脐带血单个核细胞迁移体外实验研究

发布时间：2018-01-05 22:03

本文关键词：脐带血单个核细胞迁移体外实验研究　出处：《山西医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的: 异基因造血干细胞(HSC)移植是根治恶性血液病的唯一方法。对于缺乏HLA匹配供者的病人,寻找可靠的HSC源非常必要。除骨髓外,胎儿组织如胎儿肝脏、脐带或胎盘血以及成人动员的外周血中都含有HSC,但由于不同来源的HSC具有各自的特殊性,使临床应用受到局限。与成人血相比,脐血有其独特的优势:如含有较高比例的HSC;造血干/祖细胞中端粒较长;体外对细胞因子的刺激敏感;有较强的增殖能力;免疫细胞功能幼稚;容易获得;对供者无损害;病毒抗原污染机会少等。目前脐血被认为是最有前途的HSC来源。但由于一份脐血中所含的造血干/祖细胞的数量有限,脐血移植主要局限于小儿和体重轻的成人患者,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/祖细胞以拓宽临床应用范围一直是研究的热点。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—1(stromal cell-derived factor,SDF-1)及其受体(CXCR4)作为a趋化因子家族的一个成员,不但与调节机体免疫功能、维持胚胎发育、细胞增殖等有关,而且SDF-1/CXCR4在免疫细胞趋化、肿瘤转移以及造血细胞的动员和归巢等方面发挥着关键作用。为此,我们探讨了趋化因子SDF-1以及其受体CXCR4在细胞迁移中的作用,旨在为临床成功进行造血干细胞移植提供一定的理论依据。方法: 1.采脐血后应用Ficoll淋巴细胞分离液分离、收集单个核细胞,洗涤两遍后备用。 2.调整细胞浓度后将细胞接种于含有SCF、FL、TPO造血生长因子的24孔培养板,分别在培养第4、7和10天半定量换液,补充生长因子,观察细胞扩增情况。 3.将1×10~5个细胞悬浮于含0.1ml平衡液的Transwell上腔,其下腔为含有不同浓度SDF-1的0.6ml趋化液,在37℃、5%CO_2、100%湿度条件下趋化4～5小时,同时另设细胞抗原CXCR4阻断组。计数进入下腔的细胞。 4.将1×10~6个细胞接种在含生长因子SCF、FL、TPO的2ml IMDM培养基中培养。分别在培养的第0、4、7、10、14天,收集细胞,流式细胞仪检测CXCR4表达情况。同时,在第3步骤中获得的SDF-1最佳趋化浓度条件下检测细胞迁移率。 5.统计分析采用SPSS 13.0统计软件,所有检验结果采用双侧检验,P值小于或等于0.05认为差别有统计学意义。计量数据用均数±标准差((?)±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: 通过对6例标本的观察,我们发现 1.每份脐血的体积及所含有核细胞在不同个体中差异较大。6份脐血体积范围在44～91ml,平均69.17±17.43ml;有核细胞范围为(1.14～4.52)×10~8个,平均2.91±1.27×10~8个。 2.经14天重组人造血生长因子组合SCF、FL、TPO体外培养,各阶段的MNC均得到有效扩增;至14天时,总有核细胞数扩增了248倍,细胞数量显著增加。 3.脐血MNC迁移实验发现:随着SDF-1浓度增加,新鲜脐血MNC迁移率升高,但SDF-1浓度达到150ng/ml时迁移率趋于平稳;当CXCR4阻断型抗体作用后,MNC迁移率与未加SDF-1组无差异。 4.重组人造血生长因子组合SCF、FL、TPO体外培养MNC时,培养的早期,趋化因子SDF-1受体CXCR4的表达升高,同时脐血MNC迁移率也升高。但随着培养时间的延长,CXCR4表达量渐渐降低,而且脐血MNC迁移率随之降低。结论: 1.经有核细胞计数证明,在含血清、无基质细胞支持的条件下,采用重组人造血生长因子SCF、FL、TPO联合扩增系统,可以有效地对新鲜脐血MNC进行体外扩增。 2.通过Transwell Plate可以有效地模拟细胞穿越内皮现象。随着趋化因子SDF-1浓度的增高,MNC迁移率递增,但当SDF-1浓度达一定高时,细胞趋化率达稳定,继续增高SDF-1浓度,细胞迁移率变化不显著。 3.MNC迁移率与趋化因子受体CXCR4表达量间存在相关性,应用CXCR4阻断抗体后MNC趋化率与未加SDF-1无显著差异。
[Abstract]:Objective:
Allogeneic hematopoietic stem cell transplantation (HSC) is the only way to cure malignant hematological diseases. For the lack of HLA matched donors for patients, it is necessary to find a reliable source of HSC. In addition to the bone marrow, fetus liver, adult umbilical cord or placental blood and mobilized peripheral blood are contained in HSC, but because of different sources of HSC have their own specialty, the clinical application is limited. Compared with the adult blood, umbilical cord blood has its unique advantages such as: with a higher proportion of HSC; hematopoietic stem / progenitor cells in longer telomeres; stimulation of cytokine in vitro sensitivity; have a strong proliferation ability; immature immune function; easy to get; no damage to the donor; less pollution. At present the opportunity of virus antigen of umbilical cord blood is considered as the most promising source of HSC.
But because of a contained in cord blood hematopoietic stem / progenitor cell number is limited, umbilical cord blood transplantation mainly confined to children and adults with light weight, which greatly limits the clinical application of umbilical cord blood transplantation. The in vitro expansion of umbilical cord blood stem / progenitor cells to broaden the scope of clinical application has been the focus of research. But in clinical practice a large number of hematopoietic stem cell transplantation show that success depends not only on the hematopoietic stem / progenitor cell number, and the ability of homing.
Stromal cell derived factor - 1 (stromal cell-derived, factor, SDF-1) and its receptor (CXCR4) as a member of the a chemokine factor family, not only with the regulation of immune function, maintain the embryonic development, cell proliferation, chemotaxis and SDF-1/CXCR4 in immune cells, play a key role in tumor metastasis and hematopoietic stem cell mobilization homing and etc. Therefore, we explore the role of chemokine SDF-1 and its receptor CXCR4 in cell migration, designed for clinical successful hematopoietic stem cell transplantation provides a theoretical basis.
Method:
1. after umbilical cord blood, Ficoll lymphocyte separation solution was used to separate the mononuclear cells, and the cells were washed for two times.
2., after adjusting the cell concentration, the cells were seeded on 24 hole culture plates containing SCF, FL and TPO hematopoietic growth factors. They were cultured in 4,7 and 10 days respectively for quantitative exchange, supplementation of growth factors and observation of cell expansion.
3., 1 x 10~5 cells were suspended in the Transwell upper chamber containing 0.1ml equilibrium solution, the lower chamber was 0.6ml chemotactic solution containing different concentrations of SDF-1, and it was chemotactic for 4~5 hours at 37 C and 5%CO_2100% humidity. At the same time, another cell antigen CXCR4 blocking group was set up. Counting the cells entering the inferior cavity.
4. will be 1 * 10~6 cells inoculated in containing the growth factor SCF, FL, TPO 2ml IMDM medium. Cells were collected in cultured 0,4,7,10,14 days, respectively, flow cytometry was used to detect the expression of CXCR4. At the same time, in the third step the SDF-1 optimal chemoattractant concentration under the conditions of detection of cell migration rate.
Using SPSS 13 statistical software 5. statistical analysis, all test results using two-sided test, P value is less than or equal to 0.05 that the difference was statistically significant. The mean and standard deviation for the measurement data ((?) + s) said, compared with single factor analysis of variance, linear correlation analysis was used to study the relationship between the two variables.
Result:
Through the observation of 6 specimens, we found that
1., the volume of umbilical cord blood and the nuclear cells contained in each umbilical cord are quite different among different individuals. The volume range of umbilical cord blood of.6 is 44 to 91ml, with an average of 69.17 + 17.43ml, and the range of nucleated cells is (1.14 to 4.52) x 10~8, with an average of 2.91 + 1.27 * 10~8.
2., after 14 days of recombination, artificial blood growth factor combination SCF, FL and TPO were cultured in vitro, and MNC at each stage was effectively amplified. Until 14 days, the total number of nuclear cells increased 248 times, and the number of cells increased significantly.
3. umbilical cord blood MNC migration experiment showed that with the increase of SDF-1 concentration, fresh cord blood MNC migration rate increased, but the concentration of SDF-1 reached 150ng/ml migrated rate tended to be stable; when the CXCR4 blocking antibody, MNC mobility and without SDF-1 group had no difference.
4. recombinant human hematopoietic growth factor SCF, FL, MNC and TPO were cultured in vitro and cultured early expression of chemokine receptor CXCR4 and SDF-1 elevated cord blood MNC migration rate also increased. But with prolonged incubation time, CXCR4 expression gradually decreased, and the umbilical cord blood MNC mobility decreases.
Conclusion:
1., by nuclear cell count, it is proved that the recombinant human blood growth factor SCF, FL and TPO combined amplification system can effectively expand the MNC of fresh umbilical cord blood in serum and without stromal cell support.
2., Transwell Plate can effectively simulate the phenomenon of cell passing through the endothelium. With the increase of SDF-1 concentration, the mobility of MNC increased, but when the SDF-1 concentration reached a certain level, the chemotaxis rate of cells was stable, and the SDF-1 concentration increased, and the mobilities of cell migration did not change significantly.
There was a correlation between the mobility of 3.MNC and the expression of chemokine receptor CXCR4, and there was no significant difference between the chemotactic rate of MNC and the non SDF-1 after the application of CXCR4 to block the antibody.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

【共引文献】