPT因其成分齐全,营养丰富,富含不饱和脂肪酸、有机元素、矿物质和维他命等,所以具有抗病毒、抗氧化、抗过敏、抗肿瘤、提高免疫力和促进机体新陈代谢等生物学活性,是大自然的杰作。但是,MPPT对h UC-MSCs的抗衰老作用尚未见明确报道。因此,本研究通过体内外实验研究MPPT对h UC-MSCs的抗衰老作用。目的本研究拟通过体外实验检测MPPT对h UC-MSCs的抗衰老作用,通过体内实验研究MPPT对h UC-MSCs移植治疗AD的抗衰老作用及其可能的分子机制。为提高干细胞治疗效果和寻求新的AD防治策略提供科学依据和研究思路,具有重要的研究意义和应用价值。方法1体外实验研究MPPT对h UC-MSCs衰老的影响取剖宫产健康新生儿脐带,分离、传代、培养h UC-MSCs,显微镜下观察细胞生长形态,将h UC-MSCs培养至第3、10、15代,流式细胞仪检测细胞表面标记CD45、CD90、CD44、CD34、CD29;收集第3、10、15代细胞,q RT-PCR检测不同传代细胞中p16、p21、p53、p CNA、sirt2、sirt1m RNA的表达。CCK-8法分析不同浓度的MPPT对h UC-MSCs生长增殖的影响,确定MPPT的最佳作用浓度;流式细胞术检测1g/L的MPPT对h UC-MSCs细胞周期和细胞凋亡的影响;β-半乳糖苷酶染色法检测1g/L的MPPT对h UC-MSCs衰老的调控;q RT-PCR检测MPPT对h UC-MSCs中p16、p21、p53、p CNA、sirt2、sirt1m RNA表达的影响;Western Blot检测MPPT对h UC-MSCs中衰老蛋白P16、P21、P53、PCNA、Sirt2、Sirt1的表达变化的影响。2体内实验研究MPPT单独或联合h UC-MSCs治疗对AD的抗衰老作用取3月龄AD模型鼠(APP695v7171转基因小鼠)60只,全为雄鼠,随机分为6组:模型组(APP+组),MPPT组(每天200mg/kg给药)、P3h UC-MSCs移植组(P3 h UC-MSCs组)、MPPT联合P3h UC-MSCs移植组(MPPT+P3h UC-MSCs组)、P15h UC-MSCs移植组(P15 h UC-MSCs组)、MPPT联合P15h UC-MSCs移植组(MPPT+P15h UC-MSCs组)以及1组10只遗传背景相同的非转基因鼠作为正常对照组(APP-组)。APP+组和APP-组均给予相同体积生理盐水灌胃,干细胞移植组(5×105个),MPPT稀释成相同体积药液灌胃给药,MPPT组和联合给药组每日1次,连续灌胃1个月(至4月龄),定期观察小鼠的精神状态、肢体活动状况以及体重变化的情况。Morris水迷宫法检测小鼠行为学的改变;水迷宫结束后宰杀小鼠,免疫组化法检测小鼠脑组织中MAB1281的表达观察h UC-MSCs的存活情况;提取小鼠血清,采用TBA法和羟胺法检测小鼠血清中丙二醛(Malondialdehyd,MDA)和超氧化物歧化酶(Superoxide dismutase,SOD)的含量变化;解剖取小鼠脑组织,q RT-PCR和Western Blot检测脑组织中衰老相关基因p16、p21、p53、p CNA、sirt2、sirt1在m RNA和蛋白质水平的表达变化。结果1、h UC-MSCs在P3代时呈梭形贴壁细胞,大小均匀一致。P10 h UC-MSCs细胞形态呈梭形与P3无明显变化。P15 h UC-MSCs呈不规则状态,宽大扁平,易脱壁。P3、P10、P15 h UC-MSCs表面均表达CD44、CD90和CD29,不表达CD45和CD34;随着传代培养次数的增加,h UC-MSCs中p CNA、sirt2、sirt1m RNA相对表达量减少,而p16、p21、p53m RNA相对表达量增加。与P15组相比,1g/L的MPPT可促进P15 h UC-MSCs的增殖,促进细胞周期进入S期;MPPT处理组细胞的β-半乳糖苷酶阳性率明显降低,细胞中p CNA、sirt2、sirt1m RNA和蛋白的表达量增加,而p16、p21、p53m RNA和蛋白的表达量降低,差异具有统计学意义(P<0.05)。2、给药期间,各组小鼠精神状况良好,皮毛光泽无脱毛现象,活动正常,并未出现异常的行为学改变,如嗜睡、不安等;h UC-MSCs和MPPT均可以明显改善小鼠的学习记忆功能,二者联合时效果更佳(P<0.05)。一月后小鼠脑组织中联合治疗组的h UC-MSCs存活率明显多于单独移植组。在体内治疗过程中,h UC-MSCs(5×105 cells)和MPPT(200 mg/kg)的不同治疗模式的小鼠在SOD及MDA的改善方面,联合治疗组血清中SOD活性明显上升,MDA含量明显下降,具有着最佳的治疗效果。在各治疗组中脑组织中p16、p21、p53的表达明显下降,而p CNA、sirt2、sirt1的表达则显著上升;其中,联合治疗模式对于p16、p21和p53的抑制作用以及对p CNA、sirt2和sirt1的促进作用显著优于单一治疗方法(P<0.05)。结论1、MPPT通过调节h UC-MSCs中p16、p21、p53、p CNA、sirt2、sirt1等衰老相关基因的表达增强细胞活性、延缓干细胞衰老。2、MPPT促进h UC-MSCs的存活和调控衰老相关基因的表达提高h UC-MSCs移植治疗AD鼠的抗衰老作用,而且MPPT和h UC-MSCs的联合治疗明显优于MPPT或h UC-MSCs单一治疗效果。更多还原

【Abstract】 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.更多还原