本文關鍵詞： 組蛋白H_2A去泛素化酶MYSM1 間充質(zhì)干細胞 CRISPR-Cas9 基因敲除　出處：《中國人民解放軍軍事醫(yī)學科學院》2017年碩士論文　論文類型：學位論文

【摘要】：惡性腫瘤一直尚未攻克,血液系統(tǒng)惡性疾病也是如此。但是現(xiàn)在造血干細胞移植(hematopoietic stem cell transplantation,HSCT)為我們治療惡性血液病帶來了希望,經(jīng)過數(shù)年的發(fā)展,HSCT在臨床的應用愈來愈廣泛,移植技術也日漸成熟,因為移植技術的進步,越來越多的患者能夠獲得生存的機會,但是HSCT后也具有不少的并發(fā)癥,而移植物抗宿主病(GVHD)就是其中之一,在臨床上,移植術后的病人或多或少都會出現(xiàn)GVHD,輕者影響病人的生活質(zhì)量,重者引起移植失敗甚至死亡。所以很多研究致力于尋找更好的解決這個問題的方法。文獻中報道的急性GVHD(acute-GVHD,aGVHD)和慢性GVHD(chronic-GVHD,cGVHD)的發(fā)病機制都表明其是一個免疫相關的疾病。因此臨床上治療GVHD主要是激素及其他免疫抑制劑。但是,這些藥物對很多病人無效,而且有較多的副作用如感染等,MSCs治療aGVHD是目前臨床中相對具有前景的一種細胞療法,并且其副作用很少,因此探究MSCs的免疫調(diào)節(jié)能力具有較重要的臨床意義。間充質(zhì)干細胞(MSCs)具有多向分化能力,因此較多的應用于組織損傷的修復,但它具有的免疫抑制也一直受到廣大研究者的青睞,因此有些學者證明了其在臨床應用于免疫相關疾病的價值。但是目前對于MSCs具體的調(diào)節(jié)機制尚不清楚,其在臨床治療的不穩(wěn)定性也使得它的應用受到了限制。為了更好的探究其免疫調(diào)節(jié)的機制,為以后的臨床研究提供扎實的理論基礎,我們是否可以從表觀遺傳學去探討MSCs的調(diào)節(jié)機制呢?基因表達的過程相當復雜,而且受多種因素的調(diào)控,因此其調(diào)控的過程也相對復雜。核小體主要由兩部分組成,一個組蛋白八聚體,一段長約147bp的DNA,并且可發(fā)生如甲基化、去泛素化等多種修飾。那么組蛋白修飾影響基因表達的方式是什么呢?首先,它能改變組蛋白與DNA結(jié)合,使染色質(zhì)狀態(tài)發(fā)生改變,也可作用于啟動子,使其難以與轉(zhuǎn)錄因子結(jié)合,從而影響基因的表達。組蛋白H_2A是一種重要的組蛋白,它的去泛素化能夠有效地調(diào)節(jié)基因的表達。因此本研究旨在在小鼠上探討組蛋白的一種去泛素化酶MYSM1基因?qū)SCs的免疫學特性的影響,為了達到這個研究目的,我們通過采用基因編輯的技術將小鼠間充質(zhì)干細胞系中的MYSM1基因敲除,然后將培養(yǎng)基因修飾后MSCs的上清與小鼠脾臟淋巴細胞共培養(yǎng),觀察其對T淋巴細胞增殖的影響并檢測各種T淋巴細胞亞群所分泌因子的表達情況。那么如何敲除小鼠間充質(zhì)干細胞系中的MYSM1基因呢?我們擬采用CRISPR-Cas9技術將MYSM1基因從小鼠MSCs中敲除。我們首先利用CRISPR/Cas9基因編輯技術將MYSM1基因整合到CRISPR中,然后用相應的CRISPR RNAs來指導MYSM1的降解,,我們此技術借鑒于《Science》雜志中基于CRISPR-Cas9技術在細胞系中進行基因敲除的新方法,并交給公司Cas9慢病毒及帶有能夠特異剪切MYSM1基因的sgRNA序列的病毒,然后完成接下來的工作。該技術優(yōu)點在于其能夠在基因組的特異位點上進行敲除,而且敲除效率較高。同時為了篩選病毒轉(zhuǎn)染的細胞,我們在病毒中還加入了嘌呤霉素抗性基因。在公司完成病毒的生產(chǎn)后,我們開始接下來的工作。我們的研究主要分為2部分:首先,利用帶有綠色熒光蛋白(GFP)標記的Cas9慢病毒感染小鼠間充質(zhì)干細胞系C3H10T 1/2,用CRISPR-Cas9技術來達到剪切小鼠MYSM1基因的目的,并利用流式細胞術(FCM)觀察細胞表面的GFP表達情況來檢測其轉(zhuǎn)染效率。其次,在成功轉(zhuǎn)染后,收集細胞內(nèi)的RNA及蛋白質(zhì),通過定量PCR(qPCR)及蛋白印記的方法分別從MYSM1基因的轉(zhuǎn)錄和翻譯兩個層面來檢測MYSM1基因是否被成功剪切。鑒定證明成功敲除MYSM1基因后,先通過FCM驗證其對MSCs本身表面特異性標記表達的變化,然后通過與收取細胞上清與小鼠脾臟淋巴細胞共培養(yǎng)對其進行免疫調(diào)節(jié)功能的驗證。通過實驗可以得出以下結(jié)果:1.經(jīng)流式檢測細胞GFP的表達情況病毒轉(zhuǎn)染的效率高達99%;通過qPCR及蛋白印記法發(fā)現(xiàn)MYSM1基因已經(jīng)在小鼠MSCs中敲除,獲得MYSM1基因穩(wěn)定敲除的MSCs;2.通過抗體標記流式檢測發(fā)現(xiàn)敲除MYSM1基因的MSCs表面特異標記的表達與MSCs無明顯差別,且通過細胞上清與小鼠脾臟淋巴細胞共培養(yǎng)后發(fā)現(xiàn)MYSM1基因穩(wěn)定敲除的MSCs對各亞群的T淋巴細胞具有增強的抑制作用。因此,可以得出結(jié)論:我們利用病毒轉(zhuǎn)染技術及CRISPR-Cas9技術成功MYSM1基因從小鼠間充質(zhì)干細胞系中敲除,并且初步證明其在體外能夠發(fā)揮更強的免疫抑制作用。
[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.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R457.7

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本文編號：1445310

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