Telomere length and telomerase activity are related with immortalization frequency but not with replicative senescence in mammalian embryonic fibroblasts, except human embryonic fibroblasts

김현석,홍재승 한국통합생물학회 2014 Animal cells and systems Vol.18 No.6

The maximum life span of animals is proportional to a maximum population doubling (PD) number of animal fibroblasts. Human fibroblasts have about 50–70 PDs, while mouse fibroblasts have only 8–10 PDs. Although telomere shortening isone of the best candidates to explain the replicative senescence, this cannot explain why mouse fibroblasts have shorterreplicative potential than human fibroblasts even though mouse cells have longer telomere and telomerase activity. Weprepared four mammalian embryonic fibroblasts – human embryonic fibroblasts (HEFs), dog embryonic fibroblasts (DEFs),rat embryonic fibroblasts (REFs), and mouse embryonic fibroblasts (MEFs) –and then used to compare the immortalizationfrequency, p53/p21/p16 expression levels, p53 binding activity, telomere length, and telomerase activity. Immortalizationfrequency of HEFs by SV40 large T antigen was the highest followed by REF and DEFs. HEFs showed almost noimmortalization frequency. Protein levels of p21 and p16 were increased, and DNA-binding activity of p53 was increasedduring replicative senescence. On the other hand, p21 protein level was decreased and p16 was significantly increased in allimmortalized cells. Embryonic fibroblasts from mammals of shorter life span have longer telomere and stronger telomeraseactivity. These results indicate that telomere length and telomerase activity are related with immortalization frequency butnot replicative senescence in mammalian fibroblasts, except human fibroblasts. Only human fibroblasts showed replicativesenescence-related telomere shortening.

Lipopolysaccharide로 자극시킨 방사선 조사 치은 섬유아 세포에서 granulocytemacrophage colony-stimulating factor와 transforming growth factor-β1 생성

김홍식(Hong-Sik Kim),이성근(Seong-Geun Lee),김광혁(Kwang-Hyuk Kim),김욱규(Uk-Kyu Kim),김종렬(Jong-Ryoul Kim),정인교(In-Kyo Chung),양동규(Dong-Kyu Yang) 대한구강악안면외과학회 2002 대한구강악안면외과학회지 Vol.28 No.3

Purpose: Irradiation in the oral cancer patients causes early and late complications such as intraoral mucositis and fibrosis, with a various expression of GM-CSF and TGF-β. The purpose of this study was to investigate the production of GM-CSF and TGF-β1 by the irradiated human gingival fibroblasts cultivated with lipopolysaccharide. Materials and Methods: Irradiated (total dose, 60 Gy) human gingival fibroblasts were incubated with LPS. Culture supernatants that were collected at 24, 48, and 72 hours were assessed for GM-CSF and TGF-β1 by enzyme-linked immunosorbent assay. Results: 1. GM-CSF production in nomal gingival fibroblasts was increased with incubation time, but decreased with incubation time in irradiated gingival fibroblasts. GM-CSF production in both normal and irradiated gingival fibroblasts induced with LPS was higher than the control. 2. TGF-β1 production in normal gingival fibroblasts was decreased after 24 hours, but, it was increased until 48 hours in irradiated gingival fibroblasts. TGF-β1 production in normal gingival fibroblasts exposed with LPS was higher than the control. Conversely, It was lower than the control in irradiated gingival fibroblasts exposed with LPS. Conclusion: This indicates that irradiation in gingival fibroblasts may play an important role in radiation-induced intraoral mucositis and fibrosis. However, LPS decreases the production of TGF-β1 in the irradiated gingival fibroblasts.

Up-regulated macrophage migration inhibitory factor protects apoptosis of dermal fibroblasts in patients with systemic sclerosis

Kim, J.-Y.,Kwok, S.-K.,Hur, K.-H.,Kim, H.-J.,Kim, N. S.,Yoo, S.-A.,Kim, W.-U.,Cho, C.-S. Blackwell Publishing Ltd 2008 Clinical and experimental immunology Vol.152 No.2

<P>Summary</P><P>Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been demonstrated to regulate the apoptosis of several cell types. Dysregulated apoptosis of fibroblasts has been implicated in a variety of fibrotic diseases, including systemic sclerosis (SSc). In this study, we investigated the role of MIF in the apoptosis of dermal fibroblasts. The concentrations of MIF were measured in sera and in culture supernatants of peripheral blood mononuclear cells (PBMCs) and dermal fibroblasts by enzyme-linked immunosorbent assay. The degree of apoptosis was determined by colorimetric assay, and signalling pathways were examined by Western blot. The results showed that serum levels of MIF were significantly higher in patients with SSc (<I>n</I> = 47) than in healthy controls (<I>n</I> = 56). Stimulation of PBMCs by anti-CD3 and anti-CD28 increased the production of MIF by fourfold over the constitutive levels. SSc dermal fibroblasts produced higher amounts of MIF than normal dermal fibroblasts. When treated with sodium nitroprusside (SNP), SSc dermal fibroblasts showed a lower degree of apoptosis compared with normal dermal fibroblasts. Exogenous MIF (1–100 ng/ml) inhibited SNP-induced apoptosis of dermal fibroblasts dose-dependently. Both extracellular regulated kinase (ERK) inhibitor (PD98059) and protein kinase B (Akt) inhibitor (LY294002) almost completely blocked the inhibitory effect of MIF on apoptosis. Furthermore, MIF increased the expression of Bcl-2, phospho-ERK and phospho-Akt activity in dermal fibroblasts. Taken together, our data suggest that MIF released by activated T cells and dermal fibroblasts decreases the apoptosis of dermal fibroblasts through activation of ERK, Akt and Bcl-2 signalling pathways, which might be associated with excessive fibrosis in SSc.</P>

산소분압과 발생부위에 따른 켈로이드 배양섬유모세포의 혈관내피 성장인자(VEGF) 발현

문형식 ( Hyung Sik Moon ),손숙자 ( Sook Ja Son ),박건 ( Kun Park ),강희규 ( Hee Gyoo Kang ),임희정 ( Hee Joung Lim ),박향준 ( Hyang Jun Park ) 대한피부과학회 2009 대한피부과학회지 Vol.47 No.5

Background: The pathophysiological events resulting in keloid formation remain unclear. Overabundant levels of VEGF have been reported to contribute to excessive wound healing. There have been many studies describing the relationship between keloids and VEGF expression. However, there have been no reports about VEGF expression related to donor sites. Objective: We investigated VEGF expression of cultured normal and keloid fibroblasts obtained from different body areas under normoxic and hypoxic culture conditions. Methods: Normal fibroblasts from the earlobe (n=2), shoulder (n=2) and chest (n=2) as well as keloid fibroblasts from the earlobe (n=3), shoulder (n=3) and chest (n=3) were collected and cultured. VEGF expression of fibroblasts at 6 hours, 12 hours, 24 hours and 48 hours for cells maintained under normoxic and hypoxic conditions was measured by the use of RT-PCR. Paraffin-embedded tissues (normal and keloid tissue) were assayed by immunohistochemical staining. Results: For the cultured normal fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition, irrespective of the donor site and time. However, for the cultured keloid fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition for cultured shoulder fibroblasts. For each donor site, VEGF expression was highest in the shoulder, followed by the chest and earlobe for cultured normal fibroblasts, irrespective of time. For the cultured keloid fibroblasts, the highest VEGF expression occurred at 6 hours for cells in the normoxic condition and the highest VEGF expression occurred at 6 hours and 12 hours for cells in the hypoxic condition. Based on immunohistochemical staining, VEGF expression of paraffin-embedded normal tissue was lower as compared to paraffin-embedded keloid tissue. For each donor site in paraffin-embedded keloid tissue, VEGF expression was highest in the shoulder, followed by the chest and earlobe. Conclusion: Oxygen tension and the nature of fibroblasts from different donor sites are involved in keloid pathogenesis. (Korean J Dermatol 2009;47(5):539~546)

P030 : Encouraging results of wound healing in diabetic mouse: autologously cultured dermal fibroblasts showed better wound healing on the diabetic mouse skin ulcers than nonself stem cell

( Ji Hae Lee ),( Jung Min Bae ),( Eu Seok Chung ),( Hanmi Chung ),( Sae Mi Park ),( Ju Hee Lee ),( Hee Jin Jeon ),( Seung Jun Hwang ),( Gyong Moon Kim ) 대한피부과학회 2014 대한피부과학회 학술발표대회집 Vol.66 No.2

Background: The Immunologically privileged autologously cultured dermal fibroblasts of diabetic mouse may have same or better functions than normally working fibroblasts or stem cells, and previous reports showed similar functioning abilities of diabetic dermal fibroblasts compared with that of normal origin. Objectives: We would like to show that immunologically privileged autologously sampled and cultured dermal fibroblasts might show better clinical and histological, immunohistochemical results than allogeneic mesenchymal stem cells in cell transplantation therapy for diabetic skin ulcers. Methods: Every mouse was given surgery which procedure resulted in 6 mm identical full thickness wound in both back area, and A group mouse were injected allogeneic normal stem cells in right-sided wounds with left sided wounds untreated (normal control). Simultaneously, B group mouse were injected autologous diabetic fibroblasts in right-sided wounds with left sided wounds untreated (normal control). Results: Compared with bare diabetic wounds and groups of diabetic wounds treated with allogeneic stem cells as control groups, autologous fibroblasts-transplanted diabetic wounds showed better wound healing with better, faster dermal granulation tissue regeneration and vascularization than allogeneic stem cell treated wounds. Conclusion: Immunologically privileged autologous dermal fibroblasts may be good therapeutics in nonhealing chronic, diabetic skin ulcers.

Growth Factors Supplementation in Culture Medium Leads to Active Proliferation of Porcine Fibroblasts

Kim, Bella,Ko, Na-Young,Hwang, Seong-Soo,Im, Gi-Sun,Kim, Dong-Hoon,Park, Jin-Ki,Ryoo, Zae-Young,Oh, Keon-Bong The Korean Society of Animal Reproduction 2011 Reproductive & developmental biology Vol.35 No.3

Fibroblasts of large animals are easy to isolate and to maintain in vitro culture. Thus, these cells are extensively applied to donor cell for somatic cell nuclear transfer, and to substrate cells to generate induced pluripotent stem cells after transfection of requited genes to be essentially required for direct reprogramming. However, limited mitotic activity of fibroblasts to differentiate along a terminal lineage becomes restrictive for their versatile application. Recently, commercial culture medium and systems developed for primary cells are provided by manufactures. In this study, we examined whether one of the systems developed for primary fibroblasts of human are effective on porcine ear skin fibroblasts. To this end, we performed proliferation assay after five days culture in vitro of porcine fibroblasts in medium DMEM, which is generally used for fibroblasts culture, and medium M106 for human dermal fibroblasts, supplemented with various concentrations of FBS and LSGS contained mainly growth factors, respectively. Consequence was that presence of 15% FBS and 0.1 ${\times}$ concentrations of LSGS in DMEM showed most active proliferation of porcine fibroblasts.

배양액내의 단백분해효소가 인체 피부섬유아세포의 성장과 단백질 합성에 미치는 영향

이종원,오구택,김환묵,조문제 大韓成形外科學會 1998 Archives of Plastic Surgery Vol.21 No.2

Fibroblasts which play a crucial role in repair processes are mesenchymal cells in most organs, But are also involved in development of fibrosis. Although there were many reports about fibroblasts, their growth kinetics and multiple functions such as migration, chemotaxis, protein synthesis have only recently been investigated. We performed this study in order to investigate the effects of tissue proteolytic enzymes on growth of human skin fibroblasts. To investigate the effects of collagenase and trypsin on cell growth and protein synthesis, they were added to cultured skin fibroblasts. For cell growth determination, DNA and protein synthesis were measured by [³H]-leucine and [³H]-leucine and [³H]-proline incorporation. Also, we observed the effect of trypsin in various doses and fetal calf serum on growth, DNA and protein synthesis of fibroblast using the same method. Trypsin was treated with heat(56℃ or 85℃) for inactivation of cytolytic activity. To determine proteolytic activity in conditioned media and cultured supernatant, synthetic polypeptide, milk casein and gelatine agar were used as the substrate for the enzyme activity assay. The results were as follows: 1) DNA and protein synthesis of cultured fibroblasts were decreased by below 20% in collagenase or trypsin treated group compared with in control group(p<0.001). 2) DNA and protein synthesis of cultured fibroblasts in 56℃ -treated group were lower than those in 85℃-treated group, but no significant difference between two groups was found in the amount of DNA. 3) Proteolytic activity in conditioned media of 56℃-treated group of trypsin was higher than that the 85℃-treated group, but no significant difference between two groups in cultured fibroblasts was found in the amount of DNA. 4) there was a significant decrease of DNA and protein synthesis of cultured fibroblasts in serum free media compared with in serum containing media(p<0.001). 5) when fibroblasts were cultured in serum free media, the growth rates showed no significant difference depending upon various concentrations of trypsin. In conclusion, low dose of proteolytic enzyme without cytolytic activity may inhibit the fibroblast growth, DNA and protein synthesis in cluture. With above results, we suppose that modulation of fibrosis kinetics by non-specific proteolytic enzymes, such as trypsin may be helpful to the treatment or prevention of pathologic fibrosis.

골수기질세포 및 섬유아세포의 창상치유 촉진 성장인자 분비능 비교

김세현,한승규,윤태환,김우경 대한성형외과학회 2006 Archives of Plastic Surgery Vol.33 No.1

Cryopreserved fibroblast implants represent a major advancement for healing of chronic wounds. Bone marrow stromal cells, which include the mesenchymal stem cells, have a low immunity-assisted rejection and are capable of expanding profoundly in a culture media. Therefore, they have several advantages over fibroblasts in clinical use. The ultimate goal of this study was to compare the wound healing accelerating growth factor secretion of the bone marrow stromal cells with that of the fibroblasts and this pilot study particularly focuses on the growth factor secretion to accelerate wound healing. Bone marrow stromal cells and fibroblasts were isolated from the same patients and grown in culture. At 1, 3, and 5 days post-incubating, secretion of basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta(TGF-β) were compared. In TGF-β secretion fibroblasts showed 12~21% superior results than bone marrow stromal cells. In contrast, bFGF levels in the bone marrow stromal cells were 47~89% greater than that in fibroblasts. The VEGF levels of the bone marrow stromal cells was 7~12 fold greater than that of the fibroblasts. Our results suggest that the bone marrow stromal cells have great potential for wound healing accelerating growth factor secretion.

비용종 상피세포와 섬유아세포 동시배양 모델에서 시토카인의 발현

김성완,조중생,박재경,이건희,차창일 WHO COLLABORATING CENTRE FOR TRADITIONAL MEDICINE 2007 東西醫學硏究所 論文集 Vol.2007 No.-

Background and Objectives: Nasal polyposis is a chronic inflammatory disease characterized histologically by edematous fluid with sparse fibrous cells, few mucous glands and proliferation of stromal and epithelial cells. There have been many studies done separately about epithelial cells, fibroblasts, and other inflammatory cells in nasal polyps. However, there is no usable study model for the cell-to-cell interaction. Therefore, we tried to make a new model for the study of nasal polyp by coculture of epithelial cells and fibroblasts. Subjects and Method: Nasal polyp was collected during endoscopic sinus surgery. Almost half of them were used for epithelial cell culture and the remaining portion was used for fibroblast culture in the same polyp. Using a transwell insert fibroblasts were placed in the bottom and epithelial cells were placed on the insert respectively. RNA were isolated both from cpithelial cells and fibroblasts of 12,24, and 48 hours' coculture. The expressions of IL-8,IL-6, and eotaxin from coculturcd cells were compared with cells that were cultured alone by semiquantitative RT-PCR. Results: IL-8 was highly expressed in fibroblasts than in epithelial cells, especially in cocultured cells. The expression of IL-6 in fibroblasts was much higher than in epithelial cells, especially in cocultured fibroblasts. Eotaxin is weakly expressed in fibroblasts but not in epithelial cells. Conclusion : These findings indicate that cocultured cells show different expression of the mRNA of various cytokines compared with cultured cells alone. And this coculture system may be more ideal than the mono-cell culture system for the study of cell-to-cell interactions and to reveal the pathogenesis of oasal polyposis.

Inhibitors of DNA methylation support TGF-β1-induced IL11 expression in gingival fibroblasts

Sufaru, Irina-Georgeta,Beikircher, Gabriel,Weinhaeusel, Andreas,Gruber, Reinhard Korean Academy of Periodontology 2017 Journal of Periodontal & Implant Science Vol.47 No.2

Purpose: Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-${\beta}1$ (TGF-${\beta}1$). Methods: Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-${\beta}1$. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification. Results: We found that 5-aza enhanced TGF-${\beta}1$-induced interleukin-11 (IL11) expression in gingival fibroblasts 2.37-fold (P=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 (PRG4) and NADPH oxidase 4 (NOX4). Consistent with this, 5-aza caused demethylation of the IL11 gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-${\beta}$ type I receptor kinase inhibitor SB431542 impeded the changes in IL11 expression, indicating that the effects of 5-aza require TGF-${\beta}$ signaling. 5-aza moderately increased the expression of TGF-${\beta}$ type II receptor (1.40-fold; P=0.009), possibly enhancing the responsiveness of fibroblasts to TGF-${\beta}1$. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (P=0.005) and DNMT3B (P=0.002), which are enzymes responsible for gene methylation. Conclusions: These in vitro data suggest that the inhibition of DNA methylation by 5-aza supports TGF-${\beta}$-induced IL11 expression in gingival fibroblasts.