C3a与SDF-1对脐带血单个核细胞黏附作用的影响

发布时间：2018-10-13 10:40

【摘要】： 目的 造血干细胞(hematopoietic stem cell, HSC)凭借其强大的自我更新和多系分化能力而维持机体造血处于相对恒定的状态,因此,HSC移植被认为是造血系统疾病、癌症和部分遗传性疾病的重要治疗手段。目前脐血被认为是最有前途的HSC来源。但一份脐血中所含的造血干/祖细胞的数量有限,这极大地限制了脐血移植在临床的广泛应用。体外扩增脐血干/祖细胞一直是研究的热点。但大量的临床实践表明,造血干细胞成功移植不仅取决于造血干/祖细胞的数量,而且与其归巢能力密切相关。 基质细胞衍生因子(stromal cell deriver factor-1,SDF-1)涉及造血干细胞和祖细胞的动员、归巢、植入和扩增。而Reca等发现C3a增强SDF-1a依赖的迟现抗原-4(very late antigen 4,VLA-4)介导的干/祖细胞对血管细胞粘附分子-1(vascular cell adhesion molecule 1, VCAM-1)的粘附,这提示C3a与SDF-1a间的协同作用可以增加干/祖细胞粘附到血管内皮或骨髓基质的倾向,从而促进其归巢骨髓。为此,我们探讨了SDF-1对脐血单个核细胞(mononuclear cells ,MNCs)黏附作用的影响,揭示SDF-1和干细胞归巢的相关性,观察C3a与SDF-1对MNC的黏附是否有协同作用,从而为基础研究以及临床上造血干细胞移植提供依据。 方法 1.取脐血后应用Ficoll淋巴细胞分离液分离、收集单个核细胞。用含20μg/ml人纤连蛋白(fibronectin,FN)的PBS包被96孔板。将单个核细胞孵育在已包被好的培养板中,调整细胞密度为2×105/m1,加入不同浓度SDF-1,每组复设3个孔,37℃孵育2h,进行黏附率检测。 2.在含有造血生长因子和最适浓度SDF-1的培养基中体外培养,分别收集第0、4、7、10和14天MNC进行免疫表型分析和黏附率检测。 3.观察不同浓度C3a下对MNC黏附功能的影响。 4.观察C3a与SDF-1对MNC的黏附性是否有协同作用。 5.采用SPSS 13.0软件进行统计分析,所有检验结果采用双侧检验,P≤0.05认为差别有统计学意义。数据用均数±标准差( x±s)表示,组间比较采用单因素方差分析,两变量间关系研究采用直线相关分析。 结果 1.随着SDF-1浓度增加,新鲜脐血MNC黏附率升高,但SDF-1浓度达到100ng/ml时黏附率趋于平稳。 2.体外培养MNC时,培养的早期, VLA-4的表达升高,同时脐血MNC黏附率也升高。但随着培养时间的延长,VLA-4表达量渐渐降低,而且脐血MNC黏附率随之降低。 3. C3a单独对MNC黏附性无明显影响,但C3a与SDF-1共同孵育组MNC黏附功能增强。 结论 1. MNC黏附率随着SDF-1浓度MNC黏附率逐渐增高,但当SDF-1达一定浓度时,细胞黏附率趋于稳定。 2. MNC黏附率与黏附分子VLA-4表达量间存在正相关。 3. C3a单独对MNC黏附率无明显影响,但增强SDF-1对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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