| Objective1. Establish the method to obtain hUC-MSCs from human umbilical cord2. Cultured hUC-MSCs and study its biological characteristics3. Establish the method to obtain HSCs from human cord bloodMethods1. Collected neonatal umbilical cord in Sterile environment, isolated hUC-MSCs from humanumbilical cord by culture of tissue adherence;2. Take the3-5generation of hUC-MSCs to make cells growth curve, identify the cell’s surfacemarkers, induct osteogenic, induct adipogenic;3. Collected neonatal umbilical cord blood in Sterile environment; Take the method of MACS toobtain the HSCs;Results1. hUC-MSCs were obtained and grew as fibroblast-like cells. After confluence, the cell werespiral shaped.2. The Cell growth curve showed its fast growing, strong proliferative capacity, and that is inline with mesenchymal stem cell growth characteristics.3. The Cycle analysis showed that the cells were accounted for93.29%in the Go/G1phase,accounted for5.26%in G1/M phase, accounted for1.45%in S phase; It had explained most ofthe cells were in a resting stage, which retained the ability to self-renew and separation andthat’s in line with stem cell properties.4. The flow cytometry detected the cells’surface makers, it had a strong expression of CD29,CD105; but not expressing CD34, HLA-DR.5. After inducted by osteogenesis, hUC-MSCs were stained with alizarin red, which showedcalcium nodus was positive; After Inducted by adipogenic, hUC-MSCs were stained with oilred O, which showed the oil was positive.6. The flow cytometry detected the positive CD34+HSCs which were isolated through MASCsmethod from the cord blood accounted for88.3%, the untreated accounted for2.6%,thenegative accounted for1.7; ConclusionsOur laboratory obtained hUC-MSCs by tissue cultivation. It could be confirmed the hUC-MSCs through cell morphology, cell growth curve, cell cycle, the flow cytometryidentifi-cation, osteogenic adipogenic induction; we successfully isolated CD34+HSCs from cord blood by MACS... |
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