| PurposeMesenchymal stem cells (mesenchymal stem cells, MSC) is derived from the development of the early mesoderm and ectoderm, with multi-differentiation potential of hematopoietic stem cells implanted to support and promote and self-replication features of a class of cells in vivo or in vitro Induction of specific conditions, differentiate into bone, cartilage, muscle, fat, endothelium and other tissue cells phenotype, Subculture and cryopreservation after a multi-directional differentiation potential can be used as an ideal seed cells for the aging and disease caused by injury and repair tissues and organs. Mesenchymal stem cells, many drawn from the bone marrow, an invasive subjects, restricting its clinical application. Umbilical cord blood (umbilical cord blood, UCB) is the baby is delivered, the umbilical cord ligation and away from the fractured left in the placenta and umbilical cord blood is rich in stem cells, umbilical cord blood collected in regular clinical hematopoietic stem cells for the treatment of blood diseases. In recent years studies have shown that cord blood there are also MSC. Because cord blood is rich in easily collected, exclusion of small, non-ethical arguments for the advantages of cord blood derived mesenchymal stem cells as transplant cells to more attention.The lack of specificity of cord blood MSCS surface markers, these cells expressed CD29, CD44, CD73, CD90 and HLA-ABC, CD105, and CD106, did not express CD14, CD34, CD45, CD133, and HLA-DR, and can be divided as bone, cartilage, fat into muscle-like cells and adult. Some scholars have applied single-tube multi-color flow cytometry analysis of the blood staining the number of MSCs,for every one million nucleated cells, which contain 8.8±4.3 MSC cells, bone marrow samples for every one million nucleated cells were containing 13.2±5.8 MSC cells. MSC also reported in the literature the number of cord blood varied affected by many factors. We apply single-tube multi-color fluorescence staining technical analysis of different gestational age cord blood CD44, CD 105 positive and CD34, CD45-negative cells, and colony forming capacity of cultured cells, made after the analysis to compare the number of different gestational age cord blood MSC difference, and analysis of umbilical cord blood successfully cultivated MSC related factors, in order to further explore the biological characteristics of umbilical cord blood MSC basis.Methods1. single-tube multi-color flow cytometry analysis of stained(1)sterile conditions, take a normal (28 weeks to 42 weeks) umbilical cord blood 52 cases of umbilical cord blood.(2)each amount of blood were collected from all cases for single-tube multi-color flow cytometry analysis of stained.(3)Compare the number of MSCs with different terms.2.Cell culture(1)sterile conditions, take a normal full-term (37 weeks to 40 weeks) umbilical cord blood of 12 non-full-term (28 weeks to 36 weeks) 12 cases of umbilical cord blood, CAPD-1 compound anticoagulant anticoagulant, respectively, As a full-term group and non-term group.(2)each amount of blood were collected from all cases for single-tube multi-color flow cytometry analysis of stained(3)the cord blood dilution, the direct use of Ficoll mononuclear cells containing 10%serum with the concentration of L-DMEM conventional mesenchymal stem cells in adherent culture;(4) pairs of two the number of mononuclear cells was compared; wall cell types, the growth rate of observation records; right to successfully cultivate the umbilical cord blood mesenchymal stem cells, the number of cases for statistical analysis;(5)colony-forming ability of analysis and comparison;(6)pairs of isolated umbilical cord blood mesenchymal stem cells were identified.3.statistical analysis.Results1. the results of flow cytometry:CD44 (+), CD105 (+), and CD34 (-), CD45 (-) cell content in 28weeks～30 weeks was 15±0.2/1 million nucleated cells, in 31weeks～33 weeks was 11 i 0.3, in 34 weeks～36 weeks was 10±0.1/1 million nucleated cells, in 37 weeks～39 weeks was 6±0.5/1 million nucleated cells, in 40 weeks～42weeks was 2±0.3/1 million nucleated cells;in full-term group was 6.2±0.2/1 million nucleated cells, the non-full-term group was 10.3±0.2/1 million nucleated cells, there was a significant difference (P<0.01);2. two separate comparison of cord blood mononuclear cellsFull-term group and non-term group received the number of cord blood nucleated cells, respectively ((2.8±0.3) X 106, (3.5±0.5) X 107, there was a significant difference (P<0.05); two methods of separation nucleated cells, no significant difference in mortality (P> 0.05); two groups of cells cultured for 7 days later in 30cm2 culture bottle cloning of the number of adherent cells were 18±0,27±8, there was a significant difference (P<0.05).3. full-term group and non-term group of mesenchymal stem cells ComparisonFull-term group of mononuclear cells cultured adherent cells after 96 hours there were spindle cells and round cells, fibrous two forms of cells after passage 1/12 cord blood units appear ordered spiral-shaped mesenchymal-like cells The remaining 11/12 were passaged after only a few spindle cells intermingled, trypsin digestion down hard to the final aging death. Non-term group of mononuclear cells cultured for 72 hours, adherent cells, mostly fibrous mesenchymal-like cells, after passage 3/12 were isolated from the umbilical cord blood mesenchymal-like spindle cells, and the growth rate markedly higher than in full-term group. 4. isolated and cultured umbilical cord blood mesenchymal stem cells, the success rate of non-full-term group of isolated and cultured mesenchymal stem cells the overall success rate was 8.3%, full-term group was 25.0%.5. pairs of isolated umbilical cord blood mesenchymal stem cells for identification of obtained two sets of spindle cells cultured immune cells staining MSCs marker expression:CD45 (-), CD90 (+).Conclusion1. cord blood contains mesenchymal stem cells, the non-full-term umbilical cord blood mesenchymal stem cells in the middle of content is more than full-term umbilical cord blood mesenchymal stem cells in the middle of content.2. non-full-term umbilical cord blood mesenchymal stem cells combined with the success rate is higher than full-term umbilical cord blood vaccination success rate.3. non-full-term umbilical cord blood mesenchymal stem cells grow faster than full-term umbilical cord blood mesenchymal stem cells in the growth rate.
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