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Objective:To evaluate the skin development and repair process of X-ray radiation damage in rat with non-invasive two-photon excitation fluorescence (TPEF) imaging technology in vivo. Methods:Totally 24 SD rats were randomly divided into four groups including X-ray irradiated group (25, 35 and 45 Gy) and non-irradiation control group. At different times after irradiation, the degree of skin injury was evaluated, and the pathological changes of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and collagen fiber fluorescence signals in epidermal cells were detected in vivo by TPEF imaging technology. Results:At 10 d post-irradiation, the skin of irradiation groups showed erythema and desquamation. At 15-20 d post-irradiation, the skin of radiation groups developed progressive exudation, edema and ulcers with increasing radiation dose. On day 25, the skin began to repair in the 25 Gy group, however, the skin of other groups still had exudation and ulcers. On day 10, NAD(P)H fluorescence signal in epidermal cells of irradiation groups decreased and the fluorescence signal of collagen fibers in papillary layer and reticular layer of irradiation groups reduced, which were significantly lower than that of normal control group ( t=24.145, 28.303, 26.989, 6.654, 7.510, 7.997, P<0.05). On day 30, fluorescence signal of NAD(P)H and collagen fibers in epidermal cells and dermis began to repair, the cell from stratum granulosum, stratum spinosum, and stratum basale in the 25 Gy group showed fluorescence signal, the other groups did not show. The fluorescence signal of collagen fibers in the 25 Gy group were gradually increased in papillary layer and reticular layer, however, they were significantly lower than normal control group ( t=115.133, 17.431, P<0.05), the skin of 45 Gy group did not show fluorescence signal of collagen fibers. Conclusions:The damage and repair process of epidermal cells and dermal collagen fiber can be detected noninvasively by TPEF imaging technology after X-ray irradiation in vivo.
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BACKGROUND: There are various methods for the treatment and repair of deep second-degree burn wounds, which have diverse effects and have no uniform standards. Therefore, new technologies need to be developed. OBJECTIVE: To observe the therapeutic effect of controlled debridement and traditional treatment on deep second-degree burn wounds. METHODS: A total of 80 patients with deep second-degree burns from June 2015 to June 2018 were enrolled and divided into a positive control group and a controlled debridement group, with 40 patients in each group. The positive control group was coated with Bangerkang burn bacteriostatic cream, and the oil gauze was pressure-wrapped. The dressing was changed at 3, 6, 9, and 12 days after the injury. In the controlled debridement group, epidermal cell suspension was prepared according to the requirements of wound preparation and was sprayed onto the wound surface. The wound was then covered with Recell Kit self-contained protective film. The film was removed to keep the wound dry on the 3rd day after operation, and then the wound was observed at 6, 9, and 12 days after operation. The study protocol was approved by the Ethics Committee of the Xijing Hospital of the Air Force Military Medical University. All patients volunteered to participate in the study and sign an informed consent. Patient information was registered online and appropriate treatment was performed according to a random (software online) assignment. RESULTS AND CONCLUSION: At 3, 6, 9, and 12 days after operation, the bacterial content, wound pain score, wound infection score and pro-inflammatory factor level in the controlled debridement group were significantly lower than those in the positive control group (P < 0.05). Moreover, there was no complication in both groups. These findings reveal that epidermal cell implantation combined with controlled debridement for deep second-degree burn wounds can achieve remarkable outcomes, which can significantly accelerate wound healing, reduce infection and alleviate the suffering of patients.
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Objective@#To explore the effects of dendritic epidermal T cells (DETC) on proliferation and apoptosis of epidermal cells in wound margin of mice and its effects on wound healing.@*Methods@#Twenty-eight healthy specific pathogen free (SPF) C57BL/6 wild-type (WT) male mice aged 8-12 weeks and 60 SPF T lymphocyte receptor δ-knockout (TCR δ-/-) male mice aged 8-12 weeks were selected to conduct the following experiments. (1) Eight WT mice were selected to isolate epidermal cells and primarily culture DETC according to the random number table. Morphological observation and purity identification of DETC by flow cytometer were detected immediately after culture and on culture day (CD) 15 and 30, respectively. (2) According to the random number table, 5 WT mice and 5 TCR δ-/- mice were selected and enrolled into WT control group and TCR δ-/- group. Round full-thickness skin defect with diameter of 6 mm was made on the back of each mouse. The wound healing condition was observed immediately after injury and on post injury day (PID) 2, 4, 6, 8, 10, and the percentage of residual wound area was calculated. (3) Mice were selected to group and reproduce model of full-thickness skin defect as in experiment (2). On PID 3, the tissue of wound margin was collected for hematoxylin eosin staining, and the length of new epithelium was measured. (4) Mice were selected to group and reproduce model of full-thickness skin defect as in experiment (2). On PID 3, epidermal tissue of wound margin was collected to determine expression of proliferating cell nuclear antigen (PCNA) using Western blotting for evaluation of proliferation of epidermal cell. (5) Mice were selected to group and reproduce model of full-thickness skin defect as in experiment (2). On PID 3, epidermal tissue of wound margin was selected and digested into single-cell suspension, and apoptosis of cells was detected by flow cytometer. (6) Forty TCR δ-/- mice were selected to carry out the same treatment as in experiments (2)-(5). According to the random number table, these mice were enrolled into TCR δ-/- control group and TCR δ-/-+ DETC group, with 5 mice in each group for each experiment. Round full-thickness skin defect was made on the back of each mouse. DETC in the number of 1×105 (dissolution in 100 μL phosphate with buffer purity above 90%) were injected through multiple points of wound margin of mice in TCR δ-/-+ DETC group immediately after injury, and equal volume of phosphate buffer was injected into mice of TCR δ-/- control group with the same method as above. Data were processed with one-way analysis of variance for repeated measurement, t test, and Bonferroni correction.@*Results@#(1) Along with the culture time elapse, the number of dendritic structures of DETC increased gradually. The percentage of T lymphocytes was 4.67% and 94.1% of these T lymphocytes were DETC. The purity of DETC on CD 15 was 18.50% and the purity of DETC on CD 30 was 98.70%. (2) Immediately after injury, the wound healing condition of mice in WT control group and TCR δ-/- group was similar. The wound healing speed of mice in TCR δ-/- group was slower than that in WT control group on PID 2-10. The percentages of residual wound area of mice in TCR δ-/- group on PID 2, 4, 6, 8, and 10 were increased significantly compared with those in WT control group (t=3.492, 4.425, 4.170, 4.780, 7.318, P<0.01). (3) The length of new epithelium of mice in TCR δ-/- group on PID 3 was (359 ± 15) μm, which was obviously shorter than that in WT control group [(462±26) μm, t=3.462, P<0.01]. (4) Immediately after injury, wound condition of mice in TCR δ-/-+ DETC group and TCR δ-/- control group was similar. Compared with TCR δ-/-+ DETC group, the wound healing speed of mice in TCR δ-/- control group were obviously slower on PID 2-10. The percentages of residual wound area of mice in TCR δ-/-+ DETC group on PID 2, 4, 6, 8, and 10 were decreased significantly compared with those in TCR δ-/- control group (t=2.308, 3.725, 2.698, 3.707, 6.093, P<0.05 or P<0.01). (5) On PID 3, the length of new epithelium of mice in TCR δ-/-+ DETC group was (465±31) μm, which was obviously longer than that in TCR δ-/- control group [(375±21) μm, t=2.390, P<0.05]. (6) On PID 3, PCNA expression of epidermal cell in wound margin of mice in TCR δ-/- group was 1.25±0.04, which was obviously lower than that in WT control group (2.01±0.09, t=7.415, P<0.01). (7) On PID 3, PCNA expression of epidermal cell in wound margin of mice in TCR δ-/-+ DETC group was 1.62±0.08, which was significantly higher than that in TCR δ-/- control group (1.05±0.14, t=3.561, P<0.05). (8) On PID 3, apoptosis rate of epidermal cell in wound margin of mice in TCR δ-/- group was (16.1±1.4)%, which was higher than that in WT control group [(8.1±0.6)%, t=5.363, P<0.01]. (9) On PID 3, apoptosis rate of epidermal cell in wound margin of mice in TCR δ-/-+ DETC group was (11.4±1.0)%, which was obviously lower than that in TCR δ-/- control group [(15.4±1.4)%, t=2.377, P<0.05].@*Conclusions@#DETC participates in the process of wound healing though promoting the proliferation of epidermal cells in wound margin and inhibit the apoptosis of these cells.
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Objective: To explore the possible mechanism of human adipose-derived stem cells (hADSCs) promoting seawater immersion wound healing in vitro. Methods: Human epidermal cell line HaCaT cells and artificially simulated seawater were used to establish an in vitro model of cell damage induced by seawater immersion. hADSCs were isolated from human adipose tissues, and a co-culture system of HaCaT cells and hADSCs was established. The proliferation and migration abilities of HaCaT cells were detected by cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation detection kit and cell scratch test. The activation levels of epidermal growth factor receptor (EGFR)/extracellular-regulated protein kinase (ERK) signaling pathway were detected by Western blotting and real-time quantitative PCR. Results: The proliferation of HaCaT cells cultured with the medium containing 10% artificial seawater was significantly inhibited compared with the cells cultured without artificial seawater (P0.05). The expression of EGFR/ERK signaling pathway in seawater-cultured HaCaT cells was significantly inhibited compared with the cells cultured without seawater and those co-cultured with hADSCs and seawater (P0.05). Conclusion: Seawater can block the activation of EGFR/ERK signaling pathway and inhibit the proliferation and migration of HaCaT cells. hADSCs can promote the activation of EGFR/ERK signaling pathway and reduce the inhibition effect of seawater against proliferation and migration of HaCaT cells.
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Objective To explore the possibility of primary culture of human adipose-derived stem cells in vitro and differentiation induction into epidermal cells.Methods Adipose-derived stem cells were primarily cultured by enzyme digestion method.The expression of CD29,CD34,CD44,CD49d,CD80 and CD106 was detected by immunohistochemical staining.ADSCs differention into epidermal cells in vitro were under induction medium.Proliferation activity and morphology of cells of induction group and control group were observed,and the expression of CKs in the two groups were also analyzed.Results ADSCs were successfully isolated and cultured from human liposuction tissue.It was revealed by immunofluorescence staining that ADSCs possessed specified expression of surface antigen of stem cells; ADSC successfully differentiated towards epidermal cells in vitro and possesed considerable high-level reproductive activity,cell morphology and expression of CKs also shown the tendency of differentiated into epidermal cells.Conclusions ADSCs can be isolated and cultured from human liposuction tissue,and can proliferate persistently.ADSCs possess specified expression of surface antigen.ADSCs can also be differentiated in vitro to the direction of epidermal cells.
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PURPOSE: The purpose of this study was to investigate the transplantation results of human amniotic membrane (HAM), epidermal cells, or marrow mesenchymal stem cells (MSCs) in healing a skin defect. MATERIALS AND METHODS: Defects (full-thickness) in rabbits were treated with HAM alone (group A), HAM injected with cultivated epidermal cells (group B), HAM injected with cultivated MSCs (group C), or Vaseline gauze (group D). Tissue granulation, regeneration, re-epithelization and healing time were measured. Defects and healed area were calculated 2 weeks after surgery. RESULTS: The mean healing area was 67.5%, 81.7%, 83.2% and 49.5% in each group, with all treatment groups significantly different than group D (p<0.01), and groups B and C compared higher than group A (p<0.05). The healing time of groups A, B, and C was 5.7 to 6.4 days faster than that of group D (p<0.01). Histologic analysis showed that the new epidermis covered nearly the whole wound surface in group B and C, and contained granulated tissue with fibroblasts, capillaries, and collagen. CONCLUSION: HAM grafts injected with cultivated epidermal cells or MSCs promoted healing of skin defects.
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Humans , Rabbits , Amnion , Bone Marrow , Capillaries , Collagen , Epidermis , Fibroblasts , Mesenchymal Stem Cells , Petrolatum , Regeneration , Skin , TransplantsABSTRACT
Aim To investigate the effects of aloe coarse polysaccharid on proliferation and the intracellular calcium level and the leakage rate of lactate dehydrogen-ase(LDH) of the epidermal cellsin vitro.Methods The epidermal cells were treated with aloe coarse po-lysaccharid of75,150,300,600 and 1 200 mg.L-1respectively.The proliferation of epidermal cells wasobserved interms of the proliferation curve and the pop-ulation doubling/day(PD/d).The concentration ofintracellular calcium and the leakage rate of LDHwerealso measured.Results In aloe coarse polysaccharidgroup,a dose-dependent and statistically significant(P
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Objective To investigate the dedifferentiation of epidermal cells into their progenitor stem cells induced by basic fibroblasts growth factors(bFGF) in vitro.Methods HEKa cells obtained from Cascade were found flattening and formation of cell-to-cell contacts after 6 to 7 passages,which resembled differentiated epidermal cells in vivo.To examine the effect of growth factors on the cell proliferative alterations,bFGF(100 ng/ml) was added into the culture medium for different periods(6,12,24,48,or 72 h),then the cell proliferation was measured by MTT assay.Phenotypic changes and the cell-fate determination of HEKa cells after bFGF treatment were detected by immunocytochemical assays,flow cytometry and RT-PCR analysis.HEK cells with no intervention treatment were used as a control.Results MTT assay proved that the optimal culture condition to induce the dedifferentiation of epidermal cells into their progenitors was to culture HEKa cells for 36 to 48 h when the addition of bFGF was 100 ng/ml.After treatment with bFGF for 48 h,clusters of round-shaped cells appeared around differentiated epidermal cells,and expanded progressively thereafter.These cells were smaller in shape and with larger nuclear/cytoplasm ratio,and had not only clonogenicity but also ability to form a cutaneous ridge-like structure.Immunohistochemical staining revealed that the expression levels of ?1 integrin,CK19 and CK14 were up-regulated,while the expression of CK10 was significant down-regulated after bFGF treatment.Flow cytometry indicated that there were more CK19-positive and CK14-positive cells in the treatment group than in the control(74.77% vs 15.74%,and 87.14% vs 67.26%respectively),but much lesser CK10-positive cells(4.56% vs 98.56%).Additionally,the mRNA expression levels of ?1 integrin,CK19 and CK14 were up-regulated after bFGF treatment,but that of CK10 was down-regulated.Conclusion bFGF can reverse the differentiated process of epidermal cells and induce them to produce immature,stem-like cells,which can proliferate and be used in the wound repair and regeneration of skin tissues.
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AIM: To investigate the mechanism by which human amnion induced mouse embryonic stem (ES) cells to differentiate into epidermal like cells. METHODS: ES-BALB/c cells were cocultured with human amnion in transwells for 4-5 days, and those cultured alone without amnion were taken as control group. The morphological differentiation were observed. The committed differentiation of ES cells into epidermal like cells were detected by integrin-?_1, CK19, CK15 and involucrin immunohistochemistry, respectively. RESULTS: After 4-5 days of coculture, ES cells differentiated into single layer of epidermal like cells, fitted tightly, with polyhedral in shape. The immunohistochemical staining results showed that, most of the cells were integin-?_1 positive, only a few cells were CK19 and CK15 positively stained. Most of the cells in control group died, the survived ones were different in morphological shapes, and no integrin-?_1, CK19 and CK15 positive cells were found. CONCLUSION: Soluble substances secreted by human amnion may play an important role in inducing the differentiation of mouse ES cells into epidermal like cells. [
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Objective To study the shape differences of upper epidermal cells in leaves of medicinal plants of Swertia L. and establish the new method for identifying them. Methods The epidermis were mounted in ordinary technique, and then scanned and binarized through HPIAS-1000 image analytic system. The waveness of the anticlinal walls (SFC) and the ratio of the Ferets diameter (SLF) of upper epidermal cells were detected. The leaf epidermal cells of 21 kinds of raw materials in 12 plants of Swertia L. from various regions were examined. Results The precision and reproducibility of software system were good, and the shape characters of the upper epidermal cells of the middle lamina in the third node arising from ground are the stablest inside the same species by the magnification 20?10 under microscope. The SFC and SLF of the upper epidermal cells of the same species of plants are relatively constant, conversely that of different species of plants are obviously different and distinguishable from each other. Conclusion The proposed method is a useful technique for identifying traditional Chinese medicinal materials originated from herbs and leaves of plants. HPIAS-1000 is simple, rapid, accurate, and practical for shape analysis of upper epidermal cells of plant leaf.
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Using anti-T6 and anti-HLA-DR monoclonal antibodies, this study was designed to attain what would do to epidermal Langerhans cell (LC) markers in psoriatic patients when two remedies, etretinate and UVB, having controversial effects on LC were put to use simultaneously. In normal and psoriatic subjects, HLA-DR+ LC was approximately 80% of T6+ LC and a single dose of UVB equivalent to minimal erythema dose (MED), reduced LC membrane markers to approximately 30% of non-irradiated control. The recovery of LC membrane markers, after a single dose of UVB exposure were significantly faster in the group of etretinate treated psoriatic subject than only UVB irradiated psoriatic control. Taken together, seemed to exert prompt recovery of normalization of the number of LC from the depletion following the UVB.
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Adult , Female , Humans , Male , Middle Aged , Antibodies, Monoclonal , Antigens, Differentiation, T-Lymphocyte/analysis , Drug Therapy, Combination , Etretinate/therapeutic use , HLA-DR Antigens/analysis , Immunohistochemistry , Langerhans Cells/drug effects , Psoriasis/pathology , Ultraviolet TherapyABSTRACT
The epidermis, which is composed mainly of keratinocyt;es, undergoes continual proliferation and differentiation, To study keratinocyte differentiation, it is necessary to separate the keratinocytes at their sucessive stages of maturation. We separate the keratinocytes by layers by using Percoll and obtain the relatively pure fractions of basal cells, spinous cells, and granular cells, 76. 8%, 67. 2%, 30%. respectively.
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Humans , Epidermis , KeratinocytesABSTRACT
It has been shown that epidermal Langerhans cells (LCs) were antigen presenting cells in cutaneous cell-mediated immuneresponse,and they were implicated in skin allograft rejection,If LCs can be depleted from skin grafts,the survival period of the allografts may be prolonged. Therefore,we observed the changes of LCs in cultured human epidermal cells (ECs) by ATPase staining,HLA-Dr monoclonal antibody indirect immunofluoresence and enzyme labelling avidin-biotin complex (ABC) staining.And the capacity of ECs to stimulate allogeneic lymphocyte proligeration before and after cultures was testedby mixed epidermal cell-lymphocyte culture response (MEcLR).We found that LCs in original skin explants were significantly decreased and deformed three days after culture.LCs were reduced more than 90 percent after 7 days of culture.LCs in original skin explants were approximately eliminated 14 days or more of culture.LCs were never seen within epidermal outgrowth in culture.The Ecs gained from the original skin explants which were cultured after 7 days and from epidermal outgrowth lost their ability to stimulate allogeneic lymphocyte transformation in MEcLR.These results indicate that LCs bearing HLA-Dr antigen in human are able to be depleted from epidermal grafts by epidermal cell culture,and suggest that the ECs cultured in our culture system can prolong their survival period as they are used as allografts.
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Objective To study the effects of podophyllotoxin solid lipid nanoparticles (POD-SLN) on the proliferation of human epidermal cells in vitro. Methods POD-SLN was prepared by using microemulsion technique, the morphology of POD-SLN was examined by transmission electron microscope (TEM), and its particle size and Zeta potential were studied by Zetasizer analyzer. High-performance liquid chromatography (HPLC) was employed to determine the entrapment efficiency of podophyllotoxin (POD) in the nanoparticles, and its stability was observed. Human epidermal cells were treated with different concentrations (0.1-1 000 ?g/L) of POD-SLN, and the proliferation of human epidermal cells was studied at different time points after exposure (6h, 12h, 24h, 48h). The cytotoxic effects of POD-SLN, POD liposome, free POD, blank solid lipid nanoparticles (SLN) and control groups on human epidermal cells were assessed using a colorimetric MTT cell viability assay. Results POD-SLN displayed spherical or elliptical in shape, and it was stable. The average particle size of POD-SLN was 87.2?10.3nm, Zeta potential was 25.3?0.8mv and the entrapment efficiency of POD in the nanoparticles was 83.2%?2.5%. POD-SLN inhibited the proliferation of human epidermal cells in a concentration- and time-dependent manner. At the same concentration, the effect of PDP-SLN on anti-proliferation was stronger than that of POD liposome and POD. The inhibition of human epidermal cells after 48h exposure to PDP-SLN, POD liposome, and POD reached 91.05%, 77.02% and 68.46% respectively, at the highest concentration of 1000?g/L, and the IC50 were 2.11?g/L, 16.65?g/L and 101.42?g/L, respectively. Blank SLN had no effect on the proliferation of human epidermal cells. Conclusion This formulation and technology are stable and practical. POD-SLN can significantly inhibit the proliferation of human epidermal cells in vitro and the inhibitory effect was better than that of POD liposome and POD.