Maintenance of chronicity signatures in fibroblasts isolated from recessive dystrophic epidermolysis bullosa chronic wound dressings under culture conditions

BACKGROUND: Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused by variants in the COL7A1 gene, coding for type VII collagen (C7), an important component of anchoring fibrils in the basement membrane of the epidermis. RDEB patients suffer from skin fragility starting with blister formation and evolving into chronic wounds, inflammation and skin fibrosis, with a high risk of developing aggressive skin carcinomas. Restricted therapeutic options are limited by the lack of in vitro models of defective wound healing in RDEB patients. RESULTS: In order to explore a more efficient, non-invasive in vitro model for RDEB studies, we obtained patient fibroblasts derived from discarded dressings) and examined their phenotypic features compared with fibroblasts derived from non-injured skin of RDEB and healthy-donor skin biopsies. Our results demonstrate that fibroblasts derived from RDEB chronic wounds (RDEB-CW) displayed characteristics of senescent cells, increased myofibroblast differentiation, and augmented levels of TGF-ß1 signaling components compared to fibroblasts derived from RDEB acute wounds and unaffected RDEB skin as well as skin from healthy-donors. Furthermore, RDEB-CW fibroblasts exhibited an increased pattern of inflammatory cytokine secretion (IL-1ß and IL-6) when compared with RDEB and control fibroblasts. Interestingly, these aberrant patterns were found specifically in RDEB-CW fibroblasts independent of the culturing method, since fibroblasts obtained from dressing of acute wounds displayed a phenotype more similar to fibroblasts obtained from RDEB normal skin biopsies. CONCLUSIONS: Our results show that in vitro cultured RDEB-CW fibroblasts maintain distinctive cellular and molecular characteristics resembling the inflammatory and fibrotic microenvironment observed in RDEB patients' chronic wounds. This work describes a novel, non-invasive and painless strategy to obtain human fibroblasts chronically subjected to an inflammatory and fibrotic environment, supporting their use as an accessible model for in vitro studies of RDEB wound healing pathogenesis. As such, this approach is well suited to testing new therapeutic strategies under controlled laboratory conditions.

The Effect of Equine Placental Extract on Anti-skin Aging Activity / 日本補完代替医療学会誌

In the present study, we assessed the antiaging effect of equine placental extract (ePE) on dermal fibroblasts and found that it markedly suppressed the appearance of β-galactosidase-positive cells among the senescent cells induced by repeated hydrogen peroxide exposure or ultraviolet A irradiation. Moreover, the efficacy of ePE treatment was similar to that of an antioxidant, N-acetylcysteine. Thus, owing to its antioxidant effect, ePE can be used as an antiaging agent, particularly for the dermis.

LATS1-YAP pathway regulate proliferation of human skin fibroblast and synthesis of extracellular matrix / 局解手术学杂志

Objective To study the proliferation of human skin fibroblast and synthesis of extracellular matrix which were regulated by LATS1-YAP pathway. Methods They were divided into three groups:control groups, LATS1 siRNA intervention group and YAP siRNA treatment group. Using LATS1 siRNA transferred human skin fibroblasts cell lines HS27 in LATS1 siRNA intervention group, and using YAP siRNA transferred HS27 in YAP siRNA treatment group. Expression of LATS1,YAP and collageⅠwere detected by western-blot 48 h later, and the activity of HS27 cells was determined by MTT. Results Compared with control group,expression of LATS1 protein decreased while expression of YAP protein and collagenⅠprotein increased 48 h after LATS1 siRNA transfection. Expression of LATS1 protein remains un-changed and expression of YAP protein and collagenⅠprotein decreased 48 h after YAP siRNA transfection. Conclusion LATS1-YAP pathway could regulate proliferation of human skin fibroblast and synthesis of extracellular matrix. It provides a potential therapeutic targets for skin wound repair and cicatrization.

Effect of estrogen on proliferation and migration of human skin fibroblast / 中华医学美学美容杂志

Objective To explore the biological effects of estrogen (17β-E2) on the proliferation and migration of human skin fibroblast (HSFB).Methods HSFBs were isolated and cultured by enzyme digestion.The fourth generation of HSFBs were adopted; (1) the proliferating effect of diverse concentrations of 17β-E2 and 17β-E2+ ICI-182780 on HSFBs was determined with MTT method at 24,48,72,96 h; (2) the influence of 17β-E2 and ICI-182780 to HSFBs cycle distribution were determined with flow cytometry; (3) the migration effect of diverse concentrations of 17β-E2 and 17β-E2+ICI-182780 on HSFBs was determined at 24,48,and 72 hours after the creation of the scratch-wound in vitro.Results (1) The proliferating speed of HSFBs in 10-10mol/L 17β-E2 group (group A)was the highest of all at 48,72,96 h,which was higher than that in ICI-182780+10-10mol/L 17β-E2 group (group B) and control group (group C) (P＜0.01) ;(2) the HSFBs during the S phase in group A was more than that in groups B and C (P＜0.01),while the HSFBs during the G0/G1 phase was less than that in groups B and C (P＜0.01); (3) the migrating effect of HSFBs in 10-8mol/L 17β-E2 group (group D) was the highest of all at 48 h,which was higher than that in ICI-182780+10-10mol/L control group (group E)and control group (group F) (P＜0.01).Conclusions The concentration of 10-10mol/L estrogen has the strongest effect of promoting proliferation and that of 10-8mol/L has the strongest chemotaxis; ICI-182780 can abate the above effect effectively.

Influence of radiofrequency in skin collagen secretion / 中华医学美学美容杂志

Objective To evaluate the histologic changes in the dermis and the changes of the rate of type Ⅲ and type Ⅰ collagen by the radiofrequency device. Methods The effects of radiofrequency current on the dermis were observed. Ten rabbits were treated by radiofrequency, and the histologic change in the dermis were observed by H-E staining and Sirius red staining. Results After RF treatment, the fibers in the dermis appeared more compact and the quantity of the type Ⅲ (red) and type Ⅰ (green) collagen were both increased. The fibers in the dermis appeared more compact and the rate of type Ⅲ and type Ⅰ collagen was increased. It was also found that a significant proliferation of dermal collagen was observed in 8 days after treatment. As time went by, the proliferation of dermal collagen was more pronounced, and the rate of type Ⅲ was increased. Conclusion The radiofrequency current can increase the quantity of collagen in the dermis and increase the rate of type Ⅲ and type Ⅰ collagen, which may be one of the key mechanisms of facial rejuvenation by RF.

A Case of Fanconi Anemia Diagnosed by a Chromosome Breakage Test with Skin Fibroblasts

Fanconi anemia is an autosomal recessive disease that's characterized by congenital anomalies, defective hematopoiesis and a high risk of developing acute myeloid leukemia and certain solid tumors. The clinical phenotype is extremely variable; therefore, the diagnosis is frequently delayed until the pancytopenia appears. Chromosomal instability, especially on exposure to an alkylating agent, may be seen in affected patients and it is the basis for a diagnostic test. This cellular phenotype can be demonstrated in cultured T cells, B cells, fibroblasts and fetal cells cultured from both amniotic fluid and chorionic villi. But somatic mosaicism may make the diagnosis of Fanconi anemia difficult because of inconclusive chromosome breakage studies. If the test is negative in lymphocytes and yet the clinical setting is highly suspicious, then the skin fibroblasts must be assessed. Because skin fibroblasts are somatic cells, a definitive test can be performed on primary skin fibroblasts. In this report we describe a case of Fanconi anemia that was diagnosed with the use of cultured skin fibroblasts, and this was despite the normal breakage studies in the peripheral blood.

Effects of high concentration of glucose on growth and collagen synthesis of rat skin fibroblast cells / 上海第二医科大学学报

Objective To investigate the effects of different concentrations of glucose on growth and collagen synthesis of rat skin fibroblast cells cultured in vitro. Methods Rat skin fibroblast cells cultured in vitro were treated with glucose of different concentrations (30,35 and 40mmol/L) for 48 h (high glucose group 1,2 and 3). Cell proliferation was detected by MTT method,the hydroxyproline contents in culture media were determined by the commercial kit,and the mRNA expression of typeⅠand Ⅲ procollagen,matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2)were detected by RT-PCR. Cells cultured with 25mmol/L glucose were served as controls. Results With the increase of glucose concentration in culture media,the growth of skin fibroblast cells was significantly inhibited,and the hydroxyproline contents were significantly decreased (P

Experimental study on establishing eukaryotic expression vector pcDNA3.1(+)TPA and its expressing activity in human skin fibroblasts / 中南大学学报(医学版)

Objective To establish a cell line stably expressing the tissue plasminogen activator (TPA) in human skin fibroblasts so as to develop the function analysis and gene therapy of TPA in ischemic heart diseases. Methods Eukaryotic expression vector pcDNA3.1(+)TPA was constructed and transferred into human skin fibroblasts. After G418 selection, the exogenous expression and activity of TPA were observed subsequently by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay. Results Eukaryotic expression vector pcDNA3.1(+)TPA was expressed effectively in human skin fibroblasts. Quantitative ELISA showed that the expression of TPA protein of the experiment group was much higher than that of the hours). And the chromogenic substrate assay showed that the exogenous TPA activity of the experimental group was also much higher than that of the hours). Conclusion The exogenous TPA gene can be expressed effectively after pcDNA3.1(+)TPA was transferred into human skin fibroblasts, suggesting that the cell model will become an important tool in the further study of TPA function and gene therapy in ischemic heart diseases.

Colony Size Distributions according to in vitro Aging in Human Skin Fibroblasts / 대한방사선종양학회지

PURPOSE: To investigate the percentage of colonies with 16 or more cells distribution of human skin fibroblast according to in vitro aging, and to evaluate the relationship between percentage of colonies with 16 or more cells and in vivo donor age in human skin fibroblast culture. MATERIAL AND METHOD: C1, C2, C3a, and C3b human skin fibroblast samples from three breast cancer patients were used as subjects. The C1, C2, and C3a donor were 44, 54, and 55 years old, respectively. C3a and C3b cells were isolated from the same person. Single cell suspension of skin fibroblasts was prepared with primary explant technique. One hundred cells are plated into 100ml tissue culture flask and cultured for two weeks. The colony size was defined as colonies with 16 or more cells. The cultured cell was stained with crystal violet, and number of cells in each colony was determined with stereo microscope at x10 magnification. Passage number of C1, C2, C3a and C3b skin fibroblast were 12th, 17th, and 14th, respectively. RESULTS: Percentage of colonies with 16 or more cells of skin fibroblast samples decreased with increasing in vitro passage number. In contrast, cumulative population doublings of skin fibroblast sample increased with increasing in vitro passage number. Percentage of colonies with 16 or more cells also decreased with increasing population doublings in human skin fibroblast culture. There was strong correlation with percentage of colonies with 16 or more cells and population doublings in C3a skin fibroblast sample. At the same point of population doublings, the percentage of colonies with 16 or more cells of the young C1 donor was higher level than the old C3a donor. CONCLUSION: The population doublings increased with increasing in vitro passage number but percentage of colonies with 16 or more cells decreased. The results of this study imply that percentage of colonies with 16 or more cells is useful as a indicator of in vitro human skin fibroblast aging and may estimate the in vivo donor age.