【摘要】： 目的 造血干細(xì)胞(hematopoietic stem cell, HSC)憑借其強(qiáng)大的自我更新和多系分化能力而維持機(jī)體造血處于相對(duì)恒定的狀態(tài),因此,HSC移植被認(rèn)為是造血系統(tǒng)疾病、癌癥和部分遺傳性疾病的重要治療手段。目前臍血被認(rèn)為是最有前途的HSC來源。但一份臍血中所含的造血干/祖細(xì)胞的數(shù)量有限,這極大地限制了臍血移植在臨床的廣泛應(yīng)用。體外擴(kuò)增臍血干/祖細(xì)胞一直是研究的熱點(diǎn)。但大量的臨床實(shí)踐表明,造血干細(xì)胞成功移植不僅取決于造血干/祖細(xì)胞的數(shù)量,而且與其歸巢能力密切相關(guān)。 基質(zhì)細(xì)胞衍生因子(stromal cell deriver factor-1,SDF-1)涉及造血干細(xì)胞和祖細(xì)胞的動(dòng)員、歸巢、植入和擴(kuò)增。而Reca等發(fā)現(xiàn)C3a增強(qiáng)SDF-1a依賴的遲現(xiàn)抗原-4(very late antigen 4,VLA-4)介導(dǎo)的干/祖細(xì)胞對(duì)血管細(xì)胞粘附分子-1(vascular cell adhesion molecule 1, VCAM-1)的粘附,這提示C3a與SDF-1a間的協(xié)同作用可以增加干/祖細(xì)胞粘附到血管內(nèi)皮或骨髓基質(zhì)的傾向,從而促進(jìn)其歸巢骨髓。為此,我們探討了SDF-1對(duì)臍血單個(gè)核細(xì)胞(mononuclear cells ,MNCs)黏附作用的影響,揭示SDF-1和干細(xì)胞歸巢的相關(guān)性,觀察C3a與SDF-1對(duì)MNC的黏附是否有協(xié)同作用,從而為基礎(chǔ)研究以及臨床上造血干細(xì)胞移植提供依據(jù)。 方法 1.取臍血后應(yīng)用Ficoll淋巴細(xì)胞分離液分離、收集單個(gè)核細(xì)胞。用含20μg/ml人纖連蛋白(fibronectin,FN)的PBS包被96孔板。將單個(gè)核細(xì)胞孵育在已包被好的培養(yǎng)板中,調(diào)整細(xì)胞密度為2×105/m1,加入不同濃度SDF-1,每組復(fù)設(shè)3個(gè)孔,37℃孵育2h,進(jìn)行黏附率檢測。 2.在含有造血生長因子和最適濃度SDF-1的培養(yǎng)基中體外培養(yǎng),分別收集第0、4、7、10和14天MNC進(jìn)行免疫表型分析和黏附率檢測。 3.觀察不同濃度C3a下對(duì)MNC黏附功能的影響。 4.觀察C3a與SDF-1對(duì)MNC的黏附性是否有協(xié)同作用。 5.采用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)分析,所有檢驗(yàn)結(jié)果采用雙側(cè)檢驗(yàn),P≤0.05認(rèn)為差別有統(tǒng)計(jì)學(xué)意義。數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差( x±s)表示,組間比較采用單因素方差分析,兩變量間關(guān)系研究采用直線相關(guān)分析。 結(jié)果 1.隨著SDF-1濃度增加,新鮮臍血MNC黏附率升高,但SDF-1濃度達(dá)到100ng/ml時(shí)黏附率趨于平穩(wěn)。 2.體外培養(yǎng)MNC時(shí),培養(yǎng)的早期, VLA-4的表達(dá)升高,同時(shí)臍血MNC黏附率也升高。但隨著培養(yǎng)時(shí)間的延長,VLA-4表達(dá)量漸漸降低,而且臍血MNC黏附率隨之降低。 3. C3a單獨(dú)對(duì)MNC黏附性無明顯影響,但C3a與SDF-1共同孵育組MNC黏附功能增強(qiáng)。 結(jié)論 1. MNC黏附率隨著SDF-1濃度MNC黏附率逐漸增高,但當(dāng)SDF-1達(dá)一定濃度時(shí),細(xì)胞黏附率趨于穩(wěn)定。 2. MNC黏附率與黏附分子VLA-4表達(dá)量間存在正相關(guān)。 3. C3a單獨(dú)對(duì)MNC黏附率無明顯影響,但增強(qiáng)SDF-1對(duì)MNC的黏附功能。
[Abstract]:Objective Hematopoietic stem cell (hematopoietic stem cell, HSC) is considered to be a disease of hematopoietic system because of its strong ability of self-renewal and multi-lineage differentiation to maintain a relatively constant state of hematopoiesis. An important treatment for cancer and some hereditary diseases. Cord blood is currently considered the most promising source of HSC. However, the limited number of hematopoietic stem / progenitor cells in a single cord blood greatly limits the wide application of cord blood transplantation in clinical practice. In vitro expansion of umbilical cord blood stem / progenitor cells has been a hot topic. However, a large number of clinical practices show that successful transplantation of hematopoietic stem cells depends not only on the number of hematopoietic stem / progenitor cells, but also on their homing ability. Stromal cell derived factor (stromal cell deriver factor-1,SDF-1) involves the mobilization, homing, implantation and amplification of hematopoietic stem cells and progenitor cells. Reca et al found that C3a enhanced the adhesion of stem / progenitor cells to vascular cell adhesion molecule-1 (vascular cell adhesion molecule 1 (VCAM-1) mediated by SDF-1a dependent antigen-4 (very late antigen 4 (VLA-4). These results suggest that the synergism between C3a and SDF-1a can increase the tendency of dry / progenitor cells to adhere to vascular endothelium or bone marrow stroma, thus promoting homing bone marrow. Therefore, we investigated the effect of SDF-1 on (mononuclear cells, MNCs) adhesion of umbilical cord blood mononuclear cells, revealed the correlation between SDF-1 and stem cell homing, and observed whether C3a and SDF-1 had synergistic effect on MNC adhesion. So as to provide the basis for basic research and clinical hematopoietic stem cell transplantation. Method 1. After cord blood was taken, the mononuclear cells were collected by Ficoll lymphocyte isolate. The 96 well plate was coated with PBS containing 20 渭 g/ml human fibronectin (fibronectin,FN). The mononuclear cells were incubated in a well coated culture plate, the cell density was adjusted to 2 脳 10 5 / m 1, 3 holes were added to each group with different concentrations of SDF-1, and incubated at 37 鈩，

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