去泛素化酶MYSM1调控脂肪间充质干细胞的免疫调节功能及机制研究

发布时间：2018-04-25 19:48

本文选题：脂肪来源间充质干细胞 + MYSM1　；参考：《中国人民解放军医学院》2017年博士论文

【摘要】：间充质干细胞(MSCs)是一类具有自我更新和多向分化能力的成体干细胞。近年来MSCs的另一个特性即免疫调节能力逐渐成为人们关注的热点,MSCs能抑制多种淋巴细胞的功能,在许多免疫相关疾病中被广泛研究,但MSCs的免疫调节机制仍然不是十分明确。组蛋白修饰中的泛素化和去泛素化是表观遗传学中一个重要的领域。MYSM1,一种组蛋白H2A去泛素化酶,能够对小鼠体内的多种免疫细胞发育和功能产生影响。那么,MYSM1是否会调控具有免疫调节能力的MSCs的功能,目前尚未有报道,但这却为MSCs能否在临床中广泛应用提供了一个新的思路和依据。我们从以下三个部分进行这方面的研究。第一部分:小鼠脂肪来源间充质干细胞的分离、培养与鉴定实验方法:首先利用基因型鉴定方法确定小鼠基因型,然后分离野生型(wild type, WT)小鼠和敲除型(knockout, KO)小鼠的腹股沟脂肪组织来源的MSCs (ADSCs),并对这两种细胞的形态、增殖、表型和成脂成骨分化能力进行鉴定。实验结果:1、Mysm1敲除并不影响ADSCs的形态、表型和增殖。WT和KOADSCs均阳性表达 Sca-1、CD44、CD29,阴性表达 CD86、CD45、CD31、CD11b 及 MHCII 类分子;2、WT和KO ADSCs均具有成脂成骨分化能力。qRT-PCR结果显示成骨相关基因Runx2、Osterix和Ocn以及成脂相关基因Ppar-γ、Cebp-α和aP2的表达量在各自的诱导下均升高。第二部分:MYSM1调控小鼠ADSCs免疫调节功能的体内外研究实验方法:1、qRT-PCR检测Mysm1在炎症环境下的表达变化;2、通过两种ADSCs与T细胞共培养来比较WT ADSCs和KO ADSCs对T细胞增殖的影响;3、在炎症环境下,检测ADSCs表达免疫相关因子的mRNA水平以及细胞表面免疫表型的变化;4、建立IBD模型,检测WT ADSCs和KO ADSCs在体内免疫抑制能力的差别。实验结果:1、Mysm 1在TNFα+IFNγ的刺激下表达最高,并且随着其浓度及时间的增加而升高;2、WT和KO ADSCs均能抑制T细胞增殖,并且WT ADSCs对T细胞增殖的抑制能力要明显强于KO ADSCs;3、无论在有无TNFα+IFNy刺激下,WTADSCs均表达更高的抗炎因子iNOS、IL10,而KOADSCs则表达更高的炎症因子IL1β、TNFα、IFNy及IL6; PD-L1的表达在低浓度刺激下均明显升高且没有差异,ICAM-1和VCAM-1的表达也没有差异;4、体内IBD模型实验也证实了 WT ADSCs具有更强的免疫抑制能力,更有助于小鼠炎症的恢复。第三部分:MYSM1通过miR-150调控小鼠ADSCs免疫调节功能实验方法:1、qRT-PCR检测WT和KO ADSCs中miR-150的表达差异;2、KO ADSCs过表达MYSM1后,检测miR-150的表达变化;3、免疫染色质共沉淀(CHIP)实验验证MYSM1对miR-150的作用;4、C3H10T1/2过表达miR-150(C3H LV- miR-150),检测其表型、增殖能力的变化;5、通过C3H LV- miR-150和C3H LV-GFP (对照细胞)与T细胞共培养实验,比较二者对抑制T细胞增殖能力的差异;6、qRT-PCR和流式技术检测炎症环境下C3H LV- miR-150和C3H LV-GFP免疫相关因子和细胞表面免疫表型的表达差异。实验结果:1、KOADSCs中miR-150的表达明显低于WTADSCs;2、当KOADSCs过表达MYSM1时,miR-150的表达随之升高;3、CHIP显示MYSM1能结合到miR-150启动子上游1200-1000的区域;4、C3H LV- miR-150和C3H LV-GFP的表型没有明显变化,仍然显示出干细胞表型;过表达miR-150相较于对照细胞C3H LV-GFP增殖能力变快;5、C3H10T1/2过表达miR-150后,其抑制T细胞的增殖能力明显增强;6、C3H10T1/2过表达miR-150后,促炎因子IFNγ及信号抑制分子SOCS1表达降低,抑炎因子iNOS和IL10表达升高;7、C3H10T1/2 过表达 miR-150 后,PD-L1、VCAM-1、ICAM-1 表达没有明显差异。结论:MYSM1在ADSCs的免疫调节功能中发挥重要的作用,敲除Mysm1后ADSCs的免疫抑制能力明显减弱。MYSM1能够作用于miR-150的启动子区促进miR-150的表达,从而对ADSCs的免疫调控功能发挥作用,但下游的具体机制还需进一步探究。
[Abstract]:Mesenchymal stem cells (MSCs) are a kind of adult stem cells with self-renewal and multidirectional differentiation. In recent years, another characteristic of MSCs has been the focus of attention. MSCs can inhibit the function of many kinds of lymphocytes and is widely studied in many immune related diseases. However, the immunomodulatory mechanism of MSCs is still the mechanism of immunomodulating. Not very clear. Ubiquitination and ubiquitination in histone modification are an important field in epigenetics,.MYSM1, a histone H2A ubiquitinase, which can affect the development and function of various immune cells in mice. Then, whether MYSM1 will regulate the function of MSCs with immune regulation is not yet available. There are reports, but this provides a new idea and basis for the wide application of MSCs in clinical practice. We do this in the following three parts. The first part: the isolation, culture and identification of mouse fat derived mesenchymal stem cells: first of all, the gene type of mice was determined by the method of identification, and then divided into two parts. MSCs (ADSCs) from the inguinal adipose tissue derived from wild type (WT) mice and knockout (KO) mice and identification of the morphology, proliferation, phenotype and osteogenic differentiation of these two cells. Experimental results: 1, Mysm1 knockout does not affect the morphology of ADSCs, and the phenotype and proliferation of.WT and KOADSCs are both positive for Sca-1. D44, CD29, negative expression of CD86, CD45, CD31, CD11b and MHCII molecules; 2, WT and KO ADSCs all showed osteogenic differentiation ability.QRT-PCR results showed bone related genes Runx2. In vivo and in vitro research methods of regulating function: 1, qRT-PCR was used to detect the expression of Mysm1 in the inflammatory environment; 2, the effect of WT ADSCs and KO ADSCs on T cell proliferation was compared with two ADSCs and T cells; 3, in the inflammatory environment, the mRNA level of the immune related factors and the changes of the cell surface immunophenotype were detected by ADSCs; 4 The IBD model was established to detect the difference in the immune suppression ability of WT ADSCs and KO ADSCs in the body. The results were as follows: 1, Mysm 1 was expressed highest under the stimulation of TNF alpha +IFN gamma, and increased with the increase of concentration and time; 2, WT and KO ADSCs inhibited the proliferation of T cells. 3 Under the stimulation of TNF alpha +IFNy, WTADSCs expressed a higher anti-inflammatory factor iNOS, IL10, while KOADSCs expressed higher inflammatory factors IL1 beta, TNF a, IFNy and IL6, and PD-L1 expression increased obviously under low concentration and no difference, and there was no difference in ICAM-1 and expressions. 4 The third part: MYSM1 regulation of ADSCs immunoregulation in mice by miR-150: 1, qRT-PCR detection of the difference in miR-150 expression in WT and KO ADSCs; 2, KO ADSCs over expression MYSM1, detection of the changes in miR-150 expression; 3, immunochromatin coprecipitation test Verify the effect of MYSM1 on miR-150; 4, C3H10T1/2 overexpressed miR-150 (C3H LV- miR-150) to detect its phenotypic and proliferative ability. 5, the difference between the two groups was compared with that of C3H LV- miR-150 and C3H LV-GFP (control cells). C3H LV-GFP immune related factors and cell surface immunophenotype expression differences. Experimental results: 1, the expression of miR-150 in KOADSCs was significantly lower than WTADSCs; 2, when KOADSCs overexpressed MYSM1, the expression of miR-150 increased; 3, CHIP showed that MYSM1 could combine to the upstream region of the miR-150 promoter 1200-1000; 4 There was no obvious change in the phenotype and still showed the phenotype of stem cells; overexpression of miR-150 was faster than the control cell C3H LV-GFP proliferation ability; 5, C3H10T1/2 overexpressed miR-150, and the proliferation ability of T cells was significantly enhanced; and after C3H10T1/2 overexpressed miR-150, the expression of proinflammatory promoter and signal suppressor molecule SOCS1 expression decreased and anti inflammatory factor iNO was iNO. The expression of S and IL10 increased; 7, after C3H10T1/2 overexpression of miR-150, there was no significant difference in the expression of PD-L1, VCAM-1 and ICAM-1. Conclusion: MYSM1 plays an important role in the immune regulation of ADSCs, and the immunosuppressive ability of ADSCs after knocking down Mysm1 obviously decreases the expression of.MYSM1 to act on the promoter region. The immune regulatory function plays a role, but the specific mechanism of downstream needs further exploration.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

【相似文献】