【作者】 陈曦；

【导师】 李薇；

【作者基本信息】 吉林大学 ， 内科学， 2016， 博士

【摘要】 研究背景和目的:诱导多能干细胞是通过导入特定的基因或基因产物,将已分化的体细胞人工诱导重编程成为类似于胚胎干细胞(embryonicstemcell,ES)的、具有多向分化能力的、可以持续分离生长的多功能干细胞。这项技术由日本京都大学Yamanaka教授在2006年首先提出。人工诱导多能干细胞通过取自体体细胞进行体外培养,这就同时避免了ES产生的伦理和排异两大障碍。目前传统的iPSCs技术主要是通过逆转录病毒介导的转基因技术将4个细胞因子Oct4,Sox2,Klf4和c-Myc(即OSKM)导入宿主细胞中而获得iPSCs细胞。通过OSKM因子使得终末分化的体细胞重编程后成为iPSCs的效率非常低,只有大约0.05%。这种低效率对于人iPSCs应用造成了巨大的障碍。因此,人们为了改变这一低效的重编程过程做出了大量的努力,通过应用DNA甲基化抑制剂,组蛋白修饰酶抑制剂,抑制肿瘤抑制基因,维生素C,信号通路抑制剂等来提高效率。小分子化学剂丙戊酸(valproicacid,VPA)是一种组蛋白去乙酰化酶抑制剂。在临床上,丙戊酸钠被用于治疗癫痫、双向性躁狂,预防偏头痛。在应用决定性因子OSKM使体细胞重编程成为iPSCs的过程中加入VPA已被证实对重编程效率有促进作用。然而目前对于VPA如何提高重编程的诱导作用的分子机制知之甚少。为了探讨VPA在iPSCs重编程中的作用和机制,我们进行以下实验。方法:1.首先将人骨髓来源的体细胞通过慢病毒转导入OSKM因子,然后转移至鼠胚胎成纤维细胞(mouseembryonicfibroblast,MEF)饲养层细胞上,之后根据是否添加VPA,将其分成两个组。通过碱性磷酸酶(alkalinephosphatase,AP)染色验证形成的的细胞克隆是否具有干性,并比较两个组之间的细胞克隆的形成的差异,进一步通过免疫组织化学染色,证明形成的iPSCs样集落是否具有多向分化潜能。接着我们通过形态观察及衰老相关β半乳糖苷酶(senescence-associatedbeta-galactosidase,SA-β-gal)染色,比较两组细胞在iPSCs重编程的过程中细胞增殖及衰老的情况,并提出假设,VPA是否通过抑制重编程导致的衰老来达到提高诱导多能干细胞的效率。2.由于使用OSKM慢病毒感染细胞重编程是效率极低的耗时过程,最关键的是MEF饲养层细胞的存在可能妨碍后续的机理研究。对于这一点,我们通过一个简单的铜诱导早衰模型取代OSKM慢病毒感染模型来研究VPA的作用。我们通过噻唑蓝(3-(4,5-Dimethylthiazol-2-y)-2,5-diphenyltetrazoliumbromide,MTT)染色法,测定不同铜浓度下细胞的存活率,来探究最适宜的铜浓度,以求得到最佳的化学诱导早衰模型。3.之后我们通过铜诱导早衰模型从细胞水平及分子水平研究VPA对于衰老的影响。通过细胞形态观察及及SA-β-gal染色,探究VPA对化学诱导的早衰的保护作用。通过磺酰罗丹明B(SulforhohamineB,SRB)染色法检测细胞的增殖,研究VPA在改善细胞生长中的作用。通过RT-PCR和Western印迹法,检测衰老相关基因的表达水平,以探究VPA在凋亡通路中的作用。最后通过检测细胞周期,研究VPA在细胞阻滞中发挥的作用。结果:1.在OSKM慢病毒感染模型中OSKM单独处理组与OSKM+VPA处理组比较,两组都出现细胞克隆,并且后者AP染色阳性的iPSCs集落明显多于前者,差异有显著性(P<0.01)。免疫组织化学染色表明,iPSCs集落其多向分化潜能标志物如SSEA-4和Tra1-60是阳性的,证明获得的细胞克隆具有干性及多向分化的潜力。OSKM单独处理组与OSKM+VPA处理组比较,前者细胞的形态具有明显衰老的特征,同时细胞的增殖明显比后者缓慢。经过SA-β-gal染色,前者染色阳性的细胞明显多于后者。2.在铜诱导的早衰模型中,用不同剂量的硫酸铜处理细胞,细胞存活率不同。当硫酸铜的浓度增加时,细胞存活率相应降低。当细胞暴露于较低浓度硫酸铜时,存活率略有下降,而一旦超过某一浓度,细胞存活率明显下降。通过MTT法,我们最终选择500,600和625μM硫酸铜浓度分别作为HSC-L1,HSC-2和MDFs铜诱导的早衰模型的最佳浓度。3.在铜诱导的早衰模型中,我们将细胞分为4组:阴性对照组、VPA单独处理组、硫酸铜处理组、硫酸铜+VPA处理组。VPA单独处理组与阴性对照组相比,细胞形态未发生明显变化。硫酸铜处理组与阴性对照组相比,细胞数量降低,细胞形态明显衰老,SA-β-gal染色阳性细胞比例明显升高。硫酸铜+VPA处理组与硫酸铜单独处理组相比,细胞生长明显改善,衰老细胞比例明显降低。SRB法测量细胞增殖,将对照组细胞的数量作为100%,可见硫酸铜处理组较对照组细胞增殖明显降低(P<0.05),硫酸铜+VPA处理组细胞增殖较硫酸铜处理组明显升高(P<0.01)。通过RT-PCR及Western印迹法,从RNA及蛋白质水平检测凋亡基因。将β-ACTIN作为内参,硫酸铜处理组与阴性对照组相比,促凋亡基因p16,p21,小窝蛋白(caveolin-1,Cav-1),载脂蛋白J(apolipoproteinJ,APO-J),食欲素受体1(orexinreceptors1,OX-1)表达明显增强,抗凋亡基因B淋巴细胞瘤-2(B-celllymphoma2,Bcl-2)明显降低。硫酸铜+VPA处理组与硫酸铜处理组比较,促凋亡基因有所下降,抗凋亡基因有所上升。通过流式细胞术检测细胞周期。对照组G2/M期比例为14.2%,硫酸铜处理组G2/M期比例为61.2%,明显较对照组高。硫酸铜+VPA处理组G2/M期比例为44%,较硫酸铜组明显降低。对照组与硫酸铜组,硫酸铜组与硫酸铜+VPA处理组比较,G2/M期比例有显著差异(P<0.01)。结论:1.通过慢病毒介导的转基因技术将4个细胞因子Oct4,Sox2,Klf4和c-Myc(即OSKM)导入人骨髓来源细胞可以获得细胞克隆。经过AP染色以及SSEA-4和Tra1-60免疫组织化学染色,证明获得的细胞克隆具有干性及多向分化的潜力,是iPSCs细胞。在重编程的过程中出现细胞衰老,细胞增殖减慢等现象,补充VPA后细胞衰老比例下降,增殖速率提高,iPSCs集落明显增加,说明VPA通过保护细胞免受重编程触发的衰老来促进iPSCs集落的产生。2.OSKM慢病毒感染细胞与用亚细胞毒性浓度的铜处理细胞都会出现细胞增殖受抑,细胞体积增大,衰老相关β-半乳糖苷酶活性升高等衰老相关特征,故铜诱导的早衰模型可以代替OSKM慢病毒感染模型。经MTT检测,500,600和625μM硫酸铜浓度分别为HSC-L1,HSC-2和MDFs造衰老模型的最佳浓度。3.在铜诱导的早衰模型中,丙戊酸可以通过抑制促凋亡基因表达,促进抗凋亡基因表达,降低细胞周期阻滞来抑制细胞的衰老,促进细胞增殖。进而证明丙戊酸通过以上途径保护细胞免受重编程触发的衰老来促进iPSCs集落的产生。更多还原

【Abstract】 Background and purpose:Induced pluripotent stem cells(i PSCs) are a type of pluripotent stem cell,which can be generated directly from somatic cells by introducing a specific gene or gene product. This type of pluripotent stem cell is similar to embryonic stem cell, which can differentiate to many kinds of cells and can continue grow. The i PSCs technology was pioneered by Shinya Yamanaka’s lab in Kyoto,Japan,who showed in2006.Induced pluripotent stem cells were taken from the somatic cells and cultured in vitro, which at the same time avoiding the ethical and rejection issues. Currently the i PSCs technology is mainly mediated by retrovirus, which transfers four transcription factors Oct4, Sox2, Klf4, c-Myc(OSKM) into a host cell to obtain i PSCs. Reprogramming of the terminally differentiated-somatic cells into i PSCs by cocktail factors OSKM is a very inefficient process. The efficiency is about 0.05%.The low efficiency caused a huge obstacle for human i PSCs application. For this,tremendous efforts have been attempted to improve the reprogramming process by using DNA methylation inhibitors, histone modification inhibitors, inhibition of tumor suppresser genes, vitamin C and signaling pathway inhibitors.Small molecular chemical valproic acid(VPA) is a kind of histone deacetylase inhibitor. Clinically, VPA has been used for the treatment of epilepsy, bipolar mania and migraine prophylaxis. During the period of somatic cell reprogramming by defined factors, treatment of cells with VPA has been demonstrated to have the pluripotency-promoting activity. However, currently little is known about the molecular mechanism as how VPA improves the induction of pluripotency. we performed the following experiments in order to investigate the effect and mechanism of VPA in i PSCs reprogramming.Methods:1. We infected the bone marrow-derived cells with lentiviruses containing cocktail factors(Oct4, Sox2, Klf4 and c-Myc, OSKM).We collected the cells and transferred them to 100 mm dishes on mitomycin C-inactivated mouse embryonic fibroblast(MEF) feeder cells. Cells were divided into two groups depending on whether adding valproic acid or not. We verified whether the cell clones had the stem cell properties or not by alkaline phosphatase(AP) staining and compared the difference between two groups, then investigated the pluripotency of i PSCs-like colonies by immunohistochemical staining. We compared cell proliferation and senescence in the process of reprogramming of two groups by observing the cell morphology and senescence-associated β-gal staining. In this study, we assumed whether VPA enhanced i PSCs induction from human bone marrow-derived cells by suppressing reprogramming-induced senescence stress.2. Cell reprogramming using OSKM lentiviruses is a time-consuming process with extremely low efficiency. Most critically, the presence of MEF feeder cells may interfere with subsequent mechanistic studies. For this, we used a simple copper-induced premature senescence model to replace the OSKM lentivirus model to study the role of VPA. We detected cell survival in different concentration of copper by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay to explore the optimum concentration of copper in order to get the best model of chemical-induced premature senescence.3. Then we investigated the effect of valproic acid at the cellular and molecular level on chemical-induced premature senescence model. We explored the protective effect of valproic acid on chemical-induced premature senescence by the cell morphology and senescence-associated β-gal staining. Cell proliferation was detected by sulforhohamine B(SRB)assay to study the effect of valproic acid on improving cell growth. We detected the expression level of aging-related genes in order to explore the role of valproic acid in the apoptotic pathway by RT-PCR and Western blot. At last, we study the valproic acid effect on cell cycle arrest by cell cycle assay.Results:1. In the OSKM lentivirus model we obtained cell clones in both groups. VPA promoted the generation of AP-positive i PSCs colonies(P<0.01)as compared with the OSKM group. Immunohistochemical staining showed that the pluripotency markers such as SSEA-4 and Tra1-60 were positive in i PSCs colonies. These proved that the obtained cell clones had the stem cell characteristics and multi-directional differentiation potential. Lentivirus-infected cells exhibited typical senescence and reduced cell growth. The β-galactosidase activity in lentivirus-transfected cells increased compared with that in the VPA treated group.2. In the copper-induced premature senescence model, cell viability is different after exposure to different doses of copper sulfate. With increasing concentrations of copper sulfate, the cell viability was accordingly decreased. When the cells were exposed to lower concentrations of copper sulfate, the survival rate decreased slightly, but once exceeded a certain concentration, cell viability decreased significantly.By MTT assay, we chose 500, 600 and 625 μM as the optimal concentration of copper sulfate concentration for HSC-L1, HSC-2 and MDFs copper-induced premature senescence model.3. In the copper-induced premature senescence model, we divided the cells into four groups: negative control group, VPA treated alone group, copper sulfate treated alone group, copper sulfate + VPA treated group.The cells of VPA treated alone group did not change significantly compared with the control group. The number of cells reduced and cell morphology was significantly aging in copper sulfate treated alone group compared with negative control group. And also β-gal positive cells were significantly increased. Cell growth improved significantly and the proportion of senescent cells reduced obviously in copper sulfate + VPA treated group compared with the copper sulfate treated alone group. We detected cell proliferation by SRB assay. For comparison, all values were normalized using the control group as 100%. Cell proliferation of copper sulfate treated alone group significantly decreased compared with the negative control group(P<0.05).Cell proliferation of copper sulfate + VPA treated group was significantly higher than that of copper sulfate treated alone group(P<0.01).We detected apoptotic genes at RNA and protein levels by RT-PCR and Western blot.β-actin was used as the internal control. The expression of pro-apoptotic genes such as p16, p21, caveolin-1(Cav-1),apolipoprotein J(APO-J), orexin receptors 1(OX-1) in copper sulfate treated alone group was significantly enhanced compared with the negative control group, and the anti-apoptotic gene B-cell lymphoma 2(Bcl-2)was significantly reduced. The pro-apoptotic genes of copper sulfate + VPA treated group declined compared with the copper sulfate treated alone group, and anti-apoptotic gene increased.We detected cell cycle by flow cytometry. G2/M phase ratio was 14.2% in control group.In copper sulfate treated alone group G2/M phase ratio was 61.2%, which is significantly higher. Compared with copper sulfate treated alone group, the G2/M phase ratio of copper sulfate + VPA treated group was 44%,which is significantly lower. The comparison of G2/M phase proportion between control group and copper sulfate treated alone group, between copper sulfate treated alone group and copper sulfate + VPA treated group were significantly different(P<0.01).Conclusions:1. We obtained cell clonies by transferring four transcription factors Oct4, Sox2,Klf4 and c-Myc(OSKM)into a host cell mediated by lentivirus. We demonstrated that the obtained cell clones had stem cell properties and multidirectional differentiation potential by AP staining, SSEA-4 and Tra1-60 immunohistochemical staining. Cell senescence occured and cell proliferation decreased in the process of reprogramming.After adding the VPA the cell senescence ratio decreased,cell proliferation rate increased and i PSCs colonies significantly increased.It proved that VPA enhanced i PSCs induction from human bone marrow-derived cells by suppressing reprogramming-induced senescence stress.2. We found the aging-related characteristics, such as cell proliferation decreased,cell volume increased and aging-related β- galactosidase activity increased in both OSKM lentiviral model and copper-induced premature senescence model.For this, we used copper-induced premature senescence model to take the place of OSKM lentiviral models. By MTT assay, we chose 500, 600 and 625 μM as the optimal concentration of copper sulfate concentration for HSC-L1, HSC-2 and MDFs copper-induced premature senescence model.3. In copper-induced premature senescence model, VPA can inhibit cell senescence and increase cell proliferation by inhibiting the apoptosis pathway and reducing G2/M blockage. We proved that VPA enhanced i PSCs induction from human bone marrow-derived cells by suppressing reprogramming-induced senescence stress.更多还原