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Cell therapy has gone through 10 years of history, at first scientist extensive research on the immune cells, such as cytokine-induced killer (CIK) cells to treat cancer has considerable prospects; the scientific community of the human umbilical cord mesenchymal stem cells (Human The umbilical cord mesenchymal stem cell, hUC-the MSC) of more concern, hUC-MSC than embryonic stem cells (ESC) and better clinical application potential of bone marrow mesenchymal stem cells (BM-the MSC), etc., and thus become human regenerative medicine and tissue engineering research hotspot. The purpose of this study is to explore more effective hUC-MSC isolation and culture, and further study of the biological characteristics of hUC-MSC, through the establishment of the optimization of the experimental method for regenerative medicine research and clinical applications operating standards and reference. METHODS: The experiment randomly selected full term healthy umbilical cord, take the hand mechanical separation method and the combination of three enzymes digestion from umbilical cord glial release of human umbilical cord mesenchymal cells; complete medium with optimized purification train each generation; MTT assay. cord mesenchymal cell growth and proliferation activity and growth curve; flow cytometry to umbilical its growth cycle and surface markers; RT-PCR amplification of gene P53, C-myc and OCT-4; RESULTS: The cultured cord mesenchymal cell morphology of spindle-shaped, fibrous-like, and a water-like or whirlpool-like growth; growth and proliferation of faster the 7th generation umbilical cord mesenchymal cells index doubling time less than 30h; 80% or more cells in the G0/G1 phase; cultured mesenchymal cells in the umbilical cord uniform and stable high expression of CD29, CD70, CD105, CD90 and CD44, while almost no expression of CD45, CD34 and HLA-DR; expression of P53 gene in generations umbilical cord mesenchymal cells, C-myc gene significant expression of the 4th generation and the generation of 18, OCT-4 gene in different algebraic expression; conclusions: 1) 1h detachable 30cm Wharton's jelly, access to a large number of umbilical cord mesenchymal cells (9 × 10 ~~ 4 / cm), the culture tests prove morphological characteristics hUC-MSC proliferative activity of surface antigens consistent is the original hUC-MSC; 2) detected by RT-PCR generations umbilical cord mesenchymal cells P53, C-myc and OCT-4 gene, found that less than 10 generations of culture hUC-MSC status than normal. The significance of this study: Tip in clinical application hUC-MSC, a comprehensive inspection of its oncogenes and tumor suppressor genes and aging apoptosis system detects that it is necessary; experimental clinical need for safe and reliable for the short-term culture amplified MSC provide research-based, to provide a reference for in vivo transplantation therapy to avoid hUC-MSC produce tumorigenic.
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