小分子化合物调节多种细胞周期蛋白促进巨核细胞的多倍体化

发布时间：2018-06-07 04:37

本文选题：巨核细胞 + 多倍体化　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景血小板是人体血液中重要的有形成分之一,其在止血、伤口愈合及炎症反应等生理和病理过程中起着至关重要的作用。同时临床的许多相关疾病都可导致血小板的减少,其中血液系统疾病、癌症放疗及大剂量化疗、严重感染、终末期肝肾疾病等所引起的血小板减少,自身难以恢复,严重危害患者的生命健康,这时必须反复预防和治疗性输注血小板。然而目前的血液成分供应却远远达不到临床所需。大量的研究表明体外利用干细胞制备血小板是一种可行的方式,这将为血小板的临床应用获得更多的来源。在血小板的形成过程中,有许多因素影响血小板的增殖、分化、成熟、释放等,其中巨核细胞多倍体化的过程对于最终血小板的释放量有着重要的意义。研究提示,多种小分子化合物能通过对细胞骨架的调控促进巨核细胞多倍体化的形成。因此,我们的研究希望通过优化小分子化合物的添加获得更多多倍体的巨核细胞,为未来规模化的血小板制备提供新策略和技术支持。实验方法首先我们建立了白血病细胞系向巨核细胞分化的培养体系,使用低浓度的佛波酯(PMA)对其进行诱导分化,并使用高浓度的PMA对其进行多倍体化的诱导;通过流式细胞技术检测细胞表面标志及其细胞周期的变化,筛选出可获得更多多倍体的诱导体系,并用于后续的研究。接下来我们分别将四种不同的小分子化合物尼克酰胺(NIC)、RHO-ROCK通路抑制剂(RRI)、Src通路抑制剂(SI)、Aurora-B通路抑制剂(ABI)单独加入到细胞系诱导体系及脐带血单个核细胞中,并再一步采用流式细胞技术检测细胞周期的变化,并对巨核细胞特异性表面标志进行检测,同时通过CCK-8及其细胞凋亡检测进一步分析细胞的增殖及凋亡情况,筛选出有利于细胞多倍体化,同时又不影响细胞凋亡的小分子化合物作为优选的靶分子。进一步检测不同组合的小分子对多倍体形成的影响,并筛选优化出最佳的小分子组合。最终,使用Q-PCR及其Western Blot技术对优选的小分子组合及促进白血病细胞系和造血祖细胞来源的巨核细胞多倍体化的机制进行深入探讨。结果在优化细胞系的培养中,采用PMA(0.625ng/ml)进行诱导分化3天,再采用PMA(1Ong/ml)对细胞系进行多倍体化诱导9天所形成的多倍体量达到15%以上,并与其它两种方式具有显著统计学差异。使用不同的四种小分子化合物后,在细胞系及脐带血单个核细胞中均显示SI和ABI两种小分子可以明显的促进巨核细胞的多倍体化,并分别达到了 29%以上,原代细胞中四倍体提高到14%,然而ABI对于原代细胞有明显的促凋亡作用,凋亡比例达到了 70%,而SI在不影响细胞凋亡的情况下,促进多倍体的形成及特异性标志CD61的表达,SI是优选的小分子化合物。进一步检测其小分子组合的结果显示,RRI+SI组合不仅可以提高细胞多倍体的形成,同时两种小分子的加入促进了向巨核系分化的特异性表面标志的表达阳性率可提高54.65%,且不影响其细胞凋亡,从形态上也可以明显地观察到细胞多倍体。通过对组合小分子作用机制的探讨,结果显示SI和RRI可以降低p53及p21基因的表达,从而明显地提高cyclin D1、cyclin E和cyclin B1几种细胞周期蛋白的表达,最终促进了多倍体的形成。结论体外制备血小板是未来重要的血小板来源,目前的研究表明可以通过小分子化合物的应用促进多倍体的形成,通过比较之间对多倍体的促进作用并寻找更有效的小分子组合。从我们的实验结果显示,NIC促进多倍体的形成效果并不明显,同样的我们也发现ABI尽管促进多倍体化明显,但是其凋亡作用使多倍体最终形成效率明显的下降。我们的研究显示RRI+SI的组合不管是加入到细胞系的培养体系,还是在原代细胞培养的应用中均可有效地提高巨核细胞多倍体的形成。对于四种小分子的作用机制研究尚未有深入的研究,我们研究发现RRI及SI两种小分子可以明显地调节多种细胞周期蛋白,从而调节巨核细胞多倍体的形成。我们的结果进一步验证了小分子化合物组合在体外促进血小板形成并最终应用于临床的可行性。
[Abstract]:Background platelets are one of the most important forming parts of the human blood, which play a vital role in the physiological and pathological processes of hemostasis, wound healing, and inflammatory reactions. Many related clinical diseases can cause thrombocytopenia, including blood system diseases, cancer radiotherapy and large dose chemotherapy, severe infection, end-stage. Thrombocytopenia caused by liver and kidney diseases, which are difficult to recover and seriously harm the life and health of the patients, must be repeatedly prevented and therapeutic infusion of platelets. However, the current supply of blood components is far from the needs of the clinic. A large number of studies have shown that the use of stem cells to prepare platelets in vitro is a feasible way. In the process of platelet formation, there are many factors affecting the proliferation, differentiation, maturation and release of platelets. The process of polyploidy of megakaryocytes is of great significance to the release of the final platelets. The regulation of the frame promotes the formation of megakaryocyte polyploidy. Therefore, our research hopes to provide more polyploidy megakaryocytes by optimizing the addition of small molecular compounds to provide new strategies and technical support for the preparation of future large-scale platelet preparation. Culture system, using low concentration of PMA to induce differentiation, and use high concentration of PMA to induce polyploidy. Through flow cytometry detection of cell surface markers and cell cycle changes, screening out more polyploid inducers, and for subsequent research. Four different small molecular compounds, Nik amide (NIC), RHO-ROCK pathway inhibitor (RRI), Src pathway inhibitor (SI) and Aurora-B pathway inhibitor (ABI), were added to the cell line induction system and umbilical cord blood mononuclear cells alone, and the cell cycle changes were detected by flow cytometry, and the specific surface of megakaryocyte The markers are detected and the cell proliferation and apoptosis are further analyzed by CCK-8 and cell apoptosis, and small molecular compounds, which are beneficial to cell polyploidy and do not affect apoptosis, are selected as the preferred target molecules. Further detection of the effects of small molecules on the formation of polyploidy is further detected. Finally, Q-PCR and its Western Blot technique were used to explore the mechanism of the preferred small molecular combination and the mechanism of promoting the polyploidy of megakaryocytes derived from leukemic cell lines and hematopoietic progenitor cells. Results in the optimization of cell line culture, PMA (0.625ng/ml) was used to induce differentiation for 3 days, and then PMA was used. (1Ong/ml) the amount of polyploidy formed in the induction of polyploidy of the cell line for 9 days is more than 15%, and has significant statistical difference with the other two methods. After using different four small molecular compounds, all of the two small molecules of SI and ABI in the cell lines and umbilical cord blood mononuclear cells can obviously promote megakaryocyte. Ploidy, and more than 29%, the tetraploid in primary cells increased to 14%. However, ABI had obvious apoptosis inducing effect on primary cells, the proportion of apoptosis reached 70%, while SI promoted the formation of polyploid and the expression of specific marker CD61 without affecting apoptosis, and SI was the preferred small molecule compound. The results of small molecular combinations showed that RRI+SI combination could not only improve the formation of cell polyploidy, but the addition of two small molecules promoted the positive rate of the expression of specific surface markers to the megakaryocytes by 54.65%, without affecting the apoptosis of cells. The results show that SI and RRI can reduce the expression of p53 and p21 genes, thus obviously improve the expression of several cell cyclin in cyclin D1, cyclin E and cyclin B1, and eventually promote the formation of polyploid. Conclusion the preparation of platelets in vitro is an important source of platelet in the future. It is possible to promote the formation of polyploidy through the application of small molecular compounds, by comparing the promotion of polyploidy and finding more effective small molecular combinations. Our experimental results show that the effect of NIC on the formation of polyploidy is not obvious. We also found that although ABI promotes polyploidy obviously, its apoptosis is also found. Our study shows that the combination of RRI+SI can effectively improve the formation of polyploid megakaryocyte in the culture system of cell lines or in the application of primary cell culture. Research on the mechanism of action for four small molecules has not yet been studied in depth. It is found that two small molecules of RRI and SI can regulate a variety of cell cycle proteins to regulate the formation of polyploidy in megakaryocytes. Our results further verify the feasibility of the combination of small molecule compounds to promote platelet formation in vitro and eventually to be used in clinical practice.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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