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  本文關(guān)鍵詞：THAP11在造血細(xì)胞分化中的作用研究　出處：《天津大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： THAP11 紅系分化 巨核系分化 K562細(xì)胞 TF-1細(xì)胞 慢病毒

【摘要】：THAP11是THAP蛋白家族的最新成員，前期研究表明THAP11是調(diào)控細(xì)胞生長、胚胎形成和ES細(xì)胞多能性的一個轉(zhuǎn)錄因子，THAP11通過下調(diào)c-Myc抑制細(xì)胞增殖。c-Myc是調(diào)控紅系巨核系分化的關(guān)鍵轉(zhuǎn)錄因子，并且表達(dá)譜數(shù)據(jù)顯示THAP11在造血干細(xì)胞、多能祖細(xì)胞和人臍帶血CD34+CD38-細(xì)胞中高表達(dá)，提示THAP11可能參與造血細(xì)胞分化，但目前對于THAP11在造血分化中的作用尚未報道。 本論文對THAP11對造血細(xì)胞分化的影響進(jìn)行了深入研究。首先，，通過檢測THAP11在人臍帶血CD34+細(xì)胞向紅系和巨核系分化過程中的表達(dá)變化，發(fā)現(xiàn)THAP11在向紅系分化中表達(dá)下調(diào)，而在巨核分化中表達(dá)上調(diào)，提示THAP11參與紅系/巨核系分化的調(diào)控。接著我們構(gòu)建了THAP11過表達(dá)及RNAi的第三代慢病毒載體，通過病毒包裝獲得慢病毒顆粒，建立了過表達(dá)和敲低THAP11的K562和TF-1細(xì)胞穩(wěn)定株。采用hemin誘導(dǎo)K562細(xì)胞向紅系分化模型，發(fā)現(xiàn)THAP11過表達(dá)可抑制紅系分化，表現(xiàn)為抑制GPA和HBA的上調(diào)，聯(lián)苯胺染色陽性細(xì)胞比例降低，而敲低THAP11則促進(jìn)紅系分化。利用EPO誘導(dǎo)TF-1細(xì)胞向紅系分化的模型，得到相同的結(jié)果。采用PMA誘導(dǎo)K562細(xì)胞向巨核系分化，發(fā)現(xiàn)THAP11過表達(dá)可促進(jìn)巨核系標(biāo)志物CD61的表達(dá)和多倍體形成，與之相反，敲低THAP11則抑制了巨核分化。初步研究表明THAP11可上調(diào)GATA-2和Fli1基因，下調(diào)c Myc和c-Myb基因的表達(dá)，提示THAP11對造血細(xì)胞分化的影響可能是通過調(diào)控多種基因表達(dá)來進(jìn)行的。另外，過表達(dá)THAP11明顯抑制CD34+細(xì)胞的增殖能力，而敲低THAP11促進(jìn)CD34+細(xì)胞的生長，提示THAP11可能參與了CD34+細(xì)胞的生長與增殖。 以上結(jié)果表明THAP11是一種新的造血細(xì)胞分化調(diào)控分子，可抑制紅系分化，促進(jìn)巨核系分化。
[Abstract]:THAP11 is the newest member of THAP protein family. Previous studies have shown that THAP11 is a transcription factor regulating cell growth, embryogenesis and es cell pluripotency. THAP11 inhibits cell proliferation by down-regulating c-Myc. C-Myc is the key transcription factor to regulate the differentiation of erythroid megakaryocytes and the expression profile data show that THAP11 plays an important role in hematopoietic stem cells. The overexpression of CD34 CD38- cells in pluripotent progenitor cells and human umbilical cord blood suggests that THAP11 may be involved in hematopoietic cell differentiation. However, the role of THAP11 in hematopoietic differentiation has not been reported. In this paper, the effect of THAP11 on hematopoietic cell differentiation was studied. By detecting the expression of THAP11 in the differentiation of human umbilical cord blood CD34 cells into erythroid and megakaryocyte, it was found that the expression of THAP11 was down-regulated in the differentiation of human umbilical cord blood CD34 cells into erythroid and megakaryocyte. The upregulation in megakaryocyte differentiation suggested that THAP11 was involved in the regulation of erythroid / megakaryocyte differentiation. Then we constructed the third generation lentivirus vector of THAP11 overexpression and RNAi. Lentivirus particles were obtained by virus packaging and stable K562 and TF-1 cell lines with overexpression and knockdown of THAP11 were established. K562 cells were induced into erythroid differentiation model by hemin. It was found that the overexpression of THAP11 could inhibit the differentiation of erythroid cells, which could inhibit the upregulation of GPA and HBA, and decrease the proportion of benzidine staining positive cells. K562 cells were induced to differentiate into megakaryocytes by PMA, and the same results were obtained by using the model of EPO inducing TF-1 cells to differentiate into erythrocytes. It was found that overexpression of THAP11 could promote the expression of megakaryotic marker CD61 and the formation of polyploid. Knockdown of THAP11 inhibited megakaryocyte differentiation. Preliminary studies showed that THAP11 could up-regulate the expression of GATA-2 and Fli1 genes and down-regulate the expression of c Myc and c-Myb genes. The results suggest that the effect of THAP11 on hematopoietic cell differentiation may be mediated by regulating the expression of multiple genes. In addition, overexpression of THAP11 can significantly inhibit the proliferation of CD34 cells. Knockdown of THAP11 promotes the growth of CD34 cells, suggesting that THAP11 may be involved in the growth and proliferation of CD34 cells. These results suggest that THAP11 is a new hematopoietic cell differentiation regulator, which can inhibit erythroid differentiation and promote megakaryocyte differentiation.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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