【摘要】： 目的 造血干細胞(hematopoietic stem cell, HSC)憑借其強大的自我更新和多系分化能力而維持機體造血處于相對恒定的狀態(tài),因此,HSC移植被認為是造血系統(tǒng)疾病、癌癥和部分遺傳性疾病的重要治療手段。目前臍血被認為是最有前途的HSC來源。但一份臍血中所含的造血干/祖細胞的數(shù)量有限,這極大地限制了臍血移植在臨床的廣泛應用。體外擴增臍血干/祖細胞一直是研究的熱點。但大量的臨床實踐表明,造血干細胞成功移植不僅取決于造血干/祖細胞的數(shù)量,而且與其歸巢能力密切相關(guān)。 基質(zhì)細胞衍生因子(stromal cell deriver factor-1,SDF-1)涉及造血干細胞和祖細胞的動員、歸巢、植入和擴增。而Reca等發(fā)現(xiàn)C3a增強SDF-1a依賴的遲現(xiàn)抗原-4(very late antigen 4,VLA-4)介導的干/祖細胞對血管細胞粘附分子-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/祖細胞粘附到血管內(nèi)皮或骨髓基質(zhì)的傾向,從而促進其歸巢骨髓。為此,我們探討了SDF-1對臍血單個核細胞(mononuclear cells ,MNCs)黏附作用的影響,揭示SDF-1和干細胞歸巢的相關(guān)性,觀察C3a與SDF-1對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胞移植提供依據(jù)。 方法 1.取臍血后應用Ficoll淋巴細胞分離液分離、收集單個核細胞。用含20μg/ml人纖連蛋白(fibronectin,FN)的PBS包被96孔板。將單個核細胞孵育在已包被好的培養(yǎng)板中,調(diào)整細胞密度為2×105/m1,加入不同濃度SDF-1,每組復設(shè)3個孔,37℃孵育2h,進行黏附率檢測。 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進行免疫表型分析和黏附率檢測。 3.觀察不同濃度C3a下對MNC黏附功能的影響。 4.觀察C3a與SDF-1對MNC的黏附性是否有協(xié)同作用。 5.采用SPSS 13.0軟件進行統(tǒng)計分析,所有檢驗結(jié)果采用雙側(cè)檢驗,P≤0.05認為差別有統(tǒng)計學意義。數(shù)據(jù)用均數(shù)±標準差( 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濃度達到100ng/ml時黏附率趨于平穩(wěn)。 2.體外培養(yǎng)MNC時,培養(yǎng)的早期, VLA-4的表達升高,同時臍血MNC黏附率也升高。但隨著培養(yǎng)時間的延長,VLA-4表達量漸漸降低,而且臍血MNC黏附率隨之降低。 3. C3a單獨對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。 結(jié)論 1. MNC黏附率隨著SDF-1濃度MNC黏附率逐漸增高,但當SDF-1達一定濃度時,細胞黏附率趨于穩(wěn)定。 2. MNC黏附率與黏附分子VLA-4表達量間存在正相關(guān)。 3. C3a單獨對MNC黏附率無明顯影響,但增強SDF-1對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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