發(fā)布時間：2018-06-23 02:35

  本文選題：CD34~+CD38~-細胞 + 細胞分選��； 參考：《中南大學》2009年博士論文

【摘要】： 第一部分臍帶血來源的CD34~+CD38~-細胞的體外純化和向各系的誘導分化 目的:建立一個可行的從臍帶血中分選出CD34~+CD38~-細胞的方法,并在體外摸索出有效的粒系、紅系和巨核系的分化體系,為后續(xù)實驗提供可靠的細胞標本。 方法:①采用免疫磁珠分選法(magnetic activated cell sorting,MACS)正性分選出CD34~+細胞,再通過二次負性分選選出CD34~+CD38~-的細胞并用流式細胞術(shù)檢測其純度和用臺盼藍拒染法檢測細胞活率。②在體外應用SCF+IL-3+G-CSF或EPO或TPO細胞因子的組合分別誘導CD34~+CD38~-的細胞向粒系、紅系以及巨核系分化。用細胞計數(shù)法繪制其各系細胞的增殖曲線及用流式細胞術(shù)檢測誘導分化的效率。 結(jié)果:①用抗CD34磁珠第一次分選CD34~+細胞后,CD34~+細胞的純度可達95.24±1.03%;第二次分選后,CD34~+/CD38~-細胞的純度為90.23±2.52%。分選前后細胞活力為均可達99%以上。②體外誘導分化14天時,粒系細胞數(shù)增加了1186.67±106.1倍,紅系細胞數(shù)增加了894.67±48.22倍,巨核系細胞數(shù)增加了627±49.65倍。③流式細胞術(shù)檢測的結(jié)果表明,在誘導分化的第14天,CD15~+細胞的比率為91.49%,CD235a~+細胞的比率為95.55%,CD41a~+細胞的比率為86.52%。 結(jié)論:我們建立了有效的MACS分選方法和體外誘導方法,這為我們后續(xù)的實驗提供了可靠的細胞標本來源。 第二部分微小染色質(zhì)免疫共沉淀方法的建立 目的:染色質(zhì)免疫共沉淀(chromatin immunoprecipitation assay,ChIP)是目前研究蛋白質(zhì)與DNA相互作用的強有力的技術(shù)之一。然而目前的ChIP實驗方法最大缺陷要求大量的細胞數(shù),而我們分選的細胞很難達到這個要求。因此我們的目的是建立一種能在少量細胞中進行的ChIP實驗方法,稱為微小染色質(zhì)免疫共沉淀(miniChIP)。 方法:綜合國外相關(guān)文獻,在傳統(tǒng)ChIP方法的基礎上建立miniChIP實驗方法。并通過運用傳統(tǒng)的ChIP實驗方法和在此基礎建立的miniChIP實驗方法對誘導前后的MEL細胞中表達的β珠蛋白基因的不同位點的組蛋白4的乙�；�(acH4)水平進行研究,證實新建的miniChIP實驗法方法的可靠性和特異性。β珠蛋白基因的不同位點包括高敏位點2(HS2)、βmaj基因啟動子區(qū)和Ey基因啟動子區(qū)。 結(jié)果:在未處理的MEL細胞中,用miniChIP實驗方法觀察到HS2和βmaj基因啟動子區(qū)域存在一定的acH4水平,而Ey基因的啟動子區(qū)域則檢測到極低水平的acH4水平。MEL細胞經(jīng)過誘導后,HS2位點和βmaj基因啟動子區(qū)域的acH4水平大大增加,分別增加了2.87和2.26倍。Ey基因的啟動子區(qū)acH4水平幾乎沒有變化。這與我們用傳統(tǒng)ChIP實驗方法觀察到的實驗結(jié)果一致,也與以前別的研究者用傳統(tǒng)ChIP實驗方法得出的結(jié)果相吻合。 結(jié)論:在傳統(tǒng)ChIP實驗方法的基礎上建立了一種可以在少量的細胞中進行的ChIP實驗方法稱miniChIP。并在誘導前后的MEL細胞中對此方法進行了驗證,證實了miniChIP方法的可行性和可靠性。 第三部分造血干細胞髓系分化過程中相關(guān)基因的組蛋白修飾特征 目的:觀察造血分化相關(guān)的轉(zhuǎn)錄因子和基因在CD34~+CD38~-細胞中以及分化后細胞中的組蛋白修飾特征,探討染色質(zhì)構(gòu)象在造血干細胞多潛能性特性維持和系特異分化中的可能作用。 方法:①采用qRT-PCR的方法檢測了造血分化相關(guān)轉(zhuǎn)錄因子和基因在不同類型細胞中的mRNA表達水平。不同類型細胞包括CD34~+CD38~-細胞和誘導分化后的CD15~+細胞、CD235a~+細胞、CD41a~+細胞。造血分化相關(guān)轉(zhuǎn)錄因子和基因包括早期造血相關(guān)轉(zhuǎn)錄因子HOXA9;粒系分化相關(guān)轉(zhuǎn)錄因子PU.1,粒系特異基因MPO、CD11b;紅系巨核系分化相關(guān)轉(zhuǎn)錄因子GATA-1、紅系特異基因EPOR和巨核系特異基因CD41a:淋系分化相關(guān)轉(zhuǎn)錄因子GATA-3、PAX5,淋系特異基因CD3、CD79a。②我們用miniChIP-qPCR實驗方法在不同類型細胞中觀察并比較了造血分化相關(guān)轉(zhuǎn)錄因子和基因的啟動子區(qū)的6種組蛋白修飾的變化。這6種不同的組蛋白修飾分別為活化性組蛋白修飾包括組蛋白3的乙�；�(acH3)、組蛋白4的乙�；�(acH4)、組蛋白3第4位賴氨酸的二甲基化(H3K4me2)、組蛋白3第4位賴氨酸的三甲基化(H3K4me3)和抑制性組蛋白修飾包括組蛋白3第9位賴氨酸的三甲基化(H3K9me3)、組蛋白3第27位賴氨酸的三甲基化(H3K27me3)。 結(jié)果:①在CD34~+CD38~-細胞中各系分化相關(guān)轉(zhuǎn)錄因子和基因存在低水平的mRNA表達或不表達,而與早期造血相關(guān)的轉(zhuǎn)錄因子HOXA9顯示高表達。當CD34~+CD38~-細胞向粒系特異分化后,粒系相關(guān)基因PU.1、MPO、CD11b表達明顯增加;CD34~+CD38~-細胞向紅系特異分化后紅系相關(guān)基因GATA-1、EPOR表達明顯增加;CD34~+CD38~-細胞向巨核系特異分化后巨核系相關(guān)基因GATA-1、CD41a表達顯著增加。同時CD34~+CD38~-細胞系特異分化后非系相關(guān)基因未能檢測到,以及HOXA9基因的表達顯著下降;②在CD34~+CD38~-細胞中各系分化相關(guān)轉(zhuǎn)錄因子和基因都有一定水平的acH4的修飾和稍低水平的acH3的修飾以及高水平的H3K4me2的修飾,但H3K4me3修飾水平很低。③在CD34~+CD38~-細胞中各系分化相關(guān)轉(zhuǎn)錄因子和基因都具有低水平H3K9me3和H3K27me3的修飾;④隨著CD34~+CD38~-細胞系特異分化后,該系特異基因的acH3、acH4和H3K4me2水平略為增加,但H3K4me3的水平明顯增加,同時H3K9me3和H3K27me3修飾仍維持在低水平。非系特異基因的acH3和acH4修飾水平降低,H3K4me3修飾仍維持在低水平,同時有H3K9me3或/和H3K27me3水平的顯著增加;⑤在CD34~+CD38~-細胞向終末細胞分化后,與早期造血相關(guān)的轉(zhuǎn)錄因子HOXA9的啟動子區(qū)上活化性組蛋白修飾包括acH3、acH4、H3K4me2、H3K4me3顯著降低,同時抑制性組蛋白修飾包括H3K9me3和H3K27me3明顯增加。結(jié)論:①在富含造血干細胞的CD34+CD38-細胞中各系分化相關(guān)基因具有一定水平的H3、H4的乙�；揎�、高水平的H3K4me2修飾以及低水平的H3K4me3修飾和低水平的組蛋白抑制性修飾。這些基因表現(xiàn)為低水平表達或者檢測不到表達。②當CD34+CD38-細胞系特異分化后,系相關(guān)基因的acH3、acH4、H3K4me2修飾水平維持不變或者略微增加,但H3K4me3修飾水平明顯增加,同時保持低水平的抑制性組蛋白修飾,基因表現(xiàn)為高轉(zhuǎn)錄狀態(tài),而非系相關(guān)基因的啟動子區(qū)上acH3、acH4的修飾水平降低,H3K4me3水平任然維持在低水平狀態(tài),但富集了高水平的抑制性的組蛋白修飾標志,基因表現(xiàn)為沉默。③當CD34+CD38-細胞向終末細胞分化后,與早期造血相關(guān)的基因HOXA9的啟動子區(qū)上H3K4me3的修飾水平降低,但抑制性組蛋白修飾標志顯著增加,表現(xiàn)為基因表達的沉默。
[Abstract]:The first part is the purification and differentiation of CD34~+CD38~- cells from cord blood in vitro.
Objective: to establish a feasible method for the selection of CD34~+CD38~- cells from umbilical cord blood, and to find out the effective granulocyte, red and megakaryocyte differentiation system in vitro, and provide reliable cell specimens for subsequent experiments.
Methods: (1) CD34~+ cells were selected by magnetic activated cell sorting (MACS), CD34~+CD38~- cells were selected by two negative sorting, and the purity of the cells were detected by flow cytometry and the cell viability was detected by trypan blue staining. (2) the group of SCF+IL-3+G-CSF or EPO or TPO cytokines was used in vitro. The cells of CD34~+CD38~- were induced to differentiate into granulocyte, red and megakaryocytes respectively. The cell proliferation curves were plotted by cell counting and the efficiency of differentiation by flow cytometry was used to detect the differentiation.
Results: after the first separation of CD34~+ cells with anti CD34 magnetic beads, the purity of CD34~+ cells could reach 95.24 + 1.03%. After the second separation, the cell viability of the CD34~+/CD38~- cells was more than 99% before and after the separation of 90.23 + 2.52%.. (2) the number of fine cell lines increased by 1186.67 + 106.1 times, and the number of erythroid cells increased. The number of megakaryocyte cells increased by 627 + 49.65 times by 894.67 + 48.22 times. The results of flow cytometry showed that the ratio of CD15~+ cells was 91.49%, the ratio of CD235a~+ cells was 95.55% and the ratio of CD41a~+ cells was 86.52%. in the fourteenth days of induced differentiation.
Conclusion: we have established an effective MACS sorting method and an in vitro induction method, which provides reliable source of cell specimens for our subsequent experiments.
The second part is the establishment of micro chromatin immunoprecipitation method.
Objective: chromatin immunoprecipitation assay (ChIP) is one of the powerful techniques to study the interaction of protein and DNA. However, the maximum number of cells in the current ChIP test method requires a large number of cells, and the cells of our separation are difficult to reach this requirement. Therefore, our aim is to establish a kind of energy. The ChIP method in a few cells is called small chromatin immunoprecipitation (miniChIP).
Methods: the miniChIP experimental method was established on the basis of the traditional ChIP method, and the level of histone 4 (acH4) in the different loci of the beta globin gene expressed in MEL cells before and after induction was studied by using the traditional ChIP experiment method and the miniChIP experiment on the basis of the traditional method. The reliability and specificity of the new miniChIP method, including the Gao Min locus 2 (HS2), the promoter region of the beta maj gene and the promoter region of the Ey gene, are found in the new method.
Results: in the untreated MEL cells, a certain acH4 level existed in the promoter region of the HS2 and beta maj genes with the miniChIP method, while the Ey gene promoter region detected that the acH4 level.MEL cells of the extremely low level of.MEL were induced, and the acH4 level of the HS2 and beta maj gene promoter regions was greatly increased by 2.8, respectively. The acH4 level in the promoter region of the 7 and 2.26 times.Ey gene was almost unchanged. This was in agreement with the experimental results observed by the traditional ChIP experiment and the results obtained by other researchers in the traditional ChIP experiment.
Conclusion: Based on the traditional ChIP experimental method, a ChIP test method, called miniChIP., which can be used in a small number of cells, was established and verified in the MEL cells before and after induction. The feasibility and reliability of the miniChIP method were confirmed.
The third part is about the histone modification characteristics of hematopoietic stem cells during myeloid differentiation.
Objective: To observe the histone modification characteristics of the transcription factors and genes related to hematopoietic differentiation in CD34~+CD38~- cells and in the differentiated cells, and to explore the possible role of chromatin conformation in the maintenance and differentiation of hematopoietic stem cells in multipotential and lineage differentiation.
Methods: qRT-PCR was used to detect the mRNA expression level of hematopoietic differentiation related transcription factors and genes in different types of cells. Different types of cells include CD34~+CD38~- cells and induced CD15~+ cells, CD235a~+ cells, CD41a~+ cells. Hematopoietic differentiation related transcription factors and genes include early hematopoietic transcription related transcription. Factor HOXA9, granulocyte differentiation related transcription factor PU.1, granulocyte specific gene MPO, CD11b, erythroid megakaryocyte differentiation related transcription factor GATA-1, erythroid specific gene EPOR and megakaryocytic specific gene CD41a: lymphoid differentiation related transcription factor GATA-3, PAX5, lymphoid specific gene CD3, CD79a. 2, we use miniChIP-qPCR experimental methods in different types. The changes in the 6 histone modifications of the promoter region of the hematopoietic differentiation related transcription factors and genes were observed and compared. The 6 different histone modifications were activated histone modification including histone 3 acetylation (acH3), histone 4 acetylation (acH4), histone 3 fourth lysine two methylation (H3K4me2), histone 3 The trimethylation (H3K4me3) and the inhibitory histone modification of fourth lysine include trimethylation (H3K9me3) of histone 3 ninth bits lysine, and histone 3 twenty-seventh methylation of lysine (H3K27me3).
Results: (1) there was a low level of mRNA expression or non expression in the differentiation related transcription factors and genes in the CD34~+CD38~- cells. The expression of the transcription factor HOXA9 related to the early hematopoiesis was highly expressed. When the CD34~+CD38~- cells differentiated into the granulocyte specific differentiation, the expression of PU.1, MPO, CD11b in the granulocyte related genes increased obviously; CD34~+CD38~- cells turned to the red system. After specific differentiation, the expression of GATA-1 and EPOR increased significantly, and the expression of megakaryocyte related genes GATA-1, CD41a expression was significantly increased after the specific differentiation of CD34~+CD38~- cells to megakaryocyte, and the non lineage related genes were not detected and the expression of HOXA9 gene decreased significantly after the specific differentiation of CD34~+CD38~- cell lines; and (2) in CD34~+CD38~- cells The transcriptional factors and genes of differentiation in each line have a certain level of acH4 modification and slightly low level of acH3 modification and high level of H3K4me2 modification, but the level of H3K4me3 modification is very low. (3) the transcription factors and genes of each lineage differentiation in CD34~+CD38~- cells are modified with low level H3K9me3 and H3K27me3; (4) along with CD34~+C After the specific differentiation of D38~- cell lines, the level of acH3, acH4 and H3K4me2 of the specific genes increased slightly, but the level of H3K4me3 increased significantly, while H3K9me3 and H3K27me3 modification remained at a low level. The level of acH3 and acH4 modification of non specific genes decreased, and H3K4me3 modification remained at a low level, while H3K9me3 or / and H3K27me3 levels were at the same time. After the differentiation of CD34~+CD38~- cells to terminal cells, the activation histone modification on the promoter region of the early hematopoiesis related transcription factor HOXA9, including acH3, acH4, H3K4me2, H3K4me3, was significantly reduced, while the inhibitory histone modification including H3K9me3 and H3K27me3 increased significantly. Conclusion: (1) CD34+C rich in hematopoietic stem cells The genes related to differentiation of D38- cells have a certain level of H3, acetylation of H4, high level of H3K4me2 modification, low level H3K4me3 modification and low level histone inhibition. These genes are expressed at low level or not detected. 2. After the specific differentiation of CD34+ CD38- cell lines, the a of the related genes The level of cH3, acH4, H3K4me2 modification remained unchanged or slightly increased, but the level of H3K4me3 modification increased significantly, while the low level of inhibitory histone modification was maintained, and the gene expression was high transcriptional, while the acH3, acH4 modification level of the non line related genes was lower and the H3K4me3 level remained at a low level, but the enrichment of the H3K4me3 level was at a low level. After the differentiation of CD34+CD38- cells to terminal cells, the level of H3K4me3 modification on the promoter region of the early hematopoietic gene HOXA9 decreased, but the inhibitory histone modification markers increased significantly, showing the silence of gene expression.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329

【共引文獻】

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2 任立晨;發(fā)育階段相關(guān)基因的結(jié)構(gòu)特征，，基因組特征和起源進化[D];上海交通大學;2007年

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