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.

Effects of HSP47 siRNA on biological behaviors of HTCF cells cultured <i>in vitro</i> and the expression level of TGF-β1 / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore the effects of heat shock protein 47(HSP47)siRNA on biological behaviors of human Tenon capsule fibroblasts(HTCF)cells cultured <i>in vitro</i> and the expression level of transforming growth factor-β1(TGF-β1).<p>METHODS: HTCF were cultured <i>in vitro</i> and divided into blank control group, empty vector group and transfection group. In transfection group, interfering siRNA sequences were designed and synthesized based on the HSP47 gene sequences, vectors were constructed and introduced into HTCF. The empty vector group was introduced with empty vectors. The expressions of HSP47 mRNA and protein in cells were detected by RT-PCR and Western blot. The proliferation, apoptosis, invasion and migration of cells were detected by clone formation assay, flow cytometry, Transwell method and scratch test. The expressions of proliferation, apoptosis, invasion and migration proteins, and TGF-β1 were detected by Western blot.<p>RESULTS: Compared with empty vector group, expression of HSP47 mRNA and protein, clone formation rate, cell healing rate, number of invasive cells, relative expression levels of Ki67, N-cadherin and TGF-β1 were significantly decreased in transfection group(<i>P</i><0.05), relative expression level of E-cadherin protein was significantly increased(<i>P</i><0.05), but there was no difference in apoptosis rate, and relative expression levels of Bcl-2 and Bax(<i>P</i>>0.05).<p>CONCLUSION: HSP47 siRNA can reduce proliferation, invasion and migration abilities of HTCF cells by inhibiting the expression of TGF-β1 protein, without significant effects on the apoptosis of HTCF cells.

PEG-PCL Nanocapsules Containing Curcumin: Validation of HPLC Method for Analyzing Drug-loading Efficiency, Stability Testing and Cytotoxicity on NIH-3T3 Cell Line

Abstract Curcumin (CUR) shows potential use for treating cancer. However, CUR has low solubility and reduced bioavailability, which limit its clinical effect. Therefore, the development of nanocarriers can overcome these problems and can ensure the desired pharmacological effect. In addition, it is mandatory to prove the quality, the efficacy, and the safety for a novel nanomedicine to be approved. In that sense, this paper aimed (a) to prepare CUR-loaded polyethylene glycol-poly(ε-caprolactone) nanocapsules; (b) to validate an analytical method by high performance liquid chromatography (HPLC) for quantifying CUR in these nanoformulations; (c) to evaluate the physicochemical stability of these formulations; and to investigate their cytotoxicity on NIH-3T3 mouse fibroblast cells. The HPLC method was specific to CUR in the loaded nanocapsules, linear (r = 0.9994) in a range of 10.0 to 90.0 µg.mL-1 with limits of detection and quantification of 0.160 and 0.480 µg.mL-1, respectively. Precision was demonstrated by a relative standard deviation lower than 5%. Suitable accuracy (102.37 ± 0.92%) was obtained. Values of pH, particle size, polydispersity index, and zeta potential presented no statistical difference (p > 0.05) for CUR-loaded nanoparticles. No cytotoxicity was observed against NIH-3T3 mouse embryo fibroblast cell line using both the tetrazolium salt and sulforhodamine B assays. In conclusion, a simple and inexpensive HPLC method was validated for the CUR quantification in the suspensions of nanocapsules. The obtained polymeric nanocapsules containing CUR showed suitable results for all the performed assays and can be further investigated as a feasible novel approach for cancer treatment.

Early morphology observation of human periodontal fibroblast cell line in the presence of human salivary exosomes

@#Human salivary exosomes have been identified as a highly informative nanovesicle with clinical-relevant information for variation of diagnostic purposes. As a continued effort from previous studies on human salivary exosomes effect at gene expression level, this study is carried out to observe the morphology of human periodontal fibroblast (HPdLF) treated with exosomes cells under the same period of changes in genotypic level occurred. In vitro, HPdLF cells were cultured for 24 hours with 10 µg/ml of human salivary exosomes. The morphology of HPdLF cells was examined under inverted light microscopy and scanning electron microscopy (SEM) for both control samples and samples treated with human salivary exosomes, while the cell count was performed via trypan blue staining. There was no significant difference in the morphology under the inverted light microscopy and the cell number of HPdLF cells for both treated and untreated cells with exosomes. However, for SEM, the treated HPdLF with salivary exosomes showed slight observable changes on the filopodia, lamellipodia, cytoplasmic vesicles and the cytoskeleton of the cells. Even within a short period (24 hours) of culturing time for cells with human salivary exosomes, the samples showed minimal changes which positively suggested a simultaneous event of exchanging materials from human salivary exosomes to cells had occurred, hence, potentially proving that human salivary exosomes can enhance cell proliferation

Inducement effect of tetrandrine on the apoptosis of cultured human Tenon's capsule fibroblasts / 眼科研究

BackgroundExcessive conjunctival scar formation is a main cause of filtering surgical failure in glaucoma.It has been reported that the failure rate of filtering surgery is a tough problem in the research of glaucoma.Research showed that tetrandrine (Tet) suppress the proliferation of cultured human fibroblasts of Tenons capsule (TCFs) in vitro,but its possible mechanism is still unclear up to now.ObjectiveThe aim of this study is to investigate the inducement effects of Tet on the apoptosis of fibroblasts of Tenons capsule and mechanism.MethodsThe Tenon's capsule was obtained from donor eyes of Zhongshan Eye Center Eye Bank for further use.Human Tenon's capsule fibroblasts were cultured by explant culture method in mixed medium in vitro and the third to seventh generations of cells were collected for the experiment.The subcultured cells were identified by morphology observation and Vimentin staining.The apoptosis of cultured fibroblasts in Tet-treated group and control group was studied by using TUNEL (TdT- mediated dUTP nick end labling,TUNEL),flow cytometry (FCM) and transmission electron microscope.The cell number in different cellular cycle was calculated in Ted-treated group and control group.Results The cultured cells reached confluence in two weeks after cultured and presented the spindle or triangle shape with the radial-like or vortexin-like arrangement .The cells showed the positive staining for Vimentin.Apoptosis changes of cultured cell and apoptosis bodies were seen under the transmission electron microscope.Apoptotic cell nuclei were observed in Tet group by TUNEL.FCM result showed that the cells at G_0/G_1 phase decreased by 22.2%,and the cells at S and G_2/M phases increased by 20.53% and 1.6% in Ted-treated group.Significant differences in the numbers of cells in different cellular cycles and cell numbers of apoptosis were found between Tet-treated group and control group(P=0.000).ConclusionTet can inhibit the proliferation of human TCFs by inducing apoptosis in vitro.

Treatment of Diabetic Ulcer using Autologous Fibroblast-Hyaluronic Acid Complex

PURPOSE: To evaluate clinical efficacy and safety of hyaluronic acid based autologous dermal fibroblasts (Hyalograft 3D) in the treatment of diabetic foot ulcers. METHODS: A total of 28 patients with diabetic ulcers were randomized to either the control group with nonadherent foam dressings(n=14) or the treatment group with autologous tissue-engineered grafts(n=14). Weekly assessment contained vital sign checks, ulcer size measurements, and wound photos. In the 12th week, percentages of complete wound healing and mean healing times were compared. Safety was also monitored by adverse events. RESULTS: Complete wound healing was achieved in 84.6% of the treatment group and 23.1% of the control group(p<0.005). The mean times of closures for the treatment versus control groups were 6.1 weeks and 10.9 weeks, respectively. No adverse events related to the study treatment occurred. CONCLUSION: The use of hyaluronic acid based autologous fibroblast grafts was found to be a safe and effective treatment for diabetic foot ulcers.

Optimization of Wet Fixation Methods for AFM Imaging of Human Fibroblast Cells

We investigated the effect by the chemical fixative on human fibroblast cells (HFCs) in order to make nano-scale images using by the atomic force microscopy (AFM). The cell fixation needed to be optimized as prerequisite step for the preparation before analysis. AFM imaging after optimal wet fixation can provide practical, simple and fast technique for scanning living cells. In this study, AFM images - topography and amplitude - and the optic images of HFCs which were fixed with phosphate buffered saline (PBS), 2:1 ethanol:acetic acid, 4% glutaraldehyde and 37% formaldehyde were compared respectively. The final effect by washing with PBS or distilled water (D.W.) was examined after 4% glutaraldehyde fixation. To determine the optimal fixation method for HFCs, we performed quantitative and qualitative analysis by the height profile, the presence of artifacts and the morphology of well-conserved fibroblastic topography image by AFM. From AFM image which showed fibroblastic cellular morphology and differential height value of cytoplasm (670+/-47 nm, n=10) and nucleus (847+/-32 nm, n=10) in HFCs, we proposed that wet fixation by 4% glutaraldehyde, followed by final washing with PBS, could be the most suitable preparation for AFM imaging of HFCs, which enable us to approach easily on living cells with the least shrinkage.

The study on the effect of nanoemulsion for the prevention and treatment of gingival inflammation

We have found out the relationship of nanoemulsion containing nano vitamin C, E and propolis and gingival disease. We've confirmed effect of nanoemulsion through the experiment of in vivo and in vitro. We tested cell viability of gingival fibroblast cells by MTT assay and mRNA appearance of interleukin-1 beta, using mouse that was guided inflammation. Anti-microbacterial activity for Antibacterial effect's experiment was carried out by using S.aureus and E.coli. In addition, inflammation tissue has been observed with scanning electrical microscopy. In this study, expression of interleukin-1 beta was decreased after adding nanoemulsion containing nanovitamin C, E and propolis. We've also obtained good results from the test of Antibacterial effect against S.aureus and E.coli. Also, swelling of inflammation tissues observed by scanning electrical microscopy has gone down. In conclusion, we have gained confidence that nanoemulsion containing nano vitamin C, E and propolis has very high Antibacterial effect against bacteria in oral. And it made us guess that inflammation of gingival reduces after decreasing interleukin-1 beta. Thus, we expect that nanoemulsion containing nano vitamin C, E and propolis gives good effects to patient having gingival disease.

Effects of Recombinant Human Epidermal Growth Factor on the Proliferation and Radiation Survival of Human Fibroblast Cell Lines in Vitro / 대한방사선종양학회지

PURPOSE: To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. MATERIALS AND METHODS: Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. RESULTS: Number of fibroblast was significantly more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. CONCLUSION: rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing.

Effect of triptolide on fibroblast cells / 医学研究生学报

Objectives:To study the effect of triptolide(Tri) on fibroblast cells in vitro culture, morphologically and kinetically. Methods:Light microscope(LM)and electron microscope(EM)were employed to detect the morphological changes in Tri group. Cell DNA content and apoptosis were assayed by flow cytometry (FCM). Results:In Tri group, fibroblast cells showed less density, slimmer body, abnormally and typical apoptosis under LM, while rarefaction and content of rough endoplasmic reticulum(RER) decrease, and apoptosis in different stages could be seen under EM. In FCM,AI elevated remarkably in Tri groups, and the effect is dose dependent, with cell cycle uninfluenced at all. Conclusions:The mechanism of triptolide on hypertrophic scars is related to its promotion on fibroblast apoptosis and inhibition of collagen excretion.

Chromosomal aberration and micronucleus formation induced by sulfur mustard / 第三军医大学学报

Objective To study the effects of sulfur mustard on chromosomal aberration in Chinese hamster fibroblast cell line (CHL cells) and the micronucleus (MN) frequency in the bone marrow cells of mice. Methods CHL cells (with and without S9) treated with different concentrations of sulfur mustard for 24 h were harvested for the preparation of chromosome. Subsequently, the frequency and the type of chromosomal aberration were examined. The KM mice were sacrificed at 24 h after subcutaneous injection of sulfur mustard. The number of micronuclei-containing polychromatic erythrocytes (PCEs) was counted by means of bone marrow smear and Giemsa staining. Results Sulfur mustard might increase the chromosomal aberration frequency of CHL cell in a dose-dependent manner. S9 had no effects on the classificaiton and the frequency of chromosomal aberration. The types of chromosomal aberration included gap, break, deletion, polyploid, and so on. Compared with that in the control group, the MN ratio in every group increased significantly (P

Cultivation and identification of porcine skin fibroblasts / 第三军医大学学报

Objectives To establish an ideal cellular model for the study of infection of porcine endogenous retrovirus in intro and in vivo and to lay the foundation for evaluating the biological safety of xenotransplantation. Methods Porcine skin fibroblasts were cultured by tissue pieces, cold trypsin, and heat trypsin, respectively. The merit and defect of these methods were analyzed. Results Porcine skin fibroblast line was established and the tissue piece method was superior to trypsin methods. Conclusion Culture of porcine skin fibroblasts with tissue pieces is superior to trypsin method, with the characteristics of low cost and easy operation. The established porcine skin fibroblast cell line supplies an ideal cell model for the study of infection of porcine endogenous retrovirus in vitro and in vivo, provides a new method for the evaluation of biological safety of xenotransplantation, and plays an important role in the skin culture model of production of cloned animals.

ESTABLISHMENT OF THE FIBROBLAST CELL LINE IN THE XERODERMA PIGMENTOSUM PATIENT AND A STUDY OF ITS BIOLOGICAL CHARACTERISTICS / 第二军医大学学报

Xeroderma pigmentosum (XP; is an inborn autosomal recessive inheritance disease with DNA repair deficiency. XP has become an ideal experimental model to study human DNA repair system and the mechanism of cancer. XP 4SH cell line in this report was obtained from the skin tissue of XP patients. After cell culture, XP 4SH cell line showed the characteristics of form and growth of fibroblast cell. The number of its chromosome was 46(2n = 46).Its aberration was characterised by dicchro-mosomes appearing in XP cells. XP cells induced by UV showed that the frequency of SCE increased significantly while the level of UDS decreased. Gene complement analysis by cell fusion suggests that XP 4SH cell line belongs to the complementation group A.