Baicalin modulates let-7i-3p/PI3K/Akt/NF-KB signaling axis to reduce NLRP3 inflammasome activation in fibroblast like synoviocytes of rheumatoid arthritis / 中国药理学通报

Aim To study the effect of baicalin on the activation of NLRP3 inflammasomes in human fibroblast like synoviocytes of rheumatoid arthritis ( HFLS-RA) and its mechanism. Methods To confirm that baicalin alleviated the activation of NLRP3 inflammasome in HFLS-RA, immunofluorescence was used to observe the expression of NLRP3 before and after baicalin treatment. Western blot was used to detect the protein expression of p-PI3K, p-Akt, NF-κB p65, NL-RP3, ASC and caspase-1 after baicalin treatment for 48 h, and ELISA was employed to detect the contents of IL-1 and IL-18 in the supernatents. In order to explore the mechanism of baicalin alleviating the activation of NLRP3 inflammasome, double luciferin and Westen blot analysis were applied to verify the corresponding relationship between let-7i-3p and PIK3CA. RT-qPCR was utilized to determine the expression of let-7i-3p and PI3K before and after baicalin intervention. let-7i-3p interference was used to verify whether baicalin mitigated the activation of enhanced NLRP3 inflammasomes. Results Baicalin (50, 100 mg · L

Effect of Ermiaosan on Function of Human Fibroblast Synovial Cells Induced by TNF-α / 中国实验方剂学杂志

Objective::To study the effects of Ermiaosan on migration, adhesion and invasion of human fibroblast-liked synovial cells(FLS) and explore its mechanism. Method::Using the human FLS as the research object, the nontoxic concentration of FLS.FLS was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay for the follow-up experiment. The transwell migration, adhesion and transwell invasion test were used to detect the migration and adhesion of the different concentration of Ermiaosan on FLS, respectively. The expression of interleukin (IL)-1 beta of FLS supernatant was detected by enzyme linked immunosorbent assay (ELISA). Protein in FLS was extracted and protein expression levels of phosphorylated Janus kinase 1 (p-JAK1), p-signal transducer and activator of transcription (STAT1) and p-STAT6 were detected by Western blot. Result::Compared with control group, tumor necrosis factor(TNF)-α (20 μg·L-1) increased the proliferation, migration, adhesion, invasion and the secretion of IL-1β of FLS (P<0.01). Ermiaosan(0.2, 0.4, 0.8 mg·L-1) had no significant effect on the proliferation of FLS induced by TNF-α for 24 h. Within 24 h, the migration, adhesion, invasion, invasion, and secretion of IL-1β of FLS cells induced by TNF-α were also decreased significantly(P<0.05, P<0.01). Compared with the blank group, TNF-α could induce abnormal elevation of p-JAK1, p-STAT1 and p-STAT6 in FLS (P<0.01), while Ermiaosan of 0.2, 0.4, 0.8 g·L-1 could significantly reduce the expression levels of p-JAK1, p-STAT1 and p-STAT6 (P<0.05, P<0.01). Conclusion::Ermiaosan can inhibit the migration, adhesion and invasion of FLS, and its mechanism may be related to the inhibition of the secretion of IL-1β, the mechanism may be related to JAK/STAT pathway.

Article Effect and Mechanism of Ganoderma lucidum Polysaccharides on Human Fibroblasts and Skin Wound Healing in Mice / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of Ganoderma lucidum polysaccharides (GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism.@*METHODS@#Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide (MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type I (CICP) and transforming growth factor-β1 (TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated.@*RESULTS@#Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts (Plt;0.05 or Plt;0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group (Plt;0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group (Plt;0.05 or Plt;0.01).@*CONCLUSION@#A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.

UVB induces morphological changes and the expression of MMPs in human fibroblasts / 基础医学与临床

Objective To observe the changes of telomere length and MMPs level in human fibroblasts induced by UVB, and to explore their roles on skin photoaging .Methods Human skin fibroblasts were extracted and cul-tured.The 5th fibroblasts were irradiated by UVB .The morphology of fibroblasts were microscoped , and the length of telomere and the mRNA expression of COL1a1 and hTERT were detected by RT-qPCR.The expression of MMP-3 and MMP-1 were detected by Western blot .Results The fibroblasts gradually became round , wrinkled and disorderly arranged after 30 mJ/cm2 UVB irradiation for 24 h.The mRNA level of COL1a1 and hTERT and the expression of MMP-3 and MMP-1 were significantly increased after UVB irradiation compared with control , and the length of telomere was shortened .Conclusions UVB may frigger the early process of photoaging by the morphologi-cal changes of human skin fibroblasts and increasing the expression of MMP-3 and MMP-1 .

Proteomic study of the effect of recombinant hFGF-10 adenovirus on HaCat cells / 药学学报

This study is to investigate the effect of recombinant hFGF-10 adenovirus on the proteome of HaCat cells, and to speculate further the possible mechanism of the effect of hFGF-10 on HaCat cells via differentially expressed proteins identified. Two-dimensional gel electrophoresis (2-DE) combined with tandem time-of-flight mass spectrometry was applied to identify the differentially expressed protein spots on the 2-DE maps of the whole-cell proteins from Ad infected and rAd-hFGF-10 infected HaCat cells. The mRNA and protein levels of the differentially expressed proteins were confirmed with semi-quantitative RT-PCR and Western blotting. The results showed that the 2-DE maps with high resolution were obtained, and four selected differentially expressed proteins involved in cell apoptosis, cytoskeleton regulation and protein degradation were identified with MALDI-TOF/TOF. The mRNA and protein levels of one of the differentially expressed proteins, VDAC2, were up-regulated in HaCat cells infected with the recombinant hFGF-10 adenovirus. The differentially expressed protein, VDAC2, may be related to the bioactivities of hFGF-10.

Effects of medical polyacrylamide hydrogel on human fibroblasts: a transmission electron microscopic study / 中华医学美学美容杂志

Objecttive To explore the effect of medical polyacrylamide hydrogel on the ultrastructure of human fibroblasts.Methods 22 hyperplastic fibropeplos around the medical polyacrylamide hydrogel in mammaries after augmentation mammoplasty by injection of medical polyacrylamide hydrogel were obtained by operations.The tissues were observed with the transmission electron microscope and at the same time photographs taken.Results Most of cell organoles dissolved and disappeared;Most of the cristaes and membranes of mitochondria coalesced or disappeared;rough endoplasmic reticulum expanded.Most of the cristaes and membranes coalesced or/and disappeared.The phenomenon of shed particle was observed in some of the rough endoplasmic reticulum.Conclusion The medical polyacrylamide hydrogel obviously injures the ultrastructures of human fibroblasts.

The regulatory effect of recombinant human fibroblast growth factor-21 on the expression of liver X receptor α and glucose transporter protein 1 in the type 2 diabetes rats / 中国生化药物杂志

Purpose To examine the regulatory effect of recombinant human fibroblast growth factor-21 on the expression of liver X receptor α and glucose transporter protein 1 in the type 2 diabetes mellitus rats.Methods The rat models of type 2 diabetic mellitus were divided into four groups at random, ic. rhFGF-21 every day, after eight weeks of these treatment, Inspect the fasting blood glucose (FBG), fructosamine(FA), triglyceride(TG), T-cholesterol(TC), high density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol(LDL-C) of these rats, then detecting the mRNA expression of LXRα and GLUT1 by RT-PCR.Results (1) rhFGF-21 can reduce blood glucose steadily to near normal levels in diabetic rats. (2) The expression of LXRα and GLUT1 level was significantly higher in the rhFGF-21 treatment group than that in the model group. (3) rhFGF-21 megadoses and middle doses decreased FA, TG, TC,and LDL-C and elevated HDL-C.Conclusion rhFGF-21 could regulate the mRNA expression of LXRα and GLUT1 in diabetes rats, increase basal level glucose transport, then reduce blood glucose, improve lipid metabolize dysfunction.

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.

AAV2-mediated versus AAV2/1-mediated enhanced green fluorescent protein transfection into human skin fibroblast in vitro / 第三军医大学学报

Objective To compare the transfection rate of AAV2-EGFP and AAV2/1-EGFP to human skin fibroblast(HFB).Methods Two recombinant adeno-associated viruses(AAV) encoding enhanced green fluorescent protein(EGFP) were constructed and transfected to HFB at multiple of infection MOI ranging from 104 to 106.Twenty-four hours after the infection,the expression rates of EGFP on cultured HFB were assessed by flow cytometry and the transfected cells were observed under fluorescence microscope.The killing effect of the virus on the infected cells was assayed by MTT.Results The transfection efficiency of AAV2-EGFP was increased as MOI increased.When MOI was 104,the transfection efficiency of AAV2-EGFP was 7.68%?1.18%;When MOI was 105,that was 52.12%?1.59%;When MOI was 106,that showed no significant increase.However,the transfection efficiency of AAV2/1-EGFP showed no obvious changes at any MOI.Conclusion AAV2 is more efficient than AAV2/1 in transfecting HFB,but neither AAV has a high transfection efficiency.

Production of IL - 6 and IL - 8 in Human Fibroblasts Stimulated with Mycoplasma Lysates and Bacterial Toxins

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS), Staphylococcus enterotoxin B (SEB), and Mycoplasma lysates on regulation of IL-6 and IL-8 production by human nasal fibroblasts. Primary cultured cells were incubated with LPS (1.0 ug/ml) from E.coli, SEB (1.0 ug/ml) from S.aureus, or Mycoplasma lysates (M.pneumoniae, Mp; M. fermentans, Mf; M. hominis, Mh, each 1.0 ug/ml). The culture supernatants were collected at 2, 6, and 24 hr and assessed for IL-6 and IL-8 production by enzyme-linked immunosorbent assay. The production of IL-6 in the culture supematant was downregulated by LPS, SEB, or Mycoplasma lysates. But IL-6 was upregulated by mixed exposure with Mp+LPS (2 hr), Mp+LPS+SEB (24 hr), Mf+LPS (24 hr), Mf+LPS+SEB (2 hr), Mh+LPS (24 hr), Mh+SEB (24 hr), or Mh+LPS+ SEB (24 hr). The production of IL-8 in the culture supematant was similar to that of IL-6 by same stimulants. But IL-8 was upregulated by mixed exposure with Mf+LPS+SEB (2 hr), Mh+LPS (24 hr), Mh+ SEB (24 hr), or Mh+LPS+SEB (24 hr). These studies show that costimulation of LPS or SEB with Mycoplasma whole cell lysates upregulates the production of IL-6 and IL-8.

Production of IL-12 from gene modified human dermal fibroblasts: A preclinical study for IL-12 cancer gene therapy

Cytokine has been used as an immune stimulator and administered to patients for a treatment of cancer. Interleukin-12 (IL-12) is a potent cytokine which acts through a variety of functions including interferon-gamma production and cytotoxic T-cell activation. Considering the toxicity of high dose systemic IL-12 administration into human, local administration of low dose IL-12 can be a more efficient strategy. In ex vivo therapy, human dermal fibroblast has been considered as a useful vehicle for transferring genes, Here we show that human dermal fibroblast transduced with retrovirus containing IL-12 gene can be manipulated to produce reasonable amount of IL-12 protein. Human dermal fibroblast was isolated from freshly harvested skin specimens by collagenase digestion, grown in primary cultures, and transduced with a retroviral vector containing genes for human IL-12 and a selectable marker Neo(R). Following selection in G418, IL-12 producing fibroblasts were tested for secreted IL-12 level by ELISA. Six specimens of human skin were processed to obtain fibroblasts. ELISA results show that 40-150 units of IL-12 was produced for 24 h from 1x10(6) cells of transduced and selected fibroblast cultures. The primary cultures were maintained for up to nine passages about 108 days. The mean +/- overall time for obtaining enough number of cells was 49 +/- 2 days. The fibroblasts continued to produce IL-12 in culture for 90 days. These preliminary results can be used for the design of ex vivo gene therapy clinical trial using human dermal fibroblast.

HIGH EXPRESSION OF RECOMBINANT HUMAN BASIC FIBROBLAST GROWTH FACTOR IN ESCHERICHIA COLI / 微生物学通报

To improve the expression level of non fusion hbFGF in E coli , the coding sequence of human bFGF gene, which had been cloned from primarily cultured human fibroblast, was mutated according to the principle of lowering the GC content and increasing the codon preference After being ligated into pET 3c and transformed into BL21(DE3), the recombinant induced by IPTG Expression level was up to 30% of the total bacterial protein The result indicated that optimizing of the TIR would promote the expression level of recombinant protein

A study on labeling efficiency,cellular viability and in vitro MRI of human fibroblast cells labeled with SPIO of different concentration / 重庆医科大学学报

Objective:To explore the efficiency of super-paramagnetic iron oxide(SPIO)of different concentrations in vitro labeling human fibroblast cells as well as the influence on cellular viability,and to investigate the characteristics of magnetic resonance imaging.Methods:Respectively mix pure SPIO(concentration of iron:200 ?g/ml) and SPIO-PLL compound of different concentrations with substrate(final concentrations of iron:150,100,50,25 ?g/ml;final concentration of PLL:7.5 ?g/ml).Then incubate it with Hfbs for 12,24,48 h and 72 h.Collect the cells.Assess cellular viability with Trypan Blue Dye Exclusion Method;test iron particles distribution in cells with Prussian blue Stain;observe the positions of iron particles with optical microscope and TEM and calculate the labeling rate of SPIO;perform MR scanning to measure the signal intensity change of the cells.Results:Labeling efficiency of SPIO-PLL compound is much higher than that of pure SPIO(P0.05).Prussian blue Stain test shows there are more or less blue dyed iron particles inside cytoplasm of each labeled cells.And those iron particles concentrate in endosome and lysosome(with electron microscope).Culture solution of concentration in the range of 25～50 ?g Fe/ml is safe for labeling stem cells with SPIO,and 95%～100% of the cells will be effectively labeled in 18～24 h.And the MR scanning shows obvious reduction of signal intensity of the labeled cells.Conclusion:Human fibroblast cells can be easily and safely labeled with self-made SPIO-PLL compound.Labeling with appropriate solution(iron content of 25 ?g/ml) is highly efficient and has little influence on cellular viability,furthermore,with SPIO labeling,the signal intensity of cells scanned with MR is dramatically reduced.

AN OBSERVATION OF A HUMAN DIPLOID FIBROBLAST CELL LINE UNDER LONG TERM PRESERVATION IN VITRO / 解剖学报

A human diploid fibroblast cell line derived from normal abdominal skin of a 17-year-old female patient with ovary carcinoma was established and designated as HSF 79-2. The cultivation of skin fragments was employed in the primary explantation. The medium used was McCoy 5A supplemented with 15～25% calf serum. The grown fibroblasts were succesively subcultured at 1 to 2 split and stopped at the 45th to 54th passage.Having reached confluence the cells still had the ability to divide during 16 months observation if the medium was changed periodically. They displayed the property of dense growth and forming heavy, multilayer sheets, which became a visible membrane to the naked eyes. Under histological examination the membrane had the appearance of connective tissue. Part of the cells were subcultured again after 3,6, and 12 months maintenance in the same culture flask. Their growth character, ploidy, and the generation time were similar to that of the cells passaged normally.The characteristics of this cell line mentioned above appear to be shared by other diploid fibroblasts. It might be used for preserving cell lines and as a model for studying cell motility and differentiation.