This study was aimed to investigate whether the thrombopoietin (rhTPO) may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTT method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and IV type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and IV type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70%-80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphthol AS-D chloroacetate esterase (AS-D NCE) staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson's trichrome and Gomori's staining, neither collagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type IV appeared positive stronger in experimental group than those in control. The expressions of these molecules were not dependent on culture time. By scanning electron microscopy microvilli and fibers on cell surface appeared more and more, monolayer cells evolved into multilayer cells, and newly-formed fibroblasts appeared gradually as culture time prolonged. These alterations were not different among various groups. The expressions of CD34, CD45, CD105, CD106, and CD166 were not affected obviously by rhTPO. It is concluded that rhTPO had no effects on histochemical properties of stromal cells. Fiber staining and scanning electron microscopic examinations revealed that rhTPO can not facilitate fiber formation of stromal cells. But rhTPO may be able to augment the expressions of fibronectin, laminin and collagen type IV of stromal cells. Therefore it is still necessary to follow up the patients for a long time, who have received rhTPO therapy clinically.
Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; drug effects ; Fibroblasts ; Humans ; Stromal Cells ; cytology ; drug effects ; Thrombopoietin ; pharmacology

OBJECTIVETo explore the biological effects of tetracycline on cultured human periodontal ligament fibroblasts (HPDLFs).

METHODSIncreasing concentrations of tetracycline (1, 5, 20, 100, 500, 2500 microg/ml) were added to the medium of cultured HPDLFs, respectively. After co-incubated for 2 days, cell morphology was observed under reverse microscope, meanwhile, cell proliferation activity was assayed using MTT, the total amount of protein was detected with Coumassie Bright Blue method and DNA synthesis was measured by 3H-TdR.

RESULTSOver a concentration range of 1 to 100 microg/ml, cells demonstrated a normal appearance, spindle or fusiform shaped. Moreover, at a concentration range of 20 to 100 microg/ml, tetracycline significantly enhanced the proliferating activity and biosynthesis of HPDLFs (P < 0.01). However, higher concentration (2500 microg/ml) not only changed cell morphology, but also significantly inhibited cellular activity.

CONCLUSIONThe results suggested that proper doses of tetracycline could promote proliferation and biosynthesis of HPDLFs while higher concentrations of tetracycline had cytotoxic effect.

Cells, Cultured ; Fibroblasts ; drug effects ; Humans ; Periodontal Ligament ; cytology ; drug effects ; Tetracycline ; pharmacology

BACKGROUNDThe possible role of substance P (SP) during wound healing has been the primary research focus in recent years, but its effect on the healing process after bile duct injury is little understood. This study aimed to investigate the effects of SP on growth of fibroblast-like cells derived from rabbit bile duct.

METHODSFibroblast-like cells derived from rabbit bile duct were identified and divided randomly into control and experimental groups. SP-treated cells at different concentrations of 10(-9)-10(-5) mol/L and control group were incubated, respectively, for 48 hours. After incubating, the effects of SP on cell proliferation were assessed by cell counts and MTT test. Apoptosis rate (AR) of cells was measured by flow cytometry.

RESULTSCultured rabbit bile duct cells were fibroblast-like in morphology, and these cells were stained positively for vimentin and negatively for desmin. After SP was added to nonconfluent cells for 48 hours, cell numbers were significantly increased in experimental groups than in controls (P < 0.05). The maximum stimulation of cell proliferation was achieved at SP of 10(-5) mol/L. Bile duct fibroblast-like cells in the SP group showed a higher proliferating activity and lower AR than those in the control group or in the SP + Spantide group (P < 0.05). Spantide partly inhibited the effects of SP on fibroblast-like cells. Examination under transmission electron microscopy revealed rough endoplasmic reticulum and prominent Golgi complexes after SP treatment.

CONCLUSIONSSP has a growth regulatory property on cultivated bile duct fibroblast-like cells in vitro, suggesting that SP may involve in wound healing after bile duct injury by promoting wound fibroblast proliferation and inhibiting apoptosis and participate in pathological scar formation.

Animals ; Bile Ducts ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; Male ; Rabbits ; Substance P ; pharmacology

OBJECTIVETo study the inhibitory effect of Typhonium gigantewm Engl. (AEoTGE) on the proliferation and apoptosis of KFB in vitro and to survey the death rate.

METHODSSamples of hypertrophic scars were collected and cultured. Only 4-8 passage cells were selected for experiment. Inverted microscope and transmission electron microscope were used to observe the morphogenesis and ultrastructure of KFB. The KFB cells were treated with AEoTGE in different concentrations(3. 125,6.250, 12.500, 25.000, 50. 000,100.000 g/L) for 24 hours. The effect of AEoTGE on the proliferation and the IC50 of KFB was observed with MTT assay and EdU. The effect of AEoTGE on apoptosis of KFB was detected by flow cytometry.

RESULTSIt showed that AEoTGE could inhibit the proliferation of KFB in an concentration-dependent style within the range of 3. 125-100.000 g/L. The AEoTGE could obviously increase the apoptosis rate of the KFB compared with blank control group(P <0.05). The IC50 of AEoTGE was 35 g/L. FITC-Annexin V/PI showed that apoptosis rate of KFB in the AEoTGE group was (72. 07 +/- 0. 70)% , while it was 23. 5% in blank control group (P < 0. 05).

CONCLUSIONSAEoTGE could significantly inhibit the proliferating activity and induce apoptosis of KFB after co-culture for 24 hours. The IC50 is 35 g/L and the rate of apoptosis is (72.07 +/- 0.70)%.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; cytology ; drug effects ; pathology ; Humans ; Keloid

OBJECTIVETo investigate the possibility of placenta mesenchymal stem cells (PMSCs) differentiation into dermal fibroblast, and the potency of PMSCs used in cutaneous wound healing and stored as seed cells.

METHODSEnzyme digestion method was used to obtain PMSCs, and PMSCs were amplified after culture in vitro. Flow cytometry assay, osteogenic and adipogenic differentiation were done for MSCs identification. The induction medium composed of DMEM/F12 + 50 microg/ml VC + 100 ng/ml connective tissue growth factor (CTGF) was added into the 24-well plate for 16 days induction period. Pictures were taken to record morphologic change. Immunofluorescence tests were performed to detect Vimentin, FSP-1, collagen I , collagen III, desmin and laminin expression before and after induction. At the same time osteogenic and adipogenic differentiation were used to assay the differentiation ability change after induction. The induced dermal fibroblasts were frozen in liquid nitrogen and recovery and trypan blue was used to detect cell viability.

RESULTSAfter CTGF induction, PMSCs got obvious fibroblasts morphology, the protein level of Vimentin, FSP-1, collagen I, collagen III and Laminin increased, PMSCs started to express Desmin, the dermal fibroblasts specific proteins, and osteogenic and adipogenic differentiation ability was diminished. PMSCs were successfully induced into dermal fibroblasts, and these induced cells could get a high cell viability ( more than 90% ) after recovery.

CONCLUSIONSPMSCs could be induced into dermal fibroblasts by CTGF in vitro. PMSCs have the potential application in skin wound healing, and can be used as seed cells of dermal fibroblasts.

Cell Differentiation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; pharmacology ; Female ; Fibroblasts ; cytology ; drug effects ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Placenta ; cytology ; Pregnancy

Effects of carboxymethyl-chitosan (CM-Chitosan) with different molecular weight on the proliferation of skin fibroblasts and keratinocytes were examined in vitro; bFGF and EGF, as controls, were seperately used for comparison. Chitosan with different molecular weight was prepared by acid degradation and oxidation degradation; CM-Chitosan with different molecular weight was synthesized from corresponding Chitosan. Microscopy and MTT method were applied to evaluate the different effects. The results demonstrated that CM-Chitosan with different molecular weight promoted the proliferation of skin fibroblasts and keratinocytes at 1-1000 ppm, and the concentration at 100 ppm had the strongest effects. The effects of low molecular weight CM-Chitosan were greater than those of high molecular weight CM-Chitosan. CM-Chitosan (Mn= 3KD) had the strongest promotive effects on skin fibroblasts and keratinocytes; it had equivalent effects when compared with bFGF and EGF.
Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chitosan ; analogs & derivatives ; chemical synthesis ; pharmacology ; Fibroblasts ; cytology ; drug effects ; Humans ; Keratinocytes ; cytology ; drug effects ; Mice ; Molecular Weight ; Skin ; cytology

OBJECTIVETo explore the effects of different concentrations of putrescine on the proliferation, migration and apoptosis of human skin fibroblasts (HSF).

METHODSHSF cultured in the presence of 0.5, 1.0, 5.0, 10, 50, 100, 500, and 1000 µg/ putrescine for 24 h were examined for the changes in the cell proliferation, migration, and apoptosis using MTS assay, Transwell migration assay, and flow cytometry, respectively.

RESULTSCompared with the control cells, HSF cultured with 0.5, 1.0, 5.0, and 10 µg/ putrescine showed significantly increased cell proliferation (P<0.01), and the effect was the most obvious with 1 µg/ putrescine, whereas 500 and 1000 µg/ putrescine significantly reduced the cell proliferation (P<0.01); 50 and 100 µg/ did not obviously affect the cell proliferation (P>0.05). Putrescine at 1 µg/ most significantly enhanced the cell migration (P<0.01), while at higher doses (50, 100, 500, and 1000 µg/) putrescine significantly suppressed the cell migration (P<0.05); 0.5, 5.0, and 10 µg/ putrescine produced no obvious effects on the cell migration (P>0.05). HSF treated with 0.5, 1.0, 5.0, and 10 µg/ putrescine obvious lowered the cell apoptosis rate compared with the control group (P<0.01), and the cell apoptosis rate was the lowest in cells treated with 1 µg/ putrescine; but at the concentrations of 100, 500, and 1000 µg/, putrescine significantly increased the cell apoptosis rate (P<0.01), while 50 µg/ml putrescine produced no obvious effect on cell apoptosis (P>0.05).

CONCLUSIONLow concentrations of putrescine can obviously enhance the proliferation ability and maintain normal migration ability of HSF in vitro, but at high concentrations, putrescine can obviously inhibit the cell migration and proliferation and induce cells apoptosis, suggesting the different roles of different concentrations of putrescine in wound healing.

Apoptosis ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; Flow Cytometry ; Humans ; Putrescine ; administration & dosage ; pharmacology ; Skin ; cytology ; Wound Healing

Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo animal experiments for many years. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes prepared in 24-well plates were exposed to varying concentrations of DTAF (10(-4), 10(-3), 10(-2), 1, 10, 10(2) microgram/ml). Exposure times of 1 hour and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after initiation of exposure to DTAF using a Coulter counter. Keratocyte proliferation was not affected by 1-hour exposure to DTAF, but keratocyte proliferation measured 3 days after initiation of exposure to DTAF for 24 hours was inhibited in a dose-dependent manner (p = 0.02) and was significantly inhibited at concentrations of 10 and 100 microgram/ml (p < 0.05). Fluorescent microscopy showed binding of DTAF to keratocytes. We have demonstrated that prolonged exposure to DTAF inhibits proliferation of cultured keratocytes. These results suggest that DTAF-induced cytotoxicity may alter net production of collagen in the corneal stroma in animal models.
Cell Count ; Cell Division/drug effects ; Cells, Cultured ; Corneal Stroma/cytology/*drug effects ; Fibroblasts/cytology/drug effects ; Fluoresceins/*pharmacology/toxicity ; Fluorescent Dyes/*pharmacology/toxicity ; Humans ; Microscopy, Fluorescence

OBJECTIVETo study the influence of Benzo (a) pyrene on cell cycle distribution of quiescent diploid human embryonic lung fibroblast (HELF) cells.

METHODSHELF cells were synchronized at G0 phase of cell cycle by 0.5% serum starvation for 48 hours and identified by flow cytometry (FCM). Cells were treated with 20 micromol/L benzo (a) pyrene for 4 h and detected for the changes of cell cycle distribution 0 h, 24 h, 48 h after treatment respectively. HELF cells were treated with 0, 5, 10, 20 micromol/L Benzo (a) pyrene for 24 h and detected for cell cycle regulators p53, p21 and p16 expression changes using Western Blotting method. On the other hand, the dynamic changes of these regulators were also been detected within 24 h after 20 micromol/L Benzo (a) pyrene treated for 4h.

RESULTS0.5% Serum starvation for 48 hours could effectively synchronize HELF at G0 stage and G0 reached 78%. Well-modulated control cells entered into cell cycle to synthesize DNA and cells at S phase reached 43.9% 24 h after serum re-stimulate, while 20 micromol/L B (a) P treated cells were arrested in G1 stage. Control cells entered into the G1 stage of next cell cycle another 24 h later, B (a) P treated cells recovered from G1 arrest, 26.5% of which reached S phase, having a delay of almost 24 h compared with controls. After a series of B (a) P concentrations acting for 24 h, we found that P53 and P21 expression increased dramatically. On the other hand, P53 and P21 increased 4 h after treatment, P53 recovered to normal level after 12 h while P21 kept increasing in 24 h. P16 initially decreased and became normal at 24 h.

CONCLUSIONB (a) P induced quiescent HELF cells undergoing a reversible G1 arrest related with p53-p21 pathway.

Benzo(a)pyrene ; toxicity ; Carcinogens ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Diploidy ; Fibroblasts ; cytology ; drug effects ; Flow Cytometry ; G1 Phase ; drug effects ; Humans ; Lung ; cytology ; embryology

OBJECTIVETo investigate the effect of local application of bFGF combined with sucralfate on the cell proliferation during continuous tissue expansion (CTE).

METHODSNine white pigs were selected to undergo the continuous tissue expansion in this study and treated with bFGF and sucralfate, respectively as the following groups: group 1 with both bFGF and sucralfate, group 2 only with bFGF, group 3 with only sucralfate, and group 4 with saline as control. Fifteen samples were taken in each pig for immunohistochemistry analysis 1-14 days and 6 weeks after the operation.

RESULTSIn the group with both bFGF and sucralfate, the epidermic basal cells proliferated significantly after the operation and reached top level in 3 days, which was statistical higher than the control group, but the multiplication of basal cell was the lowest 14 days after the operation, still more than the control group. In dermal layer, proliferation of fibroblasts, vessel endothelial cells, hair follicles epidermic cells and sweat gland epicytes was also significant higher in the group with both bFGF and sucralfate than that the control group and reached top level 7 day after the operation, but the proliferation of cells decreased obviously 14 days after the operation, still higher than the control group. The mitotic activity of cells returned to the basal level in 42 days. There were no significant differences among the group 2, group 3 and group 4.

CONCLUSIONLocal application of both bFGF and sucralfate could be more effect to induce cells multiplication during early skin expansion to facilitate the growth of neoformed skin soft tissue.

Animals ; Cell Proliferation ; drug effects ; Endothelial Cells ; cytology ; drug effects ; Fibroblast Growth Factors ; pharmacology ; Fibroblasts ; cytology ; drug effects ; Sucralfate ; pharmacology ; Swine ; Time Factors ; Tissue Expansion ; Tissue Expansion Devices