Skin Fibroblast Cell Line Research Articles

Potential roles of interleukin‐17A in the development of skin fibrosis in mice

Although transforming growth factor β (TGFβ) and connective tissue growth factor (CTGF) have been considered to play central roles in the pathogenesis of systemic sclerosis (SSc), other cytokines may also be crucial for the development of SSc. The aim of this study was to examine the roles of T helper cytokines in the development of skin fibrosis. To compare the roles of Th1, Th2, and Th17 cytokines, we examined bleomycin-induced SSc in mice deficient for interferon-γ (IFNγ), interleukin-4 (IL-4), and IL-17A. The mechanism by which IL-17A contributes to bleomycin-induced fibrosis was investigated in vivo and in vitro. The outcome of mice lacking IL-17A was also investigated in TSK-1 mice. The loss of IL-17A significantly attenuated bleomycin-induced skin fibrosis, whereas a deficiency of IFNγ or IL-4 did not. Leukocyte infiltration and the expression of TGFβ and CTGF messenger RNA in bleomycin-injected skin were significantly reduced in IL-17A-deficient mice compared with wild-type (WT) mice. Daily bleomycin injections induced the expression of IL-17A in the skin and potent IL-17A producers in splenic CD4+ T cells from WT mice. Furthermore, a skin fibroblast cell line expressed increased TGFβ, CTGF, and collagen after the addition of recombinant IL-17A. IL-17A deficiency also attenuated skin thickness in TSK-1 mice. This study demonstrates that IL-17A contributes to skin fibrosis in 2 mouse models of SSc. These findings suggest that inhibition of IL-17A represents a therapeutic target for antagonizing fibrotic skin disorders such as SSc.

Herbal formula Astragali Radix and Rehmanniae Radix exerted wound healing effect on human skin fibroblast cell line Hs27 via the activation of transformation growth factor (TGF-β) pathway and promoting extracellular matrix (ECM) deposition

Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and as the principal herbs in treating diabetic foot ulcer. In this study, we investigated the effect of NF3, which comprises of AR and RR in the ratio of 2:1(w/w), on skin fibroblast cell migration and the activation of selected genes and proteins related to wound healing.Human skin fibroblast cell line Hs27 was treated with NF3 at 4mg/ml for 24h, and in vitro scratch wound healing and quantitative cell migration assays were performed, respectively. The expression of transformation growth factor (TGF-β1) and bone morphogenetic protein 6 (BMP6) in Hs27 cells with or without NF3 treatment was analyzed by western blot analysis. In addition, the expression of a panel of genes involved in human TGF-β signaling pathway was analyzed in Hs27 cells upon NF3 treatment (4mg/ml, 24h) by quantitative real-time PCR (qRT-PCR). Furthermore, the expression of several genes and proteins associated with ECM synthesis was investigated by qRT-PCR analysis or/and ELISA techniques. The results suggested that NF3 promoted the migration of human skin fibroblast cells. Western blot analysis demonstrated that NF3 up-regulated TGF-β1 and BMP-6 synthesis. qRT-PCR analysis revealed that the expression of 26 genes in Hs27 cells was changed upon NF3 induction, including TGF-β superfamily ligands and down stream effectors genes, and genes involved in TGF/Smad pathway, and Ras/MAPK (non-Smad) pathway. Among the extracellular matrix (ECM)-related molecules, it was found that NF3 up-regulated the expression of type I and III collagens, fibronectin as well as TIMP-1, and down-regulated the MMP-9 expression in skin fibroblast cells. This study demonstrated that herb formula NF3 could enhance skin fibroblast cell migration and activated genes involved in TGF-β1 pathway. NF3 could regulate gene transcription for extracellular matrix synthesis via the Smad pathway, and gene transcription for cell motility via the Ras/MAPK (non-Smad) pathway.

Cytotoxic and genotoxic characterization of titanium dioxide, gadolinium oxide, and poly(lactic‐co‐glycolic acid) nanoparticles in human fibroblasts

Engineered nanomaterials have become prevalent in our everyday life. While the popularity of using nanomaterials in consumer products continues to rise, increasing awareness of nanotoxicology has also fuelled efforts to accelerate our understanding of the ill effects that different nanomaterials can bring to biological systems. In this study, we investigated the potential cytotoxicity and genotoxicity of three nanoparticles: titanium dioxide (TiO(2)), terbium-doped gadolinium oxide (Tb-Gd(2)O(3)), and poly(lactic-co-glycolic acid) (PLGA). To evaluate nanoparticle-induced genotoxicity more realistically, a human skin fibroblast cell line (BJ) with less mutated genotype compared with cancer cell line was used. The nanoparticles were first characterized by size, morphology, and surface charge. Cytotoxicity effects of the nanoparticles were then evaluated by monitoring the proliferation of treated BJ cells. Genotoxic influence was ascertained by profiling DNA damage via detection of γH2AX expression. Our results suggested that both TiO(2) and Tb-Gd(2)O(3) nanoparticles induced cytotoxicity in a dose dependent way on BJ cells. These two nanomaterials also promoted genotoxicity via DNA damage. On the contrary, PLGA nanoparticles did not induce significant cytotoxic or genotoxic effects on BJ cells.

RETRACTED: Antibacterial activity and in vitro cytotoxicity of extracts and fractions of Parkia biglobosa (Jacq.) Benth. stem bark and Ageratum conyzoides Linn. leaves

Many species of plants in African countries are widely used in the rural communities where there is little or no access to modern medicine. However, the safety and effectiveness of these medicinal plants are poorly evaluated. The stem bark of Parkia biglobosa Jacq. and leaves of Ageratum conyzoides Linn. were investigated for their antibacterial and cytotoxic activities. The plant materials were extracted with 95% ethanol, and fractionated with petroleum ether, chloroform and ethyl acetate. The antibacterial effects of the extracts and fractions of the plant materials were assayed on the bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA) and Clostridium perfringes. Ethanol extracts of P. biglobosa and A. conyzoides were screened for cytotoxicity using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Two cancer cell lines (SK-MES 1 and SK-LU 1) and one normal cell line (human skin fibroblast cell line, FS5) were used for the screening of the extracts and the fractions obtained. The ethanolic extracts and fractions of P. biglobosa and A. conyzoides showed the best activity against E. coli, S. aureus and MRSA. All fractions of A. conyzoides leaves have no activity against P. aeruginosa. Human lung cancer cell lines (SK-LU 1 and SK-MES 1) and human skin fibroblast cell line (FS5 cells) were treated with various concentrations (3.9μg/ml-2mg/ml) of the extracts and fractions for 24h. SK-MES 1 cells are more susceptible to treatment with the plant fractions. All the fractions of A. conyzoides leaves and the petroleum ether fraction of P. biglobosa were cytotoxic to SK-MES 1 cells, which to some extent may support their traditional inclusion in herbal preparations for treatment of cancer. The overall results provided evidence that the studied plant extracts might be potential sources of new antibacterial and anticancer drug.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3–18) and its analog [Phe8]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.

Effect of growth factors on the migration of equine oral and limb fibroblasts using an in vitro scratch assay

The objective of this study was to determine the effect of platelet derived growth factor BB (PDGF), epidermal growth factor (EGF), transforming growth factor β1 (TGFβ1), insulin like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) on the proliferation and migration of equine oral mucosa and leg skin fibroblast cell lines, using an in vitro scratch assay. Fibroblasts from the two sites were firstly grown to confluence and then an area of cells removed (cell void area). Cell migration alone (with the addition of the mitosis inhibitor mitomycin-C to the culture media) and proliferation and migration combined (without mitomycin-C) into the cell void area were observed at 0, 5, 10, 24 and 36 h. The presence of mitomycin-C in the culture media significantly slowed the closure of the cell void area, as mitosis was inhibited. For the oral cells only, TGFβ1 significantly slowed both migration (with mitomycin-C) and proliferation and migration combined (without mitomycin-C). For the limb cells only, both PDGF and FGF-2 significantly increased fibroblast proliferation and migration combined (without mitomycin-C). For both cell types, EGF significantly reduced migration (with mitomycin-C). IGF-1 had no effect on any of the parameters measured. It was concluded that TGFβ1, PDGF and FGF-2 have differential effects on the proliferation and migration of equine oral and limb fibroblasts. These differences in fibroblast responses to growth factors may in part form the basis of the different clinical outcomes for oral and limb wounds.

Protective and restorative effects of a Commiphora mukul gum resin and triheptanoin preparation on the CCL‐110 skin fibroblast cell line

Coenzyme Q10 (CoQ10) is a major ingredient in skin care products because of its anti-wrinkle effects, although it has some side effects especially at higher amounts. In this study, we compare the anti-wrinkle related properties of CoQ10 and a proprietary Commiphora mukul gum resin (guggul) and triheptanoin preparation (GU-TC7). GU-TC7 is prepared with a supercritical CO₂-co-solvent extraction with ethanol, standardized to 2% guggulsterones and triheptanoin, a triglyceride composed of three 7-carbon fatty acids. Treatment of CCL-110 skin fibroblasts with GU-TC7 demonstrates a mild proliferative effect compared to CoQ10 and increased type I collagen synthesis. Additionally, GU-TC7 inhibited matrix metalloproteinase-1 (MMP-1) expression in a dose-dependent manner at 20-100 μg mL⁻¹ and inhibited human elastase expression by more than 50% as compared to no elastase inhibition with CoQ10 treatment. These results suggest that GU-TC7 possesses properties that are applicable to the treatment of wrinkles and may be considered for its further evaluation in skin care products.

Transcriptional profiling of human skin fibroblast cell line Hs27 induced by herbal formula Astragali Radix and Rehmanniae Radix

Ethnopharmacological relevanceThe herbs Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. Aim of the studyChinese herbal formulus comprising Astragali Radix (AR) and Rehmanniae Radix (RR) have been shown to improve the healing of diabetic foot ulcer through enhancing the viability of primary fibroblasts in diabetic patients suffering insulin resistance. Our previous study demonstrated that the herbal formula NF3 comprising of AR and RR in the ratio of 2:1 was effective in promoting wound healing in diabetic rats, and in vitro data indicated that the wound healing effects of NF3 might be due to the regulation and coordination of inflammation, angiogenesis and tissue regeneration. However, the underlying molecular mechanism has not been well investigated. In this study, we investigated the cellular and molecular effects of the herbal formula NF3 on human skin fibroblast cells. Materials and methodsHuman skin fibroblast cells Hs27 were treated with NF3 ranging from 0 to 8mg/ml for 24h, and the cells without NF3 treatment were used as control. Cell proliferation assay and cell cycle analysis were performed. Transcriptional profiles of Hs27 cells upon NF3 treatment were acquired by using a human cDNA microarray containing 10,000 genes, and the signaling pathways differentially regulated by NF3 were identified and analyzed. ResultsNF3 promoted Hs27 cell proliferation and cell cycle progression. Microarray analysis revealed that 116 genes were differentially expressed upon NF3 treatment. Functional analysis of the genes indicated that NF3 mainly activated Wnt and angiogenesis related pathways, which are directly related to cell proliferation, angiogenesis, extracellular matrix (ECM) formation and inflammation during the process of wound healing. ConclusionThis study provides insight into the molecular mechanism of how the herbal formula Astragali Radix and Rehmanniae Radix may serve as potential therapeutics for wound healing.

Biological activity of (Lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from Rhizobium leguminosarum bv. trifolii strain TA1

Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssBpssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and (1)H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major (1)H NMR signals for O-acetyl and N-acetyl groups were 1 : 0.8 and 1 : 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1β and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

Irradiation at 636 nm Positively Affects Diabetic Wounded and Hypoxic Cellsin Vitro

This study investigated the effect of low-intensity laser irradiation (LILI) on pro-inflammatory cytokines involved in wound healing processes in diabetes and hypoxia. Diabetes is associated with impaired wound healing and a prolonged inflammatory phase. Pro-inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 are elevated in diabetes. LILI has been reported to accelerate wound healing and decrease inflammatory cytokines. A human skin fibroblast cell line (WS1) was used in vitro. Cells were exposed to various insults, namely, wounding, and a diabetic or hypoxic environment. Experimental cells were exposed to an energy density of 5 J/cm(2) using a continuous wave 636-nm diode laser at an average power of 95 mW, an illuminated area of 9.05 cm(2), and an irradiance of 11 mW/cm(2) (irradiation time, 476 sec). The effect of laser irradiation on cytokine expression was examined at 1 or 24 h post-irradiation. Cellular morphology, viability, proliferation, and cytokine expression (IL-1β, IL-6, and TNF-α) were investigated. Translocation of nuclear factor-kappa B (NF-κB) was also determined. There was a higher rate of migration in irradiated wounded cultures, and irradiated hypoxic cells showed an improvement in cellular morphology. All cell models showed an increase in proliferation. Normal wounded cells showed a decrease in apoptosis, TNF-α, and IL-1β. Diabetic wounded cells showed an increase in viability and a decrease in apoptosis and IL-1β, whereas hypoxic cells showed an increase in viability and IL-6, and a decrease in apoptosis and TNF-α. NF-κB was translocated into the nucleus post-irradiation. Phototherapy resulted in hastened wound closure, increased proliferation, and normalization of cellular function. The decrease in the different pro-inflammatory cytokines and NF-κB translocation was model and time dependent. Overall, laser irradiation resulted in a reduction in inflammatory cytokines and directed cells into the cell survival pathway.

Comparative Potential of Cultured Skin Fibroblast, Cumulus, and Granulosa Cell to Produce Somatic Cell Nuclear Transfer (SCNT) Preimplantation Embryos in Buffaloes (Bubalus bubalis) in Relation to Gene Expressions

To improve the efficiency of somatic cell nuclear transfer (SCNT)-derived embryos in buffaloes (Bubalus bubalis), skin fibroblast, cumulus, and granulosa cells were cultured up to the 15th passage and cloned embryos were produced from each cell type. At the 15th passage the cumulative population doublings (CPDs) in cumulus cells was higher (60.78) than skin fibroblast (57.12) and granulosa (56.05) cell lines. Gene expression of chromatin remodelling proteins, that is, HDAC1, DNMT1, DNMT3a, and DNMT3b, were comparable at all five passages (P-3, P-6, P-9, P-12, and P-15) groups in cumulus cells but different in skin fibroblast and granulosa cells. Cleavage and blastocyst production rate in cumulus (65.9 and 27.4%)-derived embryos was higher than skin fibroblast (63.8 and 24.3%) and granulosa (62.5 and 22.3%)-derived embryos. Expressions of HDAC1, DNMT1, and DNMT3a mRNA in cumulus-derived blastocysts were similar to IVF blastocysts (control), whereas skin fibroblast and granulosa-derived blastocysts expression was significantly different (p < or = 0.05). DNMT3b mRNA expression in all the three donor cell types and IVF control were similar. The expression pattern of these genes showed the effect of donor cell type with different epigenetic reprogramming capabilities for SCNT embryo production rate. Overall, results indicated that cumulus cells are the best nuclear donor for SCNT.

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.

Regulation of CMV promoter-driven exogenous gene expression with doxorubicin in genetically modified cells

The regulation of gene expression after the introduction of an exogenous gene is a problematic aspect of gene therapy. The purpose of this study was to use doxorubicin to regulate exogenous gene expression in a vector containing the cytomegalovirus (CMV) promoter. The pQBI25 vector, which encodes the CMV promoter and the cDNA for red-shifted green fluorescent protein (rsGFP), was transfected into a rat skin fibroblast cell line (FR cells). The pEGFP vector, encoding the CMV promoter and enhanced green fluorescent protein (EGFP) cDNA, was transfected into human hepatoma HepG2 cells. FR-pQBI25 cells were then continuously exposed to doxorubicin and methotrexate for 96 and 48 h, respectively; HepG2-pEGFP cells were continuously exposed to doxorubicin for 48 h. The levels of c-fos, c-jun and rsGFP mRNA, as well as the levels of rsGFP protein, in the FR-pQBI25 cells were found to be significantly higher following exposure to doxorubicin. However, the level of rsGFP protein was not changed by exposure to methotrexate. The level of EGFP protein in the HepG2-pEGFP cells was also significantly higher following exposure to doxorubicin. To examine the effect of cessation of doxorubicin exposure, FR-pQBI25 cells that had been exposed to doxorubicin for 48 h were re-plated in fresh medium without doxorubicin for a further 48 h. The increased levels of c-fos, c-jun and rsGFP mRNA and rsGFP protein seen after treatment with doxorubicin had reduced by 48 h after the cessation of exposure to doxorubicin. These findings suggest that CMV-driven exogenous gene expression may be regulated by doxorubicin.

Effects of anticancer agents and scavengers on CMV-promoter-driven exogenous gene expression in genetically modified cells

Abstract Objectives This study aimed to investigate whether the levels of rsGFP mRNA and the fluorescence levels of cytomegalovirus (CMV)-promoter-driven rsGFP (red-shifted green fluorescent protein) could be changed by using anticancer agents and also to examine the effects of co-treatment with anticancer agents and scavengers. Methods The pQBI25 vector, which encodes the CMV promoter and the cDNA for rsGFP, was transfected into FR cells (rat skin fibroblast cell line). FR-pQBI25 cells were then exposed to doxorubicin, 5-fluorouracil, methotrexate or paraquat with or without scavengers such as N-acetyl cysteine (NAC) and edaravone for 48 h. Key findings The levels of rsGFP mRNA were found to be significantly higher following doxorubicin, 5-fluorouracil and paraquat treatment but were not changed by methotrexate. These levels of rsGFP mRNA were found to be significantly lower after paraquat/edaravone co-treatment compared with paraquat alone. The fluorescence levels of rsGFP were found to be significantly higher following doxorubicin and paraquat treatment but were not changed by 5-fluorouracil and methotrexate. The levels were also found to be significantly lower after paraquat/edaravone co-treatment compared with paraquat alone and also after doxorubicin/NAC co-treatment compared with doxorubicin alone. Conclusions These findings suggest that CMV-promoter-driven exogenous gene expression may be partly regulated by reactive oxygen species.

제주 재래식된장의 생리적 특성

제주재래식된장의 생리적 특성을 측정하기 위하여 제주도내 전통된장업체로부터 된장제품 4종(JTD1-4)을 수거하여, 유기용매로 추출, 분획하여 총 페놀 함량, 항산화활성(DPPH 유리기 소거능, superoxide 유리기 소거능), ACE 및 α-glucosidase 저해능, 항염 및 항암효과를 측정하였다. 에틸아세테이트 분획물에서 총 페놀 함량(201.8～305.4 ㎍/ g)과 DPPH 유리기 소거활성(37.5～59.9%)이 가장 높았다. Superoxide anion radical 소거활성은 부탄올 분획물에서 56.3～85.1%로 가장 높았는데, JTD1(81.7%)과 JTD4(85.1%)의 소거활성은 양성대조구로 사용한 ascorbic acid의 소거활성(82.2%)과 유사하였다. ACE 저해활성은 메탄올 추출물에서 23.6～74.5%로 가장 높았다. α-Glucosidase 저해활성은 디클로로메탄 분획물에서 72.0～80.2%로 양성대조구인 acarbos의 57.4%보다 높았다. RAW 264.7 세포를 대상으로한 NO 생성 저해활성(IC<SUB>50</SUB>: 17.3～29.0 ㎍/mL)은 에틸아세테이트 분획물이 가장 높았지만, 세포독성은 모든 분획물에서 관찰되지 않았다. 에틸아세테이트 분획물의 세포증식 저해활성은 정상세포주인 피부섬유아세포(HS-68)에서 JTD1-3은 100 ㎍/mL 이하의 농도에서 나타내지 않았으나, JTD4는 21%의 세포독성을 나타내었다. 대장암 세포주(HT-29)에 대해서는 JTD1과 JTD2는 세포증식이 저해되지 않았으나, JTD3과 JTD4는 50 ㎍/mL 이상에서 증식저해 효과를 나타내었다. 유방암 세포주(MCF-7)는 100 ㎍/mL에서 JTD3은 30.9%, JTD4는 34.7%의 세포증식 저해효과를 보였다.

394 CHARACTERIZATION OF BOVINE ADULT-DERIVED ADIPOSE STEM CELLS FOR USE IN NUCLEAR TRANSFER

Adipose tissue is an abundant source of adult-derived cells that have displayed multipotent properties in vitro. The goal of this research was to study the characteristics of bovine adipose-derived adult stem (ADAS) cells to determine the feasibility for use in NT. Adipose tissue was isolated from the brisket of adult cattle postmortem. Cells were isolated by incubation for 2 h with 0.25% collagenase solution, separation of stromal cells by centrifugation, and selection by adherence to plastic. The lifespan and growth characteristics for culture conditions were determined by a 2 × 2 factorial with DMEM or DMEM:F12 and with or without growth factor (GF) supplementation.A two-way ANOVA, followed by multiple pair-wise comparisons using Tukey’s test when applicable, was used to detect differences in population doublings (PD) until senescence for media treatments and GF supplementation. Dulbecco’s modified Eagle’s medium with GF supported significantly less (PD) (P &gt; 0.05) than DMEM : F12. The average lifespan was approximately 30 PD, with a cell length of 48 h until passage 8 (P8). As cells approached replicative senescence, the cell cycle length was inconsistent. Two ADAS and one adult-derived skin fibroblast cell lines from different animals were subjected to differentiation conditions for adipocytes, chondrocytes, and osteoblasts at P2, P6, and P11. Differentiation was confirmed by histological staining. Passage 2 ADAS cells differentiated more efficiently than did P6, P11, or skin fibroblasts. Global levels of DNA methylation and histone acetylation were analyzed from P1 to P6 in 3 sets of cell lines consisting of ADAS and skin cells from the same animals by immune staining and flow cytometry. There was no significant difference (P &gt; 0.05) between cell types by one-way ANOVA. Nuclear transfer was performed using ADAS cells as donor cells and commercially supplied oocytes. Mature, enucleated oocytes were reconstructed with either adult skin fibroblasts or ADAS cells. The percentage of cleaved and blastocysts from ADAS cells (62% and 8%, n = 163) and skin fibroblasts cells (42% and 8%, n = 170) were not different (P &gt; 0.05) by chi-square analysis. Interspecies NT was attempted with eland (Taurotragus oryx) ADAS cells and enucleated bovine oocytes. Two groups of enucleated oocytes were reconstructed with bovine (n = 234) and eland (n = 290) ADAS cells. There was no significant difference between the number of cleaved embryos (38% and 39%) or blastocysts formed by chi-square analysis. A total of 3 interspecies embryos (1%) and 5 bovine embryos (14%) developed to blastocysts. Bovine ADAS cells are not more efficient than bovine adult-derived skin fibroblasts as donor cells, but they do represent a viable option for use in NT because of their higher in vitro development. Eland ADAS cells resulted in development to the blastocyst stage after interspecies NT.

Development of a Cytotoxic T-Cell Assay in Rabbits to Evaluate Early Immune Response to Human T-Lymphotropic Virus Type 1 Infection

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell lymphoma/leukemia (ATL) following a prolonged clinical incubation period, despite a robust adaptive immune response against the virus. Early immune responses that allow establishment of the infection are difficult to study without effective animal models. We have developed a cytotoxic T-lymphocyte (CTL) assay to monitor the early events of HTLV-1 infection in rabbits. Rabbit skin fibroblast cell lines were established by transformation with a plasmid expressing simian virus 40 (SV40) large T antigen and used as autochthonous targets (derived from same individual animal) to measure CTL activity against HTLV-1 infection in rabbits. Recombinant vaccinia virus (rVV) constructs expressing either HTLV-1 envelope surface unit (SU) glycoprotein 46 or Tax proteins were used to infect fibroblast targets in a (51)Cr-release CTL assay. Rabbits inoculated with Jurkat T cells or ACH.2 cells (expressing ACH HTLV-1 molecule clone) were monitored at 0, 2, 4, 6, 8, 13, 21, and 34 wk post-infection. ACH.2-inoculated rabbits were monitored serologically and for viral infected cells following ex vivo culture. Proviral load analysis indicated that rabbits with higher proviral loads had significant CTL activity against HTLV-1 SU as early as 2 wk post-infection, while both low- and high-proviral-load groups had minimal Tax-specific CTL activity throughout the study. This first development of a stringent assay to measure HTLV-1 SU and Tax-specific CTL assay in the rabbit model will enhance immunopathogenesis studies of HTLV-1 infection. Our data suggest that during the early weeks following infection, HTLV-1-specific CTL responses are primarily targeted against Env-SU.

Loss of mutant mitochondrial DNA harboring the MELAS A3243G mutation in human cybrid cells after cell-cell fusion with normal tissue-derived fibroblast cells

Mutant mitochondrial (mt) DNA variants are related to human disease and have been investigated using cytoplasmic hybrid (cybrid) cells generated from human tumor cells in which mutant mt maintenance depends on the cell line. It is, however, unclear whether human intercellular fusion of non-tumorous cells influences the maintenance of disease-related mutant mt. A preliminary experiment of cell-cell fusion between a human skin fibroblast cell line from a Lesch-Nyhan syndrome patient and an osteosarcoma cybrid cell line harboring the mitochondrial tRNALeu(UUR)A3243G mutation showed a decrease of A3243G mutant mtDNA in fused cells during passages. In order to confirm the decrease of mutant mtDNA, we performed cell-cell fusion experiments using another human lung fibroblastic cell line. When the hygromycin-resistant osteosarcoma cybrid cell line was fused with the fibroblasts without any A3243G mtDNA mutations, the proportion of A3243G mutant mtDNA in the hybrid cells gradually decreased during cell culture and almost completely disappeared in all hybrid clones at the end of 15 passages. These results indicated that A3243G mutant specific mtDNA decreases in the hybrid background when normal fibroblast-derived cell contents, including the nucleus and mt, were introduced. Thus, we are hypothesizing that the non-tumorigenic fibroblast cellular components induce a difference in replication efficacy between the mtDNAs with and without the A3243G mutant sequence, which may be related to the decrease of disease-related mutant mtDNA in the hybrid cells.

Synthesis and in vitro antiproliferative activity of diphenyl(sulphonylpiperidin-4-yl)methanol derivatives

A series of novel diphenyl(piperidin-4-yl)methanol derivatives 10(a–n) were synthesized and characterized by 1H NMR, LC/MS, FTIR, and elemental analyses. All the synthesized compounds were evaluated for cell proliferation by MTT assay. The antiproliferative effects of the synthesized compounds were tested against viable human skin fibroblast cell line and carcinoma cell lines, namely HeLa cells, HT-29 cells, MCF-7 cells, and HepG-2 cells in comparing the positive and negative control. Among the synthesized compounds, (10b) and (10g) have been identified as potent antiproliferative agents.

Synthesis, structure elucidation and antitumour activity of N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid

New N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid ( 2– 12) were designed and prepared by the condensation reaction of exo- S-ethyl-7-oxabicyclo-[2.2.1]-hept-5-ene-2,3-dicarbonyl isothiosemicarbazide ( 1) with primary amines. The chemical structure of all compounds was confirmed by IR, 1H NMR, 13C NMR spectra, the X-ray crystallography (for compounds 8, 11, 12) and elemental analysis. Moreover, compounds 9– 11 were screened for their anticancer activity. Compounds 9 (in concentrations of 0.32 mM and 0.16 mM), 10 (in concentrations of 0.28 mM and 0.14 mM), and 11 (in concentrations of 0.35 mM and 0.17 mM) were found to be evidently effective in vitro against lung cell line (IC50). The distinctly marked antiproliferative effect of compounds 9 and 10 in breast carcinoma cells in vitro was ascertained. Moreover, the lowest cytotoxicity of compound 9 in concentrations of 0.16 mM and 0.03 mM against the normal skin fibroblast cell line and breast carcinoma cell in vitro after 24- and 48-h periods of incubation was noticed in this study.