利用ES细胞建立可诱导的白血病小鼠模型

发布时间：2018-04-23 03:31

本文选题：胚胎干细胞 + PML/RARα基因　；参考：《北京协和医学院》2012年硕士论文

【摘要】：背景：胚胎干细胞(embryonic stem cells, ESCs)是从囊胚内细胞团分离出来的多潜能干细胞,具有多向分化能力。病毒携带外源基因感染ES细胞后,通过囊胚注射获得具有胚系遗传的转基因动物。可用此方法分析外源基因的功能及调节。急性早幼粒细胞白血病(acute promyelocytic leukemia, APL)是髓系白血病的一种,其特异性的细胞遗传表现为t(15；17),即15号和17号染色体分别断裂并发生易位,形成PML/RARa融合基因。PML/RARa融合蛋白阻止细胞分化,致使骨髓中堆积大量异常早幼粒细胞。另外,还通过干扰正常PML蛋白的功能促进白血病细胞恶性增殖及寿命延长。因此,建立一个体内模型研究PML/RARa基因对APL的作用机制是十分重要的。目前,仍缺少一个可诱导的APL动物模型研究此机制。目的：建立一个Tet-on系统调控的hPML/RARa转基因小鼠模型。利用此模型,研究PML/RARa融合蛋白对造血干祖细胞(hematopoietic stem and progenitor cells, HSCs/HPCs)的影响。方法：我们利用慢病毒载体将人PML/RARa基因(hPML/RARa)导入小鼠ES细胞中,已获得携带hPML/RARa基因的ES细胞,且其具有正常核型,同时通过囊胚注射已形成嵌合体小鼠。病毒载体上含有Tet-on表达调控系统,可通过强力霉素(doxycycline, dox)诱导hPML/RARa基因表达。通过集落形成实验(colony-forming cell, CFC)、长周期培养实验(long term culture, LTC)、液体培养实验研究PML/RARa基因对HSCs/HPCs及造血细胞分化的影响。另外,我们通过体内长期诱导,利用流式细胞仪分析监测转基因小鼠白血病发病进程。结果：RT-PCR结果显示,携带hPML/RARa基因的ES细胞dox诱导24小时表达hPML/RARa基因；转基因小鼠骨髓细胞dox诱导3天表达hPML/RARa基因。转基因小鼠体内dox诱导10天后,CFC结果表明,hPML/RARa基因表达增加了HPCs数量,同时促进其向粒系分化。体外液体培养结果也显示hPML/RARa基因表达促进造血细胞向粒系方向分化。另外,转基因小鼠体内长期诱导8月后仍具有正常表型。结论：1.我们成功建立了一个dox诱导hPML/RARa基因表达的转基因小鼠模型。2.PML/RARa基因表达增加HPCs数量,促进造血细胞向粒系分化。3.长期dox诱导8月后,转基因小鼠还未发生APL表型,表明APL的发生可能需要更长时间或二次及多次打击。
[Abstract]:Background: embryonic stem cells (ESCs) are multipotent stem cells isolated from blastocysts. Transgenic animals with embryogenic inheritance were obtained by blastocyst injection after infection of es cells with foreign genes. This method can be used to analyze the function and regulation of exogenous genes. Acute promyelocytic leukemia (promyelocytic) is a kind of myeloid leukemia. Its specific cell genetic manifestation is tnl 15N 17, I. e., chromosome 15 and chromosome 17 are broken and translocated, respectively, to form PML/RARa fusion gene. PML / Rara fusion protein prevents cell differentiation. Causes the bone marrow to accumulate the massive abnormal promyelocyte. In addition, the malignant proliferation and longevity of leukemic cells were increased by interfering with the function of normal PML protein. Therefore, it is very important to establish an in vivo model to study the mechanism of PML/RARa gene acting on APL. At present, there is still a lack of an inducible APL animal model to study this mechanism. Objective: to establish a hPML/RARa transgenic mouse model regulated by Tet-on system. Using this model, the effect of PML/RARa fusion protein on hematopoietic stem and progenitor cells (HSCs / HPCs) was studied. Methods: human PML/RARa gene hPMLR Raa was introduced into mouse es cells by lentivirus vector. Es cells carrying hPML/RARa gene were obtained and had normal karyotype, and chimeric mice were formed by blastocyst injection. The expression of hPML/RARa gene was induced by doxycycline (dox) and doxycycline (Doxycycline). The effects of PML/RARa gene on HSCs/HPCs and hematopoietic cell differentiation were studied by colony forming assay (CFC), long term culture (LTC) and liquid culture. In addition, long-term induction in vivo and flow cytometry were used to monitor the pathogenesis of leukemia in transgenic mice. Results the expression of hPML/RARa gene was induced by dox of es cells carrying hPML/RARa gene at 24 hours and the expression of hPML/RARa gene was induced by dox in bone marrow cells of transgenic mice for 3 days. After 10 days of dox induction in transgenic mice, the results showed that the expression of hPML / RARa gene increased the number of HPCs and promoted its differentiation into granulocytes. Liquid culture in vitro also showed that hPML/RARa gene expression promoted hematopoietic cells to granulocyte differentiation. In addition, transgenic mice still have normal phenotype after long-term induction in vivo. Conclusion 1. We successfully established a transgenic mouse model of hPML/RARa gene expression induced by dox. 2. PML / Rara gene expression increased the number of HPCs and promoted hematopoietic cells to differentiate into granulocytes. After long term dox induction for 8 months, no APL phenotype was found in transgenic mice, indicating that APL may take longer, twice or more blows.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R733.7

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