Effects Of Opoiods And Their Receptors On Proliferation And Migration Of Human Epidermal Stem Cells In Vitro

Background:Skin wound repair is an important part of wound healing treatment, which concerning with a lot sorts of cells and cytokine. One of the most important part in this process is re-epithelization, which means epidermal cells proliferate and migrate from wound edge or residuary epidermis. While, epidermal stem cells (ESCs) take a very important role in skin re-epithelization process.Epidermal stem cells are specific skin stem cells, derived from the embryonic ectoderm, which have a more primitive differentiation ability, and the characteristics of immature cells, such as, the cells are small, few organelles, in a static state, and mainly locates in the basal cell layer of the skin and hair follicle outer root sheath bulge. Epidermal stem cells have self-renewal ability and powerful proliferation potential, and they are considered to be the ideal seed cells for skin tissue engineering. Epidermal stem cells are progenitors of various epidermal cells, which has capable of bidirectional differentiation. On the one hand, they can downward migration and differentiated into epidermal basal layer, format to hair follicles, on the other hand, they are able to differentiate into epidermal cells, play a key role in the repair of skin damage and the growth of hair follicle. In recent years, it has been widely recognized that the function of adult stem cells is regulated by growth factors, hormones, neurotransmitters and other factors, and the regulation of neuropeptide on various stem cells has become the focus in recent research. Opioid peptides are one kind of neuropeptides, which and their receptors widely exist in various human organs, mediating multiple signal pathways, and regulating many physiological functions.There are5opioid receptors have been proposed, which were μ, κ,δ, σ, ε, but there are only3of them widely accepted, which were p. receptor (mu-opioid receptor, MOR), κ receptor (kappa-opioid receptor, KOR) and δ receptor (delta-opioid receptor, DOR).The recent research has shown that all of the three opioid receptors are expressed in epidermal stem cells, and participate in the process of wound healing, keratinocyte proliferation and differentiation. After the skin damage, expression of opioid receptors on keratinocyte is heightened, and the number of cytokine production is increased. β-endorphin, the ligand of MOR, may stimulate the migration activities of keratinocytes, but dynorphin, the ligand of KOR, under the same conditions, just has a minimal effect. Naloxone, a μ-opioid receptor antagonist, can significantly reduce the migration of human keratinocytes. In a word, there are ample evidences on the regulation of opioid peptides and their receptors on keratinocyte differentiation, either in vitro or in vivo.The study has found that, beta-endorphin and μ-opioid receptors are mainly distributed in the peripheral nerve endings and some junction of dermis and epidermis in the normal rat skin tissue. The expression of μ-opioid receptor and beta-endorphin are enhanced instantly after skin burns damage happened. The change of β-endorphin and p.-opioid receptors in burn wound healing process prompts that they may be take an important part in skin wound healing. But there is no evidence could show that if opioid peptides and their receptors play a role in the proliferation, migration and differentiation of epidermal stem cells so far.Objective:1. To isolate, culture and identify human epidermal stem cells in vitro, observe their normal growth status, restore the seed cells for next research.2. To investigate the effect of activation or inhibition of the three opioid receptors, MOR, KOR and DOR, on the proliferation and migration of cultured human epidermal stem cell.Methods:1. The method of isolation, culture and identification human epidermal stem cells. The fresh prepuce specimens were obtained from young voluntary donations., which were washed3times with PBS, added5ml0.25%Dispase II, and stored at4℃overnight. Remove the dermis from epidermis, and mix the epidermis with4ml0.25%trypsin, after10min digestion,4ml DMEM containing10%FBS is added to termination. Triturate the admixture into single cell suspension, centrifugate them with1000r/min in5min, then add some medium of epidermal stem cells (K-SFM,30μg/ml BPE,5ng/ml hEGF) to suspend cells, which are inoculated to culture flasks that are covered with a type IV collagen-coated. Part of the cells are adherent to the wall10min later, then remove the culture medium with cells that are not adherent, add fresh medium, and put the cell in37℃,5%CO2incubator, change the culture medium every other day.Collect the cells of the second generation, make the cells seeded pieces, which are fixed with4%paraformaldehyde. After being washed with PBS, mouse anti-human integrin β1and mouse anti-human K19monoclonal antibody were added to the cells seeded in pieces, followed by incubating for1h in37℃and washing with PBS, FITC labeled Rabbit anti rat IgG (1:50) and Cy3labeled Rabbit anti mouse IgG (1:50) are added to the cells seeded in pieces, followed by incubating for1h in37℃and washing away with PBS, observe the cells in fluorescence microscope and get photographes immediately. Following sample dyeing processing, the purity of the cells is detected by flow cytometry.2. Observe the effects of opioid receptor agonists and antagonists on the proliferation of hESCs. The hESCs of the second generation are collected and seeded on the culture plates with96holes according to a density of4×103per hole. The cells were divided into7experimental groups, which belonge to3parts.The first part:To explore the effect of activation or inhibition of μ-opioid receptor on the proliferation activity in human epidermal stem cells.μ opioid receptor agonist group (group A1), culture the cells with K-SFM medium containing lnmol/L Endorphin, μ-opioid receptor antagonist group (group A2), culture the cells with K-SFM medium containing10nmol/L Naltrexone and1nmol/L Endorphin, the blank control group (group D),culture the cells with K-SFM medium only.The second part:To explore the effect of activation or inhibition of δ-opioid receptor on the proliferation activity in human epidermal stem cells.δ-opioid receptor agonist group (group B1), culture the cells with K-SFM medium containing lnmol/L [D-Ala2, D-Leu5]-Enkephalin,δ-opioid receptor antagonist group (group B2), culture the cells with K-SFM medium containing1nmol/L Naltrindole and lnmol/L [D-Ala2, D-Leu5]-Enkephalin, the blank control group (group D),culture the cells with K-SFM medium only.The third part:To explore the effect of activation or inhibition of κ-opioid receptor on the proliferation activity in human epidermal stem cells. κ-opioid receptor agonist group (group C1), culture the cells with K-SFM medium containing2nmol/L Dynorphin, κ-opioid receptor antagonist group (group C2), culture the cells with K-SFM medium containing5nmol/L5’Guanidinonaltrindoledi and2nmol/L Dynorphin, the blank control group (group D),culture the cells with K-SFM medium only.Starting by the treatment day, cells in10holes are taken from each group to do proliferation assay with MTT method in every24h. According to the20ul per hole, the MTT solution is added into each plate hole, and then them are placed in the incubator for4h. Remove the medium and MTT solution, add150ul DMSO in each hole. Shock the culture plate for10min, and the absorbance of each hole are detected by Microplate Reader.Measurement continues5consecutive days, the absorbance is recorded and cells growth curve is made to analyze the experiment result.3. Observe the effects of opioid receptor agonists and antagonists on the migration of hESCs. The hESCs were seeded on the culture plates with6holes, cultivated with K-SFM medium until the cells are covered the plate bottom completely. Scratch the full cells cover with a200μl sterile pipette tip in clean workbench to make monolayer hESCs defect model. PBS washes away the exfoliated cells, and then change the culture medium.The monolayer cell defect models are divided into7groups, which are divided to3parts, the same as the previous grouping.According to the grouping, we join in the experimental drug in K-SFM medium as same as the previous experiment.We observe the defect area and the cells that migrated to the defect regent every24h under the microscope and photographed to record the scratch width and area of wound surface. The observation and record are continued consecutive96hours. The photographs are used for accounting the area of wound changing by using Image-Pro Plus software. The rate of wound healing is calculated according to the following formula:wound healing rate=the area of being occupied by the cells that migrating to the wound surface/original wound area×100%.Results:1. Results of isolation, culture and identification of hESCs. Primary human epidermal stem cells are single or aggregate growth in the first3days, later they are showed clonal growth. They are seen cobblestone-like on the bottom of culture plates on the7th day, and9～10days later,80%of cells are fusion, which means they should be passaged. The second generation of human epidermal stem cells are identified by immunofluorescence staining. It is visible that both integrin β1and K19are positive markers, suggesting the cultured cells are human epidermal stem cells. Integrin β1positive staining cells account for (95.6±0.76)%of all cells through the flow cytometry, while K19positive cells account for (93.7±0.81)%, which suggests that the cultured cells are human epidermal stem cells, with a high purity.2. Effects of three kinds of opioid receptors agonists and antagonists on the proliferation of epidermal stem cells. The absorbance of consecutive5days is detected by MTT method, which suggests the activation of MOR or DOR could promote the proliferation of human epidermal stem cells, and the blockade of MOR or DOR, the proliferation activity of human epidermal stem cells is inhibited. No significant effect of the proliferation activity change on epidermal stem cells by activating or blocking KOR.3. Effects of three kinds of opioid receptors agonists and antagonists on the migration of human epidermal stem cells. Human epidermal stem cells in monolayer cells defect model are found to migrate to the defect region24hours after the treatmen in all groups, but the cells in MOR agonist group across the wound edge counts more than the control group, MOR antagonist group less than the control group. While, DOR agonist group counts more than the control group, DOR antagonist group less than the control group. There is no significant differences are found when compared with KOR agonist group or KOR antagonist group to control group. The exactly data of rate of wound healing after72hours is as follows, group D is81.05±0.56%, group Al is89.46±0.73%, group A2is76.10±0.32%. The rate of wound healing in group A1is significantly higher than that in group D, while, group A2is lower than group D, the differences are statistically significant (P<0.05). Group B1is85.79±0.49%, group B2is77.23±0.54%. Group B1is significantly higher than group D, and group B2is lower than group D statistically (P<0.05). Group C1is81.65±0.35%, group C2is80.14±0.71%, compared with the control group, there are no significant differences between the groups in batch3(P>0.05).Conclusions:1. We separate the epidermal cells by digestion method with neutral protease and trypsin. hESCs are purified and obtained by time difference in adherent step, as for human epidermal stem cells can adherent to type IV collagen more quickly than many other epidermal cells. The cells always grow well feeded with serum-free culture medium K-SFM.Immunofluorescence staining method is used to detect the expreson of cell surface K19and integrin β1on hESCs. The result suggests that the cells cultured in this study are human epidermal stem cells.2. This study show to us that the activation of MOR or DOR could promote the proliferation and migration of human epidermal stem cells, and the blockade of MOR or DOR, the proliferation and migration activity of human epidermal stem cells is inhibited. There is no significant effect of the proliferation or migration activity change on epidermal stem cells by activating or blocking KOR.