Isolation, Culturing And Characterization Of Mesenchymal Stem Cells Derived From Human Umbilical Cord

Cell therapy has more ten years progress. At first, scientists abundancely studied immunocyte, such as cytokine-induced killer cells theating cancer that had considerable prospect; Moreover now scientists to pay close attention to human umbilical cord mesenchymal stem cell(hUC-MSC), because hUC-MSC has more good clinical practice proficiency than embryonic stem cell and bone marrow mesenchymal stem cell, thereby become reseaching hotspot of human medicine and tissue engineering.The puepose of this study was exploring a more effective isolation and culturing method of hUC-MSC, and further studied biological characterization of hUC-MSC. Passing seting up optimized test method, may offer reference according of operating specification. Method: trial random chose twenty full term natural labour healthy fetus umbilical cords, adopted manual mechanical separating process and the combined enzyme digesting way to obtain mesenchymal cell; Umbilical cord mesenchymal cell obtained was cultured by optimized DMEM/F12; The proliferation activity of several passage umbilical cord mesenchymal cell was detected by MTT, And the growth curve of mesenchymal cell was drew; The growth cycle and surface makers of several passage umbilical cord mesenchymal cell were detected by flow cytometry; RT-PCR amplified cancer suppressor gene P53, cancer gene C-myc and maintaining stem cell undifferentiation state and pluripotency gene OCT-4; Result: the shape of umbilical cord mesenchymal cell cultured was fusiformis presents filamentary and appeared running water or vortex growing. Umbilical cord mesenchymal cell grew quickly; More than 80% umbilical cord mesenchymal cell was on G0/G1 period; Umbilical cord mesenchymal cell expressed homogeneous high CD29 CD70 CD106 CD90 and CD44, while low expressed CD45 CD34 and HLA-DR; P53 and C-myc gene appeared clear amplified bands, OCT-4 gene expressed at different passage; This research conclusions : 1)30 centimeters umbilical cord colloid was separated within one hour, obtaining 9×10~4 mesenchymal cell each centimeter umbilical cord; It possessed morphological characteristics proliferation activity, Surface markers of umbilical cord mesenchymal cell were correspond to hUC-MSC by culturing identify, Umbilical cord colloid mesenchymal cell was primary hUC-MSC; 2)The phenomenon that P53 C-myc and OCT-4 gene were detected by RT-PCR. This research significance: this reseach suggest it is very necessary to overall detect the cancer genes and cancer suppressor genes as well as senescence of hUC-MSC when hUC-MSC will be clinical application; This trial could successfully culture hUC-MSC, offered reseaching foundation for clinical therapy, offered reference according for avoiding producing tumor by transplanting hUC-MSC therapy in vivo.