小鼠MYSM1基因稳定敲除的间充质干细胞系的建立及其免疫调节作用

发布时间：2018-01-19 19:40

本文关键词： 组蛋白H_2A去泛素化酶MYSM1 间充质干细胞 CRISPR-Cas9 基因敲除　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：恶性肿瘤一直尚未攻克,血液系统恶性疾病也是如此。但是现在造血干细胞移植(hematopoietic stem cell transplantation,HSCT)为我们治疗恶性血液病带来了希望,经过数年的发展,HSCT在临床的应用愈来愈广泛,移植技术也日渐成熟,因为移植技术的进步,越来越多的患者能够获得生存的机会,但是HSCT后也具有不少的并发症,而移植物抗宿主病(GVHD)就是其中之一,在临床上,移植术后的病人或多或少都会出现GVHD,轻者影响病人的生活质量,重者引起移植失败甚至死亡。所以很多研究致力于寻找更好的解决这个问题的方法。文献中报道的急性GVHD(acute-GVHD,aGVHD)和慢性GVHD(chronic-GVHD,cGVHD)的发病机制都表明其是一个免疫相关的疾病。因此临床上治疗GVHD主要是激素及其他免疫抑制剂。但是,这些药物对很多病人无效,而且有较多的副作用如感染等,MSCs治疗aGVHD是目前临床中相对具有前景的一种细胞疗法,并且其副作用很少,因此探究MSCs的免疫调节能力具有较重要的临床意义。间充质干细胞(MSCs)具有多向分化能力,因此较多的应用于组织损伤的修复,但它具有的免疫抑制也一直受到广大研究者的青睐,因此有些学者证明了其在临床应用于免疫相关疾病的价值。但是目前对于MSCs具体的调节机制尚不清楚,其在临床治疗的不稳定性也使得它的应用受到了限制。为了更好的探究其免疫调节的机制,为以后的临床研究提供扎实的理论基础,我们是否可以从表观遗传学去探讨MSCs的调节机制呢?基因表达的过程相当复杂,而且受多种因素的调控,因此其调控的过程也相对复杂。核小体主要由两部分组成,一个组蛋白八聚体,一段长约147bp的DNA,并且可发生如甲基化、去泛素化等多种修饰。那么组蛋白修饰影响基因表达的方式是什么呢?首先,它能改变组蛋白与DNA结合,使染色质状态发生改变,也可作用于启动子,使其难以与转录因子结合,从而影响基因的表达。组蛋白H_2A是一种重要的组蛋白,它的去泛素化能够有效地调节基因的表达。因此本研究旨在在小鼠上探讨组蛋白的一种去泛素化酶MYSM1基因对MSCs的免疫学特性的影响,为了达到这个研究目的,我们通过采用基因编辑的技术将小鼠间充质干细胞系中的MYSM1基因敲除,然后将培养基因修饰后MSCs的上清与小鼠脾脏淋巴细胞共培养,观察其对T淋巴细胞增殖的影响并检测各种T淋巴细胞亚群所分泌因子的表达情况。那么如何敲除小鼠间充质干细胞系中的MYSM1基因呢?我们拟采用CRISPR-Cas9技术将MYSM1基因从小鼠MSCs中敲除。我们首先利用CRISPR/Cas9基因编辑技术将MYSM1基因整合到CRISPR中,然后用相应的CRISPR RNAs来指导MYSM1的降解,,我们此技术借鉴于《Science》杂志中基于CRISPR-Cas9技术在细胞系中进行基因敲除的新方法,并交给公司Cas9慢病毒及带有能够特异剪切MYSM1基因的sgRNA序列的病毒,然后完成接下来的工作。该技术优点在于其能够在基因组的特异位点上进行敲除,而且敲除效率较高。同时为了筛选病毒转染的细胞,我们在病毒中还加入了嘌呤霉素抗性基因。在公司完成病毒的生产后,我们开始接下来的工作。我们的研究主要分为2部分:首先,利用带有绿色荧光蛋白(GFP)标记的Cas9慢病毒感染小鼠间充质干细胞系C3H10T 1/2,用CRISPR-Cas9技术来达到剪切小鼠MYSM1基因的目的,并利用流式细胞术(FCM)观察细胞表面的GFP表达情况来检测其转染效率。其次,在成功转染后,收集细胞内的RNA及蛋白质,通过定量PCR(qPCR)及蛋白印记的方法分别从MYSM1基因的转录和翻译两个层面来检测MYSM1基因是否被成功剪切。鉴定证明成功敲除MYSM1基因后,先通过FCM验证其对MSCs本身表面特异性标记表达的变化,然后通过与收取细胞上清与小鼠脾脏淋巴细胞共培养对其进行免疫调节功能的验证。通过实验可以得出以下结果:1.经流式检测细胞GFP的表达情况病毒转染的效率高达99%;通过qPCR及蛋白印记法发现MYSM1基因已经在小鼠MSCs中敲除,获得MYSM1基因稳定敲除的MSCs;2.通过抗体标记流式检测发现敲除MYSM1基因的MSCs表面特异标记的表达与MSCs无明显差别,且通过细胞上清与小鼠脾脏淋巴细胞共培养后发现MYSM1基因稳定敲除的MSCs对各亚群的T淋巴细胞具有增强的抑制作用。因此,可以得出结论:我们利用病毒转染技术及CRISPR-Cas9技术成功MYSM1基因从小鼠间充质干细胞系中敲除,并且初步证明其在体外能够发挥更强的免疫抑制作用。
[Abstract]:Malignant tumor has not been overcome, so is the malignant hematological diseases. But now hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, HSCT) brings us hope for the treatment of malignant hematological diseases, after several years of development, HSCT is widely used in clinic and transplantation technology has become more mature, because transplant technology progress more and more patients can get the chance of survival, but HSCT also has many complications, and graft-versus-host disease (GVHD) is one of them, in clinical transplantation, patients will appear more or less GVHD, affect the patient's quality of life light, or cause of graft failure and even death. So many the research focused on methods to find a better solution to this problem. The acute GVHD reported in the literature (acute-GVHD, aGVHD) and chronic GVHD (chronic-GVHD, cGVHD) in the pathogenesis of all That is an immune related diseases. Therefore, the clinical treatment of GVHD is the main hormone and other immunosuppressive drugs. However, these drugs are ineffective for many patients, and there are many side effects such as infection, MSCs treatment of aGVHD is a kind of cell therapy in the current clinical relatively promising, and fewer side effects that has important clinical significance of immune regulation ability of MSCs. So the research of mesenchymal stem cells (MSCs) have the ability of multi-directional differentiation, so repair is mostly applied to tissue damage, but it has immune suppression also has received wide interest among researchers, so some scholars have proved that in its clinical application in immune related diseases. But the MSCs value of the specific adjustment mechanism is not clear, in the clinical treatment of instability also makes its application is restricted. In order to explore the immune regulation The mechanism, provide a solid theoretical foundation for future clinical research, we can from the epigenetic regulation of MSCs to explore the mechanism? The process of gene expression is very complex, and regulated by many factors, so the regulation process is relatively complex. Small nuclear body is mainly composed of two parts, a histone dimer eight, a period of about 147bp DNA, and can occur such as methylation, ubiquitination and other modifications to. So what are the effects of histone modification in gene expression way? First of all, it can change the group with DNA binding protein, the chromatin state changed, also can be applied to the promoter, making it difficult to combine with the transcription factor, which affects the expression of genes. Histone H_2A is an important group of proteins, its deubiquitinating can effectively regulate gene expression. Therefore, this study aims to explore a group of proteins in mice Effect of immunological characteristics of a deubiquitinating enzyme MYSM1 gene on MSCs, in order to achieve this objective, we adopted gene editing technology of murine mesenchymal stem cells in MYSM1 gene knockout, then the gene modified MSCs culture supernatant and mouse spleen lymphocytes were cultured to observe the effect of on T lymphocyte proliferation and to detect T lymphocyte subsets secreted factor expression. So how to knockout mouse mesenchymal stem cell line MYSM1 gene in? We intend to use the CRISPR-Cas9 technology of MYSM1 gene from mouse MSCs knockout. We first use CRISPR/Cas9 gene editing technology MYSM1 gene was integrated into CRISPR in the degradation, and then use the corresponding CRISPR to RNAs MYSM1, the US technology reference in the Journal based on the CRISPR-Cas9 technology in the cell lines of new gene knockout Method, and give the company Cas9 lentivirus and with specific sgRNA sequence of MYSM1 gene of shear of the virus, and then complete the following work. This technology has the advantages of enabling knockout in specific loci of the genome, and the knockdown efficiency is high. At the same time in order to obtain the virus transfected cells, we also added puromycin hormone resistance gene in the virus. The virus in the company to complete the production after we start the next work. Our research is mainly divided into 2 parts: first, the use of a green fluorescent protein (GFP) labeled Cas9 lentivirus infected mouse mesenchymal stem cell line C3H10T 1/2, using CRISPR-Cas9 technology to achieve the shear of mouse MYSM1 the purpose of gene, and the use of flow cytometry (FCM) to observe cell surface expression of GFP to detect the transfection efficiency. Secondly, the success after transfection, cells were collected in RNA and protein, the The amount of PCR (qPCR) method and Western blot respectively from two levels of transcription and translation of MYSM1 gene to detect whether MYSM1 gene was successfully identified. Shear successfully knocked out MYSM1 gene, the change of MSCs itself to verify the expression of specific marker expression by FCM, and then through the co culture supernatant and charge with the mouse spleen lymphocytes to verify immune function on it. Through the experiment we can draw the following results: 1. the expression of transfection was detected by flow cytometry GFP efficiency is as high as 99%; by qPCR and Western blot showed that MYSM1 gene in MSCs knockout mice have been obtained, stable MYSM1 gene knockout MSCs by 2.; antibody labeling flow cytometry found that knockdown of MSCs expression and MSCs specific surface markers of MYSM1 gene had no obvious difference, and the co culture supernatant and spleen lymphocytes of mice after MYSM 1 stable gene knockout MSCs has inhibitory effect on the enhancement of T lymphocyte subsets. Therefore, we can draw the conclusion: we use transfection technique and CRISPR-Cas9 technology successfully MYSM1 gene from mouse mesenchymal stem cells knockout, and preliminarily prove that it can inhibit enough to play a stronger immunity in vitro.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7

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