Preliminary Study Of Lesional Psoriatic Epidermal Stem Cells Isolation, Culture, Identity And Biological Characteristics Under Different Conditions

Objective: under different conditions, put psoriasis lesional epidermis stem cells (EpidermalStem Cells, ESCs ) in vitro separation, purification, cultivation and identification, to compareand find optimal culture conditions. Preliminary study are made about biological characteristicsof psoriatic epidermal stem cells, to provide the in-depth study basis of psoriatic lesions.Methods: 37 case of the skin lesions of psoriasis specimens, 15 cases of normal human foreskinspecimens. ESCs separation, screening, culture:①Dispase enzyme, trypsin, 4℃for night,37℃for 3 hours, these four digestion conditions are selected at random, make comparison ofseparation efficiency.②Preparation of single cell suspension, a comparison of total number ofskin cells, living cells at the unit area of psoriatic lesions and normal skin.③a.Separation ofepidermal stem cells of different concentration, different adhesion time IV collagen adhesion, tocalculate the epidermal cell adhesion rate.④Cell growth was observed under invertedmicroscope.⑤ESCs surface markers: a.Immunofluorescent cytochemical assay is used todetect the keratin 19(K19), keratin 10 ( K10 ) expression of ESCs of the original generation 12h、6d、12 d. b.Flow cytometry is used to examine the expression of early adherent cells, cellCD29 ( integrinβ1 ) , CD71, P63of primary 12 days、P 1generation 7 days. Cytometry method isadopted to determine ESCs cell growth curve.Results : ESCs separation, screening, culture:①Under 2.5g / L Dispase II 5%CO2, 4 degreesovernight conditions, true epidermal separation efficiency is high, the total efficiency is as highas 100%.②The number of psoriatic lesional epidermis cell per unit area and live cells arebigger than normal foreskin samples, the results showed significant difference (P < 0.05 ).③a.The IV type collagen adhesion 10min, can gain higher purity of ESCs can be gained; b. the50mg / L and 100mg / L concentration of collagen type IV epidermal cell adhesion rate had nosignificant difference (P > 0.05 ).④Under the inverted microscope, psoriasis ESCs presented thesilent phenomena at the first three days, four days later, speed growth starts, the largest cloningformed by a single ESCs is far greater than the prepuce group, after the culture for a few days, itis easy for psoriasis ESCs to be differentiated.⑤ESCs surface markers: a.Theimmunofluorescent cytochemical assay: K19 shows bright fluorescent green particlesdeposited on the cell surface and in the cytoplasm, expressing positive; no fluorescent particledeposition for K10. b. The examination through the flow cytometry of CD29, P63 highexpression, and CD71 low expression, in combination with immunofluorescent cells method, to determine that cultured cells are epidermal stem cells. Cell counting method for drawing ESCsgrowth curve ,showed that psoriasis ESCs doubing speed within short time.Conclusion:①Successful separation, screening, training psoriasis ESCs, the conditions are asfollows: 2.5g / L, dispase II of the enzyme 4°C overnight digestion and separation of the trueepidermis, 2.5g / L trypsin digestion system cell suspension, 50 mg / L, collagen type IVadhesion 10min screeningsuccessfully sorting ESCs, steady growth.②Lesional psoriaticepidermal cell number,ESCs proliferation,cell morphology,and different from normal skin;③thesingle cell of psoriasis ESCs demonstrates a strong cloning capability, fast growth, and easydifferentiation.