In Vitro Human Placenta Mesenchymal Stem Cells Into Cartilage Cells

ObjectiveThe mesenchyma stem cell (MSCs) belongs to mesoderm more than kind of competent cells, mainly exists in the knot contracts the organization and the organ mesenchymal, the content is richest by the myeloid tissue. In recent years, along with the people to the mesenchyma stem cell biology characteristic and the function deep research, already successfully from the person marrow, the circumference blood, the muscle, the fat, the navel blood, the amniotic fluid and the embryo organized to separate and to appraise MSCs. The placenta maintains the parent substance and the embryo oxygen and the nutrients exchange important temporary reproductive organ as the embryonic development, by stems from the cytotrophoblast and the extraembryonic mesoderm embryo dense chorion and the parent substance womb base decidua is composed together, regardless of from the dissection structure in the growth behavior, has contained a weaker embryo and tends the mature adult stem cell ingredient; The placenta after the embryo childbirth leaves namely completes the mission, becomes "abandons" the thing, cannot involve ethics morals question to its research, therefore present has become seeks for the human mesenchyma stem cell to originate and to enhance the clinical practice effect newly the research hot spot. This article the mesenchyma stem cell which originates on the placenta-derived mesenchymal stem cells (PMSCs) in vitro separation and the raise method and PMSCs in the monolayer culture to the cartilage cell direction detection differentiation's condition do elaborate.Methods1. Human placenta mesenchyma stem cell's extraction Takes the full-term c-section embryo's placenta, the peeling subfield decidua, cuts takes under the decidua to organize, PBS flushes two times, the room temperature settles 10min, will organize the block cuttings approximately 1mm³, puts in the culture dish base evenly, applies the 10% DMEM culture medium raise in 37°C the 5% CO₂ incubator, each 3d trades the fluid one time, treats the cell when reaches 80%⁹0% along the culture dish base growth area, according to conventional method transfer of generation.2. Placenta cell phenotype determinationTakes the 3rd generation of cell, digests 3 min after 0.25% trypsin, the adjustment cell number for 1×10⁶/L, after joining 5μL the mouse blood serum seals up 15min, joins the mouse anti-person PE mark the monoclonal antibody, on the ice evades the light to respond that 20min, PBS washes 2 from now on, 4°C 1000r/min centrifugal 5min, after abandoning clear add-on enters 200μL cold PBS blows bugle and beats drums mixes uniform, flows the type cell meter examination.3. Induction differentiationAfter mesenchyma stem cell pancreas enzyme digestion which, originates the 3rd generation of placenta makes the cell to hang the fluid, after the offcenter abandons clear, PBS to wash 3, respectively by 2×10⁴/cm² the density vaccination in advance in six cell nutrient fluids which smudges by 0.1% gelatin, after treating the cell pastes the wall, changes the cartilage derivative, puts in 37°C in 5% CO₂ incubator to raise, each 3d trades fluid one time.4. Toluidine blue dyeingTakes raises 21d two groups of cells, after PBS trades 2, after 4% paraformaldehyde fixed 15～30min, PBS washes 3, after 1% toluidine blues dye 2⁴h, 95% ethylalcohols to wash under 3, light microscope to observe.5. Immunocytochemistry examination the II collagen expressesThe concrete step according to the SABC dyeing reagent box instruction booklet operation, and abandons in the orifice the nutrient fluid, PBS (0.02mol/L pH7.2～7.6) the fluid washes 3, 4% paraformaldehyde fixed 30min, 0.3% H₂O₂ deactivation endocardial enzyme, after BSA seal, joins 1:200 dilution separately rabbit anti-person-anti-collagen type II, 4°C the foster over night, fosters the 1h, DAB developer coloration with goat anti-rabbit-mouse IgG, after the phenodin duplicate dyes, under the light microscope observes.ResultsThe organization block raises about 11d, obviously the few cells crawl, assumes the circular or the polygon, increases along with the cell number, the cell assumes the whirlpool shape to grow, the cell body changes tall and slender, the shape becomes the ciliary cell similarly, raises about 3w, the cell may reach 80% fusions, after the cell transfers generation pastes the wall, grows into the spindle-shaped cell, this cell passed on to 20 generations still might grow stably. Flows the type cell meter examination to demonstrate that originates the 3rd generation of cell which organizes in the person placenta expresses CD13, CD44, CD73, CD90, CD166, HLA-AB, does not express CD34, CD45, HLA-DR, originates the mesenchyma stem cell with the marrow to have the similar cell surface symbol. The cell when the induction starts, the cell multiplication speed is slow, induces when one week namely stopped multiplying, the cellular form has the change, the cell volume fill-out, the partial cells by original spindle-shaped turn the polygon gradually, the cellular form vary, the entire induction differentiation process cell has not transferred generation. The cell when induces 21d the toluidine blue dyes the obvious intercellular space matrix to assume the royal purple. The II collagen immunocytochemistry examination discovers in the cell cytoblastema to have the yellowish brown pellet masculine result.Conclusion1. The human placenta-derived mesenchymal stem cells may succeed in vitro raise and increases massively, the biology character is stable, organizes one of project fine seed cell origins. 2. This experiment under the monolayer culture condition, succeeds the human placenta mesenchyma stem cell induction splits up the cartilage cell. After induction raise forms the cartilage cell has cartilage cell's shape characteristic, and can secrete the cartilage cell specificity II Collagen.