【摘要】： 中文摘要 背景與目的高遷移率族蛋白B1(high mobility group box 1,HMGB1)是一種DNA結(jié)合蛋白,組織損傷后釋放至胞外,招募多種類型干細(xì)胞,參與受損組織修復(fù)。造血干細(xì)胞移植(haematopoietic stem cell transplantation ,HSCT)的預(yù)處理必然導(dǎo)致骨髓組織受損,釋放HMGB1,推測(cè)其可能參與造血重建。本實(shí)驗(yàn)將首次觀察預(yù)處理后骨髓基質(zhì)細(xì)胞HMGB1的釋放、HMGB1對(duì)人臍血造血干細(xì)胞歸巢和增殖分化的影響,并對(duì)其機(jī)制予以探討;以期為促進(jìn)HSCT術(shù)后造血重建尋找新靶點(diǎn)。方法體外培養(yǎng)人骨髓基質(zhì)細(xì)胞,加速器照射后,利用ELISA方法檢測(cè)培養(yǎng)上清中HMGB1含量的變化。利用MACS系統(tǒng)分選人臍血CD34+細(xì)胞,HMGB1與其共培養(yǎng)6天,通過流式細(xì)胞術(shù)檢測(cè)臍血CD34+細(xì)胞分化指標(biāo)(CD13、CD14、CD11c、CD41,CD71)的變化。利用克隆形成實(shí)驗(yàn)觀察HMGB1與對(duì)造血干細(xì)胞增殖分化的影響。應(yīng)用transwell小室趨化裝置觀察HMGB1對(duì)人臍血CD34+細(xì)胞的遷移活性的影響。流式細(xì)胞術(shù)檢測(cè)HMGB1的受體RAGE、TLR2和TLR4在人臍血CD34+細(xì)胞上的表達(dá)。利用抗-RAGE抗體、抗- TLR2抗體和抗- TLR4抗體阻斷RAGE、TLR2和TLR4,重復(fù)趨化實(shí)驗(yàn),體外觀察RAGE、TLR2和TLR4是否參與HMGB1可能誘導(dǎo)的人臍血CD34+細(xì)胞遷移。結(jié)果1.經(jīng)X線照射后骨髓基質(zhì)細(xì)胞培養(yǎng)上清中HMGB1含量增高:人骨髓基質(zhì)細(xì)胞,加速器照射(12Gy)后,培養(yǎng)上清中HMGB1含量為(4.3±0.9)ng/ml,較不照射組HMGB1含量(0.4±0.2)ng/ml升高(p0.01)。2.HMGB1表面受體檢測(cè)人臍血CD34+細(xì)胞表達(dá)HMGB1的受體RAGE(43.1±7.2)%、TLR2(36.1±6.6)%和TLR4(23.1±5.2)%。3.HMGB1促進(jìn)CD34+細(xì)胞向紅系和粒單系增殖分化:臍血分選富集的純度為(98.25±0.93)%,與HMGB1體外液體共培養(yǎng)6天后,和對(duì)照組比較,紅系(CD71)和粒單系(CD13、CD14、CD11c)標(biāo)記的表達(dá)明顯增強(qiáng),分別為CD13(18.4±3.8 vs 32.6±5.9)%、CD14(12.6±2.7 vs 25.4±4.4)%、CD11c(9.8±2.1 vs 20.3±3.9)%、、CD71(26.6±4.6 vs 47.1±7.4)%,而巨核系標(biāo)記CD41(1.1±0.4% vs 1.3±0.5%)的表達(dá)無明顯變化。同樣,克隆形成實(shí)驗(yàn)示共培養(yǎng)14天后,紅系集落、粒-巨噬細(xì)胞集落和總集落的生成較對(duì)照組明顯增多(p0.05)。4.HMGB1誘導(dǎo)臍血CD34+細(xì)胞的遷移:HMGB1在一定的濃度范圍內(nèi)隨濃度遞增其對(duì)人臍血CD34+細(xì)胞的遷移作用逐漸增強(qiáng),當(dāng)HMGB1濃度為100 ng/ml時(shí),趨化活性最強(qiáng),趨化指數(shù)為3.96±0.46,與對(duì)照組比較差異顯著(p0.01),抗-RAGE抗體可部分抑制HMGB1對(duì)臍血CD34+細(xì)胞的遷移作用。 結(jié)論照射后骨髓基質(zhì)細(xì)胞培養(yǎng)上清HMGB1含量明顯升高;HMGB1可促進(jìn)人臍血CD34+細(xì)胞向粒單核及紅系分化,并促進(jìn)紅系集落和粒-巨噬細(xì)胞集落的生成;一定濃度的HMGB1可加強(qiáng)臍血CD34+細(xì)胞遷移功能,此作用有可能通過RAGE介導(dǎo)。
[Abstract]:Background and objective High mobility group protein B1 (high mobility group box 1 (HMGB1) is a DNA binding protein that is released into extracellular cells after tissue damage and enlists many types of stem cells to participate in the repair of damaged tissues. The pretreatment of hematopoietic stem cell transplantation (haematopoietic stem cell transplantation, HSCT) inevitably results in bone marrow tissue damage, and the release of HMGB1, speculates that it may be involved in hematopoietic reconstitution. In this study, the release of HMGB1 from bone marrow stromal cells after pretreatment and the effect of HMGB1 on homing, proliferation and differentiation of human umbilical cord blood hematopoietic stem cells were observed for the first time, and the mechanism was discussed in order to find a new target for promoting hematopoiesis reconstruction after HSCT. Methods Human bone marrow stromal cells (BMSCs) were cultured in vitro. After irradiation with accelerator, the content of HMGB1 in culture supernatant was detected by ELISA. Human umbilical cord blood CD34 cells were selected by MACS system and co-cultured with HMGB1 for 6 days. The changes of differentiation index (CD13,CD14,CD11c,CD41,CD71) of CD34 cells in cord blood were detected by flow cytometry. The effects of HMGB1 on the proliferation and differentiation of hematopoietic stem cells were observed by clone formation assay. Transwell chamber chemotactic assay was used to observe the effect of HMGB1 on the migration activity of human umbilical cord blood CD34 cells. Flow cytometry was used to detect the expression of HMGB1 receptor RAGE,TLR2 and TLR4 in human umbilical cord blood CD34 cells. Using anti-RAGE antibody, anti-TLR2 antibody and anti-TLR4 antibody to block the repeated chemotaxis test of RAGE,TLR2 and TLR4, the effects of RAGE,TLR2 and TLR4 on CD34 cell migration induced by HMGB1 were observed in vitro. Outcome 1. The content of HMGB1 in the culture supernatant of bone marrow stromal cells was increased after X-ray irradiation. After 12Gy, the content of HMGB1 in the culture supernatant was (4.3 鹵0.9) ng/ml,. 2. The expression of HMGB1 receptor RAGE in human umbilical cord blood CD34 cells was detected by HMGB1 surface receptor (43.1 鹵7.2)%, and the HMGB1 content was (0.40.2) ng/ml higher than that of non-irradiated group (p0.01.HMGB1 surface receptor was (43.1 鹵7.2)%). TLR2 (36.1 鹵6.6)% and TLR4 (23.1 鹵5.2)%. 3. HMGB1 promoted the proliferation and differentiation of CD34 cells into erythroid and granulocytic lines. The purity of cord blood separation and enrichment was (98.25 鹵0.93)%. After 6 days of co-culture with HMGB1, HMGB1 was co-cultured with HMGB1 for 6 days. Compared with the control group, the expression of CD71 and CD13,CD14,CD11c markers were significantly increased, which were (18.4 鹵3.8 vs 32.6 鹵5.9)% for CD13 and (12.6 鹵2.7 vs 25.4 鹵4.4)% for CD14, respectively. The expression of CD11c (9.8 鹵2.1 vs 20.3 鹵3.9)%, CD71 (26.6 鹵4.6 vs 47.1 鹵7.4)%, and megakaryocyte marker CD41 (1.1 鹵0.4% vs 1.3 鹵0.5%) did not change significantly. Similarly, clone formation tests showed that after 14 days of co-culture, the erythroid colonies, 4.HMGB1 induced the migration of cord blood CD34 cells: the migration of HMGB1 to human umbilical cord blood CD34 cells increased gradually with the increasing concentration of HMGB1 within a certain range of concentration. 4. HMGB1 induced the migration of human umbilical cord blood CD34 cells in a certain concentration range. 4. HMGB1 induced the migration of human umbilical cord blood CD34 cells in a certain concentration range (p0.05). When the concentration of HMGB1 was 100 ng/ml, the chemotactic activity was the strongest, and the chemotactic index was 3.96 鹵0.46, which was significantly different from that of the control group (p0.01). Anti-RAGE antibody could partially inhibit the migration of HMGB1 to CD34 cells in cord blood. Conclusion HMGB1 can promote the differentiation of human umbilical cord blood CD34 cells into granulocyte monocytes and erythroid cells, and promote the formation of erythroid colony and granulocyte-macrophage colony, and increase the content of HMGB1 in the culture supernatant of bone marrow stromal cells after irradiation, and HMGB1 can promote the differentiation of human cord blood CD34 cells into granulocyte monocytes and erythrocytes. A certain concentration of HMGB1 can enhance the migration of cord blood CD34 cells, which may be mediated by RAGE.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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