UNLABELLEDThe periodontal ligament fibroblast (PDLF) was cultivated artificially as the cell to be tested, and then it was loaded with mechanical stress-strain of different values and for different times. The cell and nucleus projected areas and shapes as well as the structure of cytoskeleton were tested by use of confocal laser scanning microscope and immunity fluorescence technique. Then the relationship among the stress-strain, the time, the shape and the structure of cytoskeleton of the PDLF was detected.

RESULTSIn the trial groups of 0, 8%, 12%, 16% strain values, the cell and nucleus projected areas were proportional to strain (stress) and time. The diameter, density and order of the structure of cytoskeleton increased in the strain and time dependent fashion. In the trial group of 20% strain values, the cell and nucleus projected areas decreased with the increase of time, and the structure of cytoskeleton became disorderly. It was demonstrated in this study that the shape and structure of cytoskeleton of PDLF underwent regular changes when the PDLF was loaded with the mechanical stress-strain.

Cells, Cultured ; Cytoskeleton ; physiology ; Fibroblasts ; cytology ; physiology ; Humans ; Periodontal Ligament ; cytology ; Radioimmunoassay ; Stress, Mechanical ; Tensile Strength ; physiology

Animals ; Endothelial Cells ; metabolism ; physiology ; Fibroblasts ; metabolism ; physiology ; Humans ; Hypertension, Pulmonary ; etiology ; genetics ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; physiology

One of the differences between fetal and adult skin healing is the ability of fetal wounds heal without contraction and scar formation. Extracellular matrix (ECM) provides a substratum for cells adhesion, migration, and proliferation and can directly influence the form and function of cells. As motility is essential for many important biological events, including wound healing, inflammatory response, embryonic development, and tumor metastasis, this study was designed to compare the motilities cultured dermal fetal and neonatal fibroblasts in the extracellular matrix. The motility of cultured fetal and neonatal fibroblasts was compared using a video-microscopy system that was developed in combination with a self-designed CO2 mini-incubator. To determine migration speed, cells were viewed with a 4X phase-contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. We found that cultured fetal fibroblasts move faster than neonatal fibroblast on type I collagen (fetal fibroblast, 15.1 micrometer/hr; neonatal fibroblast, 13.7 micrometer/hr), and in fibronectin (fetal fibroblast, 13.2 micrometer/hr; neonatal fibroblast, 13.0 micrometer/hr) and hyaluronic acid (fetal fibroblast, 11 micrometer/hr; neonatal fibroblast, 9.8 micrometer/hr).
Cell Movement ; Cells, Cultured ; Comparative Study ; Extracellular Matrix/*physiology ; Fetus/physiology ; Fibroblasts/*physiology ; Human ; Infant, Newborn ; Skin/cytology/*embryology ; *Skin Physiology

UNLABELLEDObtaining human blastocysts is a prerequisite for cell replacement therapy using embryonic stem cells. We established an interspecies somatic cell nuclear transfer (iSCNT) technique for producing blastocysts without sacrificing human oocytes. Human foetal fibroblasts were used as donor cells injected into the enucleated bovine oocytes in nuclear transfer, whereas bovine foetal fibroblasts were used to produce intraspecies embryos. We also examined the fate of human and bovine mitochondrial DNA (mtDNA) during preimplantation development after nuclear transfer by PCR. PCR analysis for the detection of human and bovine mtDNA was done at the 2,8-morula, and blastocyst stages of the embryos.

RESULT2.8% interspecies embryos developed to blastocysts after cultured in an SOF medium, while blastocyst rate of intraspecies embryos were 10.1%. Both human and bovine mtDNAs existed until the morula stage, whereas only the bovine mtDNA was found at the blastocyst stage. These results indicated that interspecies cloning without using human oocytes could generate human blastocysts. Because of the incoordination between bovine mtDNA and human nuclear gene, developmental rate of interspecies embryos was significantly lower than intraspecie. Whether the embryonic stem cell could be used for cell replacement therapy need further research.

Animals ; Blastocyst ; cytology ; physiology ; Cattle ; Cloning, Organism ; DNA, Mitochondrial ; genetics ; Embryonic Development ; physiology ; Female ; Fibroblasts ; physiology ; Humans ; Nuclear Transfer Techniques ; Oocytes ; physiology ; Species Specificity

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.
Actins/biosynthesis* ; Cell Differentiation/physiology* ; Cell Movement/physiology* ; Electric Stimulation Therapy ; Electricity ; Fibroblasts/physiology* ; Humans ; Myofibroblasts/physiology* ; Proliferating Cell Nuclear Antigen/biosynthesis* ; Skin/cytology*

AIMTo explore the effects of tRNA on the growth of mammalian cells.

METHODSL929, NIH3T3, MCF-7 and PC12 cells were seeded in 96 well culture plate individually, and incubated at 37 degrees C in 5% CO2 for 4 h, the tRNAs from different species were added to the culture media individually. After certain time of incubation, the viability of the cells was evaluated by the MTT methods. Sub-confluent L929 cells were incubated with 200 microg/ml ytRNA for different times, then the cells were pooled and analyzed with flow cytometry assay.

RESULTStRNA specifically inhibited the growth of L929 cells in a dose-dependent manner. The sizes of tRNA-treated cells showed larger sizes and longer processes than those of untreated cells. Flow cytometric analysis further showed that most of tRNA-treated cells were arrested in S phase of the cell cycle.

CONCLUSIONThe cell growth inhibitory effects of tRNAs were caused mainly by their degraded fragments. The results suggested that tRNA or its degraded fragments might play important roles in regulation of cell proliferation.

Animals ; Cell Cycle Checkpoints ; physiology ; Cell Line ; Cell Proliferation ; Fibroblasts ; cytology ; Flow Cytometry ; Mice ; RNA, Transfer ; physiology

OBJECTIVETo observe the effects of supernatants of normal skin keratinocytes(NK) and scar keratinocytes(SK) on proliferation and collagen synthesis of hypertrophic scar fibroblasts(HSFB).

METHODSThe supernatant, collected from cultured NK and SK, was added to the cultivated HSFB. The MTT-method, 3H-proline incorporation and radioimmunoassay were employed to measure the cell proliferation, collagen synthesis and secretion.

RESULTSNK supernatant could inhibit HSFB proliferation and increase the collagen synthesis, but inhibit collagen secretion, as compared with the control group. On the contrary, SK supernatant could increase collagen synthesis and secretion, which had little effects on HSFB proliferation.

CONCLUSIONKeratinocytes derived from normal skin and hypertrophic scar show different effects on hypertrophic scar fibroblasts.

Cell Division ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Collagen ; biosynthesis ; Culture Media, Conditioned ; Fibroblasts ; physiology ; Humans ; Keratinocytes ; physiology

Epithelial-mesenchymal transition (EMT) is a process by which fully differentiated epithelial cells undergo a phenotypic conversion and assume a mesenchymal cell phenotype, including elongated morphology, enhanced migratory and invasiveness capacity, and greatly increased production of extracellular matrix (ECM) components. The EMTs associated with wound healing, tissue regeneration, and organ fibrosis are termed as type 2 EMT. Over the past two decades, emerging evidence suggested that injured epithelial cells, via type 2 EMT, may serve as important sources of fibroblasts and contribute to organ fibrosis, such as kidney, liver, lung and eyes. There is perhaps no doubt that adult epithelial cells can undergo EMT in vitro in response to transforming growth factor (TGF)-β1 and other inflammatory or pro-fibrotic stimuli. However, whether type 2 EMT really occurs in vivo, whethers it is actually a source of functional and activated interstitial fibroblasts and whether it contributes to tissue fibrosis have already been the subjects of heated debate. In this review, we will describe the main features of EMT, the major findings of type 2 EMT in vitro, the evidences for and against type 2 EMT in vivo and discuss the heterogeneity and pitfalls of the techniques used to detect EMT during fibrotic diseases. We suggest that in order to ascertain the existence of type 2 EMT in vivo, different proper phenotype markers of epithelial and mesenchymal cells should be jointly used and cell lineage tracking techniques should be standardized and avoid false positives. Finally, we believe that if EMT really occurs and contributes to tissue fibrosis, efforts should be made to block or reverse EMT to attenuate fibrotic process.
Animals ; Epithelial-Mesenchymal Transition ; physiology ; Fibroblasts ; cytology ; metabolism ; Fibrosis ; metabolism ; pathology ; Humans

OBJECTIVETo explore a feasible method to repair full-thickness skin defects with tissue engineered techniques.

METHODSThe skin specimens were cut from the Changfeng hybrid swines' abdomen, then keratinocytes and fibroblasts were isolated and harvested by trypsin, EDTA and type II collagenase. The cells were seeded in petri dishes for primary culture. When the cells were in logarithmic growth phase, they were treated with dispase II (keratinocytes) or trypsin (fibroblasts) to separate them from the floor of the tissue culture dishes. A biodegradable material-pluronic F-127 was prefabricated and mixed with these cells, and then the cells-pluronic compounds were seeded evenly into polyglycolic acid (PGA). Tinally the constructs were replanted to autologous animals to repair full-thickness skin defects. Histological changes were observed in 1, 2, 4 and 8 weeks postsurgery.

RESULTSThe cells-pluronic F-127-PGA compounds could repair autologous full-thickness skin defects. Histologically, the tissue engineered skin was similar to normal skin with stratified epidermis overlying a moderately thick collageneous dermis.

CONCLUSIONTissue engineered skin can repair autologous full-thickness skin defects with primary-cultured keratinocytes and fibroblasts as seed cells and PGA as a cell carrier.

Animals ; Female ; Fibroblasts ; physiology ; Male ; Polyglycolic Acid ; pharmacology ; Skin Transplantation ; Skin, Artificial ; Swine ; Tissue Engineering

A hyaluronic acid (HA) incorporated porous collagen matrix was fabricated at -70 degree C by lyophilization. The HA incorporated collagen matrix showed increased pore size in comparison with collagen matrix. Biodegradability and mechanical properties of matrices were controllable by varying the ultraviolet (UV) irradiation time for cross-linking collagen molecules. Addition of HA to collagen matrix did not effect ultimate tensile stress after UV irradiation. HA incorporated collagen matrices demonstrated a higher resistance against the collagenase degradation than collagen matrix. In an in vitro investigation of cellular behavior using dermal fibroblasts on the porous matrix, HA incorporated collagen matrix induced increased dermal fibroblast migration and proliferation in comparison with collagen matrix. These results suggest that the HA incorporated collagen porous matrix assumes to enhance dermal fibroblast adaptation and regenerative potential.
Collagen/*metabolism ; Extracellular Matrix/*metabolism ; Fibroblasts/*physiology ; Human ; Hyaluronic Acid/*metabolism ; Porosity