Regulation Of Human Keratinocyte Proliferation By The Direct Contact Effect With Human Fibroblast

The extensive skin defects caused by large, full-thickness burns threaten the lives of burn patients severely. How to cover the extensive burn wound in time is still challenging not only the victims but also the surgeons due to the short of the patient's own health skin to transplant. Experimental and clinic studies show that autograft of the cultured keratinocyte sheets (CKS) is a promising technique for skin restoration. A stamp-sized biopsy skin could be cultured and grow to a big piece of CKS, which could cover thousands-time -sized burn wounds. Unfortunately, there are some major limitations in this technique, which obstructs the application of CKS in clinic, such as the period for preparing CKS is too long; it is not inconvenient to graft the CKS because of their mechanical instability (high contractility, high friability, too thin);the healing wound is too fragile, and so on. Therefore, it is necessary for burn injury research to overcome or limit these shortcomings.Keratinocyte and fibroblasts are two main cells involved in wound healing, and human fibroblasts can produce a multitude of fiber and matrix, which was believed to accelerate the growth and proliferation of keratinocyte. Herein, adding certain ratio of fibroblasts to coculture with keratinocyte may not only improve the abradability and elasticity of CKS, but also increase the proliferation of keratinocyte. It has been proved that human fibroblast could enhance the proliferation of human keratinocyte via paracrine secretory pathway and via cell-matrix interactions, but the effect of the direct contact between these two kinds of cells on human keratinocyte proliferation is still unknown. So the effect on human keratinocytes proliferation through direct contact with fibroblasts was investigated in this study.Objectives:1. To observe the regulatory effects of the direct contact with original/subcultured fibroblasts on the proliferation of original/ subcultured human keratinocytes.2. To explore the possible mechanisms of promoting human keratinocyte proliferationby direct contact with fibroblasts. Materials and Methods:1. Isolation, culture and identification of human keratinocyte: human keratinocytes from skin specimen were isolated by a routine method with dispase and trypsin, and the isolated keratinocytes were cultivated in keratinocyte serum free medium. The cells were identified by immunofluorescence with a mouse anti-human keratin specific monoclonal antibody. MTT assay was used to survey and map the growth curve of the cells, and the cell cycle was detected with flow cytometer.2. Preparation, culture, and verification of human fibroblasts: the epidermis in skin specimen was removed with dispase firstly, then human fibroblasts were isolated with collagenase, and the isolated cells were verified by S-P method of immunocytochemistry with a mouse anti-human vimentin monoclonal antibody and a mouse anti-human keratin monoclonal antibody. The growth curve was surveyed as that for keratinocyte.3. Observation of the direct contact effect on original human keratinocyte proliferation by fibroblasts: human original keratinocytes and human fibro- blasts were co-cultured with different ratios. The keratinocyte proportions, adherence ratios, cell cycle and harvesting quantity were evaluated respect- tively and compared with that in mono-culture of human kerationocyte.4. Survey of the direct contact effect on subcultured keratinocyte proli- feration by fibroblasts: three culture systems, mono-culture system of sub- cultured human keratinocytes, indirect co-culture and direct co-culture system of human fibroblasts and sub-cultured human keratinocytes, were established. The concentrations of EGF and b-FGF in the supernatant from different culture systems were detected by enzyme-linked immunosorbent assay; the expressing levels of keratin 19 in different groups were determined by cytoimmunochemistry. The harvesting quantity and cell cycle in different groups, and the proportions of keratinocyte in direct co-culture system were measured respectively.5. Exploration of the mechanisms to the direct contact effect on sub-cultured keratinocyte by fibroblast: Inhibitors were used to explore the role of different signal pathways in this direct contact effect. PD98059 and SB20358ERK were added to the culture systems to block ERK and p38 MAPK pathways respectively. The changes ofkeratinocyte proliferation, concentrations of EGF and b-FGF, and expressing levels of keratin 19 in different culture systems and groups were checked and compared with their control groups. Results1. The cells isolated from skin specimen with the methods described above were identified as keratinocyte and fibroblast respectively, and the purities were measured as high as 99%, and the cells grew and proliferated very well.2. The proliferation of original human keratinocytes could be enhanced by the direct contact with fibroblasts in the coculture system. It was found in our experiment that the most effective seeding proportion of these two cells was 40/l(HK/HF).3. The direct contact with fibroblasts also could promote the keratinocyte harvesting quantity, keratin 19 expression and b-FGF production from subcultured human keratinocytes, while there was no change of EGF production.4. This positive regulation on keratinocyte proliferation by direct contact with fibroblasts could be attenuated partially by either blocking ERK pathway with PD98059 or blocking p38 MAPK pathway with SB20358, and it was found that PD98059 was more effective than that of SB20358. Which suggest that both ERK pathway and p38 MAPK pathway were involved in the signal transduction of direct contact of HF and HK.Conclusions:1. The direct contact with fibroblast could enhance the keratinocyte proliferation, which may be helpful for CKS preparation and application in clinic.2. ERK and p38MAPK pathways may participate in this direct contact effect on keratinocyte proliferation by fibroblast.