| Introduction: Hematopoietic inductive microenvironment(HIM) is the site of origin, proliferation, differentiation and development of hematopoietic stem cell(HSC). As one of important component of HIM, stromal cell has tight relationship not only with the self-renew, proliferation, differentiation and homing of HSC, but also with the occurrence, progress and prognosis of hematologic diseases. So, it is very important to study the effect on hematoposis of stromal cell. The research and application on stromal cell in the past focused on bone marrow stromal cells, thus, further study of human umbilical cord blood stromal cell(hUCBSC) and umbilical cord blood associated hematopoietic inductive microenvironment are needed in the future. Hematopoietic stem cell in cord blood is more primitive than it is in bone marrow and peripheral blood, and have several behaviors, including extensive source, convenient collection, light graft verus host disease (GVHD) after transplantation, high proliferation and long-term bone marrow reconstruction, etc. There is great promise for it's clinical application. Our prophase study have confirmed that there exist hematopoietic stromal precursor cell in umbilical cord blood which could effective expansion by definite cytokine combination in vitro. GM-CSF, SCF and TPO can be excreted by hUCBSC after its expansion. Based on the analysis above, in this study, several tests were used to evaluate the biological behavior of hUCBSC, eukaryotic expression vector of vascular cell adhesion molecule-1(VCAM-1)was constructed, and then it was transfected to hUCBSC in order to investigate the capability of VCAM-1 modified hUCBSC to support hematopoiesis in vitro. Methods: 1. The umbilical cord blood CD34+ cell was cultured in Dexter system to abtain hUCBSC. Immunocytochemistry and cytochemistry were used to evaluate the cell components of hUCBSC. The condition and method of subculture of hUCBSC was explored for the first time. 2. The different expansion abilities in vitro of the umbilical cord blood CD34+ cell were identified under the condition of cytokine stimulation or/and hUCBSC feeder layer. The effect on hematopoietic reconstitution of different expanded hematopoietic cell groups was compared after CFU-GM, CFU-E and CFU-Mg semi-solid culture were proceed respectively. To evaluate the potential use of hUCBSC in the field of expansion of hematopoietic stem/progenitor cells in vitro. 3. With semi-nested RT-PCR, Vascular cell adhesion molecule-1(VCAM-1) cDNA was acquired from human umbilical vein endothelial cell line HUV-EC-C and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant vector was named VCAM-1-pcDNA3.1(+) recombinant vector. 4. The recombinant vector was transfected into hUCBSC by liposome. The mRNA and protein expression levels of VCAM-1 in transfected and untransfected cells were detected by semiquantitative RT-PCR and immunocytochemistry respectively. 5.Compare the expansion abilities of hematopoietic cell in vitro under different conditions, including cytokine stimulation, hUCBSC feeder layer and VCAM-1 modified hUCBSC feeder layer etc. To evaluate the significance of VCAM-1 modified hUCBSC on hematopoiesis. Results: 1. The morphology characteristic and evaluation of hUCBSC: There are three different cell components of hUCBSC after expanded, including fibroblast liked cell, macrophage liked cell and small-sphere liked cell. Cytochemistry stain: the positive rate reached 100% in PAS stain, ALP stain manifest 26% positive. Immunocytochemistry stain: the positive rate of hUCBSC for CD68, CD31 and Fn was 93%, 95%, 98% respectively, CD34 show negative. 2. Subculture method of hUCBSC: After the density of cells were above 80% confluence, hUCBSC were subcultured with 1:1 ratio under the same culture medium and condition. With the increase of the number of subculture, cell morphous trend to uniformity. Fibroblast liked cell is the main component cell, macrophage liked cell and small-sphere liked cell decrease remarkablely. hUCBSC can be subcultured for 4 generations under preasent condition. 3. Expansion of human umbilical cord blood CD34+ cells on hUCBSC feeder layer:Human umbilical cord blood stromal cell could be used for expansion of CD34+ cells. Combining cytokines with hUCBSC feeder layer could further increase the degree of expansion(P<0.05). hUCBSC have superiority in enhancing expansion of CFU-Mg(P<0.05). 4. Establishment of VCAM-1-pcDNA3.1(+) eukaryotic expression vector: With semi-nested RT-PCR method, VCAM-1 cDNA segment1, 2 and 3 were acquired from human umbilical vein endothelial cell line HUV-EC-C repectively. SOE technique was applicated to connect segment 1, 2 to segment 4 and segment 2, 3 to segment 5. Segment 4 and segment 5 were subcloned to pMD18-T vector separately. After sequencing and plerosis, VCAM-1cDNA was cloned into eukaryotic expression vector pcDNA3.1(+) and found no difference from that in GeneBank. The sequencing result proved that the open reading frame of VCAM-1 in the recombinant vector was correct. 5. Transfection of recombinant vector and evaluation of positive clone: The VCAM-1-pcDNA3.1(+) eukaryotic expression vector was transfected into hUCBSC via liposome mediation. The positive clone was formed after 7 days. After transfected, the amount of hUCBSC decreased, cell body augmentated relatively. Fibroblast liked cells, macrophage liked cells and small-sphere liked cells is still the main cell components. Contrast group (untransfected hUCBSC) were dead after screening with G418. It was confirmed that recombinant vector had been transfected into hUCBSC with NeoR gene detection. 6. Detect the expression of VCAM-1 gene in hUCBSC before and after transfected: With semiquantitative RT-PCR and immunocytochemistry, it was certificated that the expression of VCAM-1 increased remarkablely after transfected(P<0.05). 7. Expansion of human umbilical cord blood CD34+ cell on VCAM-1 modified hUCBSC feeder layer: Based on the experiment above, VCAM-1 modified hUCBSC feeder layer and combining cytokines with VCAM-1 modified hUCBSC feeder layer were added. The result manifest that VCAM-1 modified hUCBSC feeder layer could be used for expansion of CD34+ cells. Combining cytokines with VCAM-1 modified hUCBSC could further increase the degree of expansion(P<0.01). VCAM-1 modified hUCBSC expanded CFU-Mg more effectively than untransfected hUCBSC did(P<0.05). Conclusions: 1. HUCBSC possess the fundamental character of hematopoietic stromal cells. 2. HUCBSC can be subcultured and its morphous trend to uniformity after subculture.It can be subcultured for 4 generations under present condition. 3. HUCBSC could be used for expansion of CD34+ cells. Combining cytokines with hUCBSC feeder layer could further increase the degree of expansion. hUCBSC have superiority in enhancing expansion of CFU-Mg. 4. NeoR gene can be detected in the recombinant vector transfected hUCBSC, and the expression of VCAM-1 in different level increased after transfected. It proved that the VCAM-1 expression system was established successfully. 5. VCAM-1 modified hUCBSC could be useed for expansion of CD34+ cells. they expanded CFU-Mg more effectively than untransfected hUCBSC did... |
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