▎ 摘　　要

NOVELTY - Separating, culturing and amplification of human umbilical cord mesenchymal stem cells comprises (i) cleaning the blood and debris attached to the aseptically obtained umbilical cord, soaking in alcohol, washing with cleaning solution 3 times, cutting along the inner side of the ligation ends on both sides of the umbilical cord discarding the edema or blood clotting part, cutting the remaining umbilical cord into a length of 6 plus minus 2 cm, squeezing out the blood in each section of the umbilical cord, and transferring to another petri dish containing cleaning solution; and (ii) taking out all the Wharton's jelly in the umbilical cord, washing with a physiological saline solution, cutting the Wharton's jelly into small pieces of tissue, filtering the red blood cell lysis buffer with a 0.22 mu m microfiltration membrane at room temperature, adding filtered red blood cell lysis buffer, and treating the small tissue, collecting the treated small pieces of tissue by centrifugation and washing. USE - The method is useful for separating, culturing and amplification of human umbilical cord mesenchymal stem cells. DETAILED DESCRIPTION - Separating, culturing and amplification of human umbilical cord mesenchymal stem cells comprises (i) cleaning the blood and debris attached to the aseptically obtained umbilical cord, soaking in 75% alcohol for 1 minute, washing with cleaning solution 3 times, cutting along the inner side of the ligation ends on both sides of the umbilical cord discarding the edema or blood clotting part, cutting the remaining umbilical cord into a length of 6 plus minus 2 cm, squeezing out the blood in each section of the umbilical cord, and transferring to another petri dish containing cleaning solution; (ii) taking out all the Wharton's jelly in the umbilical cord, washing 3 times with a physiological saline solution, cutting the Wharton's jelly into small pieces of tissue about 2-4 mm3, filtering the red blood cell lysis buffer with a 0.22 mu m microfiltration membrane at room temperature, adding filtered red blood cell lysis buffer with a volume 3.5 times the volume of the small tissue, and treating the small tissue for 7-9 minutes, collecting the treated small pieces of tissue by centrifugation and washing 2-3 times with phosphate buffered saline, where the red blood cell lysis buffer contains 5 g/l ammonium chloride and 0.1 mMol EDTA disodium, and pH 7.2-7.4; (iii) adding collagenase digestion solution with a volume of 2-3 times the volume of small tissues, transferring to a 37 degrees C, 5% carbon dioxide incubator, and transferring from the incubator after 8-12 hours to a clean workbench, and diluting by adding phosphate buffered saline, filtering with a sterile sieve, collecting the filtrate into a 50 ml centrifuge tube, washing with phosphate buffered saline, and centrifuging to remove residual enzymes, where digestion solution is a balanced salt solution containing 1% type IV collagenase, 0.5% hyaluronidase, 300 U/ml DNA enzyme and 2% serum substitute; (iv) isolating and culturing the primary cells, flicking the cell pellet at the bottom of the centrifuge tube, adding serum-free medium of mesenchymal stem cells to obtain a cell suspension, and spreading the cell suspension in a cell culture flask, placing the tissue in the cell culture box until the tissue fits to the bottom of the bottle, and adding primary cell culture medium for umbilical cord mesenchymal stem cells for primary culture, where culture medium for umbilical cord mesenchymal stem cells does not contain animal-derived components and human platelet lysate and the serum-free medium for mesenchymal stem cells comprises 0.1 pts. vol. beta -mercaptoethanol, 1 pts. vol. non-essential amino acid aqueous solution, 10 pts. vol. serum substitute, 89 pts. vol. alpha -MEM/DMEM-F12, and 10 ng/ml basic fibroblast growth factor; (v) after the cell confluence reaches about 80%, using 0.215 wt.% trypsin, tapping the side wall of the culture flask during the digestion period, collecting the cells by centrifugation, and dividing the collected cells according to 5000 inoculation/cm2; (vi) collecting the umbilical cord mesenchymal stem cells and performing osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments, culturing osteogenic differentiation with an osteogenic induction fluid, which includes: 20 mu g/ml insulin, 0.1 mu g/ml transferrin, 0.6ng/ml BMP-4, 5 ng/ml growth hormone, 100 mu g/ml glutathione, 1.2 mM L-glutamine, 60 mu M beta -mercaptoethanol, 0.12 mM non-essential amino acid aqueous solution, and 15 mu M nano ferric oxide and nitrogen-doped graphene mixture, and medium is Dulbecco's modified eagle medium (DMEM); and the adipogenic differentiation adopts an adipogenic induction solution, which contains: 10 mol/l dexamethasone, 5 mg/l insulin, 0.5 mmol/l 3-isobutylmethylxanthine, 60 mu mol/l indomethacin, and 10 vol.% fetal bovine serum, and the medium is DMEM; the chondrogenic differentiation adopts a chondrogenic induction culture medium containing 50 mg/ml bullacta mollusc polypeptide, 6.5 mg/l insulin, 6.5 mg/l transferrin, and 20 mu g/l transforming growth factor beta 1, DMEM-F12 medium with 0.5 mu mol/l dexamethasone, 50 mg/l vitamin C, 6 mu M isomalidin, 5% fetal bovine serum and 1% double antibody, and inducing and culturing, changing medium every 5 days, after 14 days of culture, removing cartilage induction culture medium, adding 500 mu l RNA extraction reagent, until a clear and non-sticky liquid is formed, adding 180 mu l chloroform, shaking vigorously and mixing, and allowing to stand at room temperature, centrifuging, transferring upper transparent RNA aqueous phase to a another RNase-free EP tube, adding equal volume of isopropanol and mixing, discarding the supernatant after centrifugation, and saving the precipitate, adding 60 mu l 50% ethanol and centrifuging, discarding the supernatant, drying at room temperature for 4 minutes, extracting RNA, and detecting RNA quality and concentration, reverse transcribing extracted RNA to obtain a cDNA sample, amplifying cDNA sample by PCR, staining with 1% alicin blue glacial acetic acid solution for 30 minutes at room temperature, and subjecting to agarose gel electrophoresis, and observing result by an electrophoresis gel imager. An INDEPENDENT CLAIM is also included for: a system for separating, culturing and amplification of human umbilical cord mesenchymal stem cells.