| Objectives: To Observe the inductive ability (in vivo and in vitro) of isolated dermal papilla (DP) and dermal sheath cells ( DSC) to form hair follicle, we targeted at establishing three-dimensional hair follicle(HF) model in vitro and inducing hair follicle reformation in vivo, and attempted to discuss the biological properties of HF and the influence-agents during its forming.Methods: We isolated and cultured the dermal component such as DP cells and DSC from human hair follicles and the epidermal component such as interfollicular keratinocyte(KC) and hair keratinocyte (hKC) via enzyme digestion. Then we added DP and DSC into compound chitosan-collagen-gel-base and mixed to form mesenchymal cell-population gel. After the gels coagulated under room temperature, we inoculated KC or hKC onto the surface of the gels, and a three-dimensional HF reconstructive model was founded, then incubated under 37℃. After 4 weeks culture, we took out the gels,fixed in Bouin's solution, cut into the pathological sections, stained with H.E.,　immunohistochemistry, to observe the reformation of HF. Freshly isolated HF dermal cells (DP, DSC) and HF epidermal cells(HF bulb keratinocyte ,KC and hKC) were implanted into the subcutaneous or the freshly isolated DP was grafted between the epidermal and dermal of the nude mice. After 8 weeks breeding, the nude mice were killed by the method of ansethesia. The implanted locus were autopsied and fixed in Bouin's solution ,cut into pathological sections, stained with H.E.,immunohistochemistry,to observe the reformation of HF. Mixed the DP with KC to co-culture (three-dimensional and two-dimensional), it was to observe their interactions. We used DPC, DSC and FB to produce skin equivalences (SE) with KC respectively, aimed at discussing the contraction of three SEs following the time, after about 7 days culture, the SE was sectioned and the pathology and immunochemistry study were done, we then observed the three cells' effect on the KC differentiation.Results: After 4 weeks culture, we found that there were many round-shape or oval-shape cell-masses in the three-dimensional hair follicle (HF) reconstructive model in vitro, and the K6 stained positively. The DPs were implanted into nude mice between the epidermal and dermal for 8 weeks long, we could observe the hair folliclelike structure, sodid the DP, DSC, HF bulb keratinocyte, KC and hKC implanted into the subcutaneous of the nude mice. The immunochemistry study on the structure were similar with the normal scalp HF. When the DP and KC were co-cultured in 6-well culture plates (two-dimensional), they could stimulate each other. When they were co-cultured in gel (three-dimensional), the DP has the ability to induce KC to migrate around itself and to form KC clones. The DP,DSC and FB all have the ability to promote the differentiation of KC, and the KC displayed layered-structure on or above the gel surface. But the ability of contracting the gel of them was similar, with no obviously statistics difference. Conclusions: 1. We adopted the method of low-revolution and mid-revolution crossing-centrifugation, the purity of isolated DPs was rised,we also redused the losing of DPS during the centrifugation,by this method,the DPs could cohere to the wall of the culture flask in 24 hours at 100%; 2. We froze and resuscitated the sub-organ of DP,by this method we constructed DP cell bank; 3. The cultured DSCs in vitro have the aggregative growth behaviour, its shape is similar to isolated DP,this implies that the DSC is the storage-pool and source of DPC in vivo;4. DP in vitro has the ability to stimulate the growth of KC, in gel DP can induce KC migrating and form cell-clone;DPC and DSC that lose aggregative growth behaviour both have the ability to promote the differentiation of KC, so as the DFB;5. DP interacts with auto-epidermal cell in three-dimensional model,thus many cell-masses forming,and K6 stained positively,which implies the cell-masses have the tendency to differentiate into HF;6. DP plays a center role in t...
|
PDF Full Text Request