人AB型血清培养体系体外培养扩增人脐血源基质细胞的实验研究

发布时间：2018-01-08 03:27

本文关键词：人AB型血清培养体系体外培养扩增人脐血源基质细胞的实验研究　出处：《第三军医大学》2009年硕士论文　论文类型：学位论文

【摘要】： 造血微环境(Hematopoietic inductive microenvironment,HIM)是造血干细胞产生、增殖和分化发育的场所,基质细胞是造血微环境的重要组成成分,不仅与造血干细胞的自我更新、增殖、分化和归巢现象关系密切,而且对血液系统疾病的发生、进展和预后产生重要影响。人脐血源基质细胞(human stromal cells derived from umbilical cord blood, hUCBDSCs)可促进骨髓造血微环境损伤的修复,具有取材方便、细胞增殖活性高和免疫原性弱等优点。已有研究表明,从人脐血标本中分离的基质细胞通过直接接触促进造血干细胞自我更新、增殖和分化的作用.基质细胞可以分泌多种促进造血的细胞因子如TPO、GM-CSF和SCF等,可增强造血干细胞的骨髓迁移和发育,并可以分泌细胞外基质成分(extracellular matrix ,ECM)参与造血微环境的组成。然而,脐带血来源的基质细胞存在数量有限,且其体外扩增培养必须严格依赖于特定的胎牛血清等缺点,这阻碍了人脐血源基质细胞的临床应用。理想的体外培养体系应避免使用动物血清成分带来临床应用上的风险。本研究尝试以人AB型血清培养体系体外培养人脐血基质细胞,获得了较好的效果,建立了一种稳定的体外培养扩增人脐血基质细胞方法,为人脐血基质细胞的临床应用奠定基础。主要的实验结果如下: 1.人脐血基质细胞生长状态的动态观察:在培养48～96 h后,可见少数细胞已贴壁。贴壁细胞呈不规则形并可见胞浆突起形成,大约1周后贴壁细胞开始出现明显的分裂增殖,并可见CFU-F集落形成,继续培养至CFU-F集落融合形成单层细胞。经HE染色观察,人脐血基质细胞呈梭形、圆形或不规则形,核为圆形或椭圆形,CFU-F集落内细胞排列呈放射状和涡流状。集落间细胞相互连接、交错成片。 2.15%人AB型血清和bFGF细胞因子浓度对人脐血基质细胞CFU-F的影响:15%人AB型血清能有效支持脐血基质细胞在体外的扩增培养。在0～10μg/L范围内bFGF的浓度越高,形成的集落数也越多,但当其浓度大于10μg/L时,对人脐血基质细胞形成集落数的刺激作用无明显增加。 3.脐血基质细胞表面标志的检测:应用免疫组化染色,检测了Vimentin、CD34、TdT、CD45和CK等在脐血基质细胞中的表达。生长状态良好的第1-2次传代细胞用于进行免疫组化检测,结果表明人脐血基质细胞的表型为Vimentin(+)、CD34(+)、TdT(-)、CD45(-)和CK(-)。主要结论:成功建立一种人AB型血清培养体系体外培养扩增人脐血源基质细胞的方法,为下一步的治疗性研究,如体内促进放、化疗后骨髓造血功能损伤的修复和临床脐血基质细胞治疗技术的开展,提供了前期的条件准备。
[Abstract]:The hematopoietic microenvironment (Hematopoietic inductive microenvironment, HIM) is a hematopoietic stem cell, proliferation and differentiation and development of the place, stromal cells are important components of hematopoietic microenvironment, and hematopoietic stem cell self-renewal, proliferation, differentiation and homing related phenomenon, and on the blood system diseases have an important impact on progress and prognosis. Human umbilical cord blood stromal cells (human stromal cells derived from umbilical cord blood, hUCBDSCs) can promote the repair of bone marrow hematopoietic micro environment damage, it is a convenient, high cell proliferation activity and weak immunogenicity and other advantages. Studies have shown that stromal cells isolated from human umbilical cord blood samples by direct contact to promote hematopoietic stem cell self-renewal, proliferation and differentiation. Stromal cells can secrete a variety of cells such as TPO promote hematopoietic factor, GM-CSF and SCF, can be increased Strong bone marrow hematopoietic stem cell migration and development, and can secrete extracellular matrix components (extracellular, matrix, ECM) components involved in hematopoietic microenvironment. However, stromal cells derived from umbilical cord blood has a limited number, and the in vitro culture must be strictly dependent on fetal bovine serum specific shortcomings, which hinders the clinical application human umbilical cord blood stromal cells cultured in vitro. The ideal system should avoid the use of animal serum components risks in clinical practice. This study attempts to use human AB type serum cultured human umbilical cord blood stromal cell system in vitro, to obtain good results, establish a stable in vitro culture method of human umbilical cord blood stromal cells, which the basis for the clinical application of human umbilical cord blood stromal cells. The main results are as follows:
Dynamic observation of umbilical cord blood stromal cell growth state in 1.: after 48~96 h culture, showing a few cells adherent. Adherent cells were irregular in shape and the formation of cytoplasmic protrusions, marked the beginning of the proliferation of adherent cells after about 1 weeks, and visible CFU-F colony formation, continue to develop CFU-F colony formation of monolayer cells. Fusion by HE staining, human umbilical cord blood stromal cells were spindle shaped, round or irregular shape, nucleus was round or oval, CFU-F colony cells arranged radially and swirling. Inter colony cells are connected to each other and goes into the film.
Effect of serum bFGF and cytokine concentrations of 2.15% AB of human umbilical cord blood stromal cell CFU-F: 15% human AB type serum can effectively support umbilical cord blood stromal cells in vitro culture. In 0 ~ 10 g/L concentration range of bFGF is high, the colony counts more, but when the concentration is greater than 10 g/L, on human umbilical cord blood stromal cell colony formation stimulation number were not significantly increased.
3. detection of umbilical cord blood stromal cell surface markers: immunohistochemical staining, detection of Vimentin, CD34, TdT, CD45 and CK in umbilical cord blood stromal cells. The expression of 1-2 cell growth in good condition for immunohistochemical detection results showed that the phenotype of human umbilical cord blood stromal cells (Vimentin +), CD34 (+), TdT (-), CD45 (-) and CK (-).
The main conclusion: we have successfully established a human AB type serum culture system in vitro amplification of human umbilical cord blood stromal cells, on the treatment of the next step, such as promoting the body, repair of bone marrow hematopoietic function injury and clinical treatment of umbilical cord blood stromal cells after chemotherapy development, provide the conditions of preparation.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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