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Study on the Culture and Amplification of Human Stromal Cells Derived from Umbilical Cord Blood with Human AB Type Serum

Author: GongZuo
Tutor: ZouZhongMin
School: Third Military Medical University
Course: Preventive Medicine in Military
Keywords: Hematopoietic microenvironment Human umbilical cord blood source stromal cells Human AB serum In vitro culture Cell expansion
CLC: R329
Type: Master's thesis
Year: 2009
Downloads: 8
Quote: 0
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Abstract


Hematopoietic micro-environment (Hematopoietic inductive microenvironment, HIM) is a hematopoietic stem cell generation, proliferation and differentiation and development of the place, stromal cells is hematopoietic microenvironment important composition of ingredients, not only with the hematopoietic stem cell self-update, proliferation, differentiation, and homing phenomenon is closely related to , and have an important impact on the occurrence of the disease of the blood system, progress and prognosis. Stromal cells in human umbilical cord blood source (human stromal cells derived from umbilical cord blood, hUCBDSCs) can promote the repair of the bone marrow microenvironment injury, easily obtained, and cell proliferation activity and weak immunogenicity advantages. Studies have shown that stromal cells isolated from human umbilical cord blood specimens by direct contact to promote hematopoietic stem cell self-renewal, proliferation and differentiation. Promote stromal cells can secrete a variety of hematopoietic cytokines such as TPO, GM-CSF and SCF, etc. enhance the migration and development of hematopoietic stem cells in bone marrow and secretion of extracellular matrix components (extracellular matrix, ECM) to participate in the composition of the hematopoietic microenvironment. However, there is a limited number of umbilical cord blood derived stromal cells were cultured in vitro must be strictly dependent on the specific fetal bovine serum and other shortcomings, which hinder the clinical application of the stromal cells in human umbilical cord blood source. The ideal in vitro culture system should avoid the use of animal serum components to bring on the clinical application of risk. This study attempts to human AB serum-free culture system in vitro cultured human umbilical cord blood stromal cells obtained good results, the establishment of a stable cultured in vitro expansion of cord blood stromal cells, clinical application of human umbilical cord blood stromal cells lay the foundation. The main results are as follows: 1 from human umbilical cord blood stromal cell growth state of dynamic observation: seen a few cells in culture after 48 ~ 96 h adherent. The adherent cells were irregular shape and visible cytoplasmic projections formed obvious proliferation adherent cells began to appear after about 1 week, and visible to the CFU-F colony formation, culture was continued to the CFU-F colony fused to form a cell monolayer. HE staining of human umbilical cord blood stromal cells fusiform, round or irregular shape, nuclear as round or oval, CFU-F colony cells arranged radially and swirling. The colony cells connected to each other, staggered into the film. 2.15% human AB serum and bFGF cytokine concentrations in human umbilical cord blood stromal cells CFU-F: 15% human AB serum can effectively support cord blood stromal cells cultured in vitro amplification. 0 ~ 10μg / L within the scope of bFGF in the higher the concentration, the more the number of colonies formed, but its concentration is greater than 10μg / L, the stromal cells in human umbilical cord blood, no significant increase in the stimulating effect of the number of colonies is formed. Umbilical cord blood stromal cell surface marker detection: application of immunohistochemical staining detected Vimentin in the expression of CD34, TdT, CD45 and CK in the umbilical cord blood stromal cells. Growth in good condition 1-2 passage cells were used for immunohistochemical detection, the results showed that the phenotype of human umbilical cord blood stromal cells Vimentin in () CD34 (), TdT (-), CD45 (-) and CK ( -). The main conclusion: the successful establishment of a human-type AB serum-free culture system in vitro culture method of expansion of cord blood source stromal cells, for the next therapeutic research, such as the body to promote put repair damage of bone marrow function after chemotherapy and clinical umbilical blood stromal cell therapy technology to carry out, to provide the pre-conditions to prepare.

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