Cell Line ; Humans ; Liver ; metabolism ; Membrane Potential, Mitochondrial ; Mitochondria, Liver ; metabolism ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Proteins ; pharmacology

In this study, we evaluated the effects of ascorbic acid (VC), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) on in vitro culture of sheep ovarian cortical tissue. Using 2 x 2 x 2 factor experimental design, we cultured sheep ovarian cortex fragments in 8 media with MEM (control), MEM+VC (50 microg/mL), MEM +EGF (100 ng/mL), MEM+FSH (50 ng/mL), MEM+VC+EGF, MEM+VC+FSH, MEM+EGF+FSH, MEM+VC+EGF+FSH. After 0 (non-cultured control), 2, 6, 12 days of culture, the pieces of ovarian cortex were proceed to histological and proliferating cell nuclear antigen (PCNA) examination, or observed by transmission electron microscopy (TEM). The percentages of developing follicles were increased (P < 0.05) and the percentages of healthy follicles were reduced (P < 0.05). When compared to the MEM group, the addition of FSH with VC or EGF promoted a significant increase of follicles diameter and follicles survival rate (P < 0.05), and stimulated the proliferation of granulosa cells. After 12 days of culture, medium supplemented with MEM+VC+EGF resulted the lowest proportion of developing follicles (49.3% +/- 3.2%), follicles diameter((32.3 +/- 2.3) microm), follicles survival rate (41.6% +/- 3.1%) and the proportion of PCNA stained follicles (26.4% +/- 1.2%, P < 0.05). In contrast, MEM+VC+EGF+FSH resulted the highest follicles diameter ((42.5 +/- 5.1) microm), follicles survival rate (59.7% +/- 6.1%) and proportion of PCNA stained follicles (43.5% +/- 4.1%, P < 0.05). Ultrastructural analysis confirmed the integrity of follicles cultured in VC+EGF+FSH group, while follicles cultured in MEM+VC+EGF groups showed more degeneration characters. In conclusion, the addition of VC and EGF to culture medium inhibited follicular development, VC+EGF+FSH was the most effective treatment to maintain follicular integrity and promote sheep primordial follicular activation and growth during in vitro culture.
Animals ; Ascorbic Acid ; pharmacology ; Culture Techniques ; Epidermal Growth Factor ; pharmacology ; Female ; Follicle Stimulating Hormone ; pharmacology ; Ovarian Follicle ; growth & development ; Ovary ; growth & development ; Proliferating Cell Nuclear Antigen ; analysis ; Sheep

OBJECTIVETo evaluate the inhibitory effect of deuterium-depleted water (DDW) on the proliferation of nasopharyngeal carcinoma (NPC) cells in vitro and explore the possible mechanism.

METHODSThe growth inhibition of NPC cells and preosteoblast MC3T3-E1 cells following DDW treatment was measured by MTT assay and plate colony formation assay. The changes in migration and invasion of NPC cells were evaluated using Transwell and boyden chamber assays. The protein expression of proliferating cell nuclear antigen (PCNA) was determined using Western blotting. Flow cytometry was employed to evaluate the changes in cell cycle distribution after DDW treatment.

RESULTSDDW with deuterium concentrations of 100, 75 and 50 ppm significantly suppressed the cell proliferation (P<0.05) and lowered colony formation capacity and invasiveness of the NPC cells (P<0.01). Western blotting demonstrated a down-regulated expression of PCNA in the cells by DDW. DDW also caused obvious cell cycle arrest in the NPC cells with reduced cells in S phase and significantly increased cells in G(1) phase (P<0.05). Rather than causing growth inhibition, DDW promoted the growth of normal control MC3T3-E1 cells.

CONCLUSIONDDW possesses selective biological effects to inhibit the proliferation of NPC cells in vitro, suggesting the potential of DDW as a novel nontoxic adjuvant therapeutic agent in antitumor therapy.

Carcinoma ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deuterium ; administration & dosage ; pharmacology ; Humans ; Nasopharyngeal Neoplasms ; pathology ; Proliferating Cell Nuclear Antigen ; metabolism ; Water ; chemistry

Silver-containing preparations are widely used in the management of skin wounds, but the effects of silver ions on skin wound healing remain poorly understood. This study investigated the effects of silver ions (Ag) on the proliferation of human skin keratinocytes (HaCaT) and the production of intracellular reactive oxygen species (ROS). After treating HaCaT cells with Ag and/or the active oxygen scavenger N-acetyl cysteine (NAC), cell proliferation and intracellular ROS generation were assessed using CCK-8 reagent and DCFH-DA fluorescent probe, respectively. In addition, 5-bromo-2-deoxyUridine (BrdU) incorporation assays, cell cycle flow cytometry, and proliferating cell nuclear antigen (PCNA) immunocytochemistry were conducted to further evaluate the effects of sub-cytotoxic Ag concentrations on HaCaT cells. The proliferation of HaCaT cells was promoted in the presence of 10 and 10 mol/L Ag at 24, 48, and 72 h. Intracellular ROS generation also significantly increased for 5-60 min after exposure to Ag. The number of BrdU-positive cells and the presence of PCNA in HaCaT cells increased 48 h after the addition of 10 and 10 mol/L Ag, with 10 mol/L Ag markedly increasing the cell proliferation index. These effects of sub-cytotoxic Ag concentrations were repressed by 5 mmol/L NAC. Our results suggest that sub-cytotoxic Ag concentrations promote the proliferation of human keratinocytes and might be associated with a moderate increase in intracellular ROS levels. This study provides important experimental evidence for developing novel silver-based wound agents or dressings with few or no cytotoxicity.
Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Fluorescent Antibody Technique ; Humans ; Keratinocytes ; cytology ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Reactive Oxygen Species ; metabolism ; Silver ; pharmacology

Dimethyl sulfoxide (DMSO), a well-known differentiation inducer in several myeloid cells, induces G1 phase arrest in many cell lines. In this study, we investigated the possibility of using DMSO to arrest H18 hybridoma cells to the G1 phase and monitor whether the arrest improves antibody production. We showed that DMSO in concentration ranging between 0.3% and 0.6% efficiently arrested H18 hybridoma cells in G1 phase. In our experiment, > 80% of cells grown for 36h in presence of the 0.6% DMSO were arrested in G1. Furthermore, expression levels of P27 were up-regulated tow fold during the G1 phase. Higher concentration of DMSO at 0.9% leads to cytotoxicity. Herein we show a simple way, a two-stage process for antibody production, which consists of a proliferation phase leading to the desired cell density, followed by an extended production phase during which the cells remain at G1 phase. Our observation that the addition of DMSO results in increase antibody production is of significance in further use of hybridoma cells in high density large scale cell culture.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cell Proliferation ; drug effects ; Dimethyl Sulfoxide ; pharmacology ; G1 Phase ; drug effects ; Hybridomas ; drug effects ; immunology ; Mice ; Proliferating Cell Nuclear Antigen ; analysis

The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0 approximately10 microg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10(-8) to 10(-7) mol/L and the PKC inhibitor H(7) inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H(7). These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.
Animals ; Cell Proliferation ; drug effects ; Enzyme Activation ; Ginsenosides ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Proliferating Cell Nuclear Antigen ; analysis ; Protein Kinase C ; physiology ; Spermatogonia ; cytology ; drug effects

AIMTo investigate how vasonatrin peptide (VNP) can attenuate the growth-promoting effect of hypoxia in cardiac fibroblasts cultured from neonatal rats.

METHODSThe cultured cardiac fibroblasts were divided randomly into four groups: control group, hypoxia group, hypoxia + VNP group and hypoxia + 8-Bromo-cGMP group. The growth of cardiac myocytes was measured by the means of MTT method. The effect of VNP on the intracellular level of cGMP and PCNA were measured by the means of radioimmunoassay and immunohistochemistry stain respectively.

RESULTSHypoxia (24 h) significantly increased the MTT A490nm value of cardiac fibroblasts (P < 0.05 vs control group). Both VNP (10(-7) mol/L) and 8-Bromo-cGMP (10(-3) mol/L) decreased MTT A490 nm value in cardiac fibroblast (P < 0.05 vs hypoxia group). VNP (10(-7) mol/L) increased the intracellular level of cGMP (P < 0.05 vs control and hypoxia group). Hypoxia (24 h) significantly increased the expression of proliferating cell nuclear antigen (PCNA) in cardiac myocytes (P < 0.05, vs control group), but VNP (10(-7) mol/L) decreased it.

CONCLUSIONVNP can attenuate hypoxia-induced growth-promoting effect in cardiac fibroblasts which is associated with the changes of cGMP and PCNA.

Animals ; Animals, Newborn ; Atrial Natriuretic Factor ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Cyclic GMP ; metabolism ; Myoblasts, Cardiac ; cytology ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of butyrylchitosan on the expression of proliferating cell nuclear antigen ( PCNA) in fibroblast proliferation of rabbit eyes after filtering operation.

METHODSTwenty-four New Zealand rabbits were randomly divided into 2 groups, with 12 rabbits in each group. Rabbits in one group received butyrylchitosan under scleral patch of trabeculectomy in right eyes and trabeculectomy in left eyes (trabeculectomy group). Rabbits in the other group received mitomycin C (MMC) in trabeculectomy in right eyes (MMC group) and without operation in left eyes. Rabbits were killed 1, 4, and 12 weeks after operations. Immunohistochemical staining was used to detected PCNA expression in fibroblast.

RESULTSAfter use of butyrylchitosan, the PCNA expression significantly decreased compared with trabeculectomy group (P < 0. 001). PCNA expression in MMC group was significantly lower than in trabeculectomy group (P <0. 001).

CONCLUSIONUsing butyrylchitosan under scleral patch of trabeculectomy decreases PCNA expression in proliferating cell and inhibits the scarring at filtering site.

Animals ; Cell Proliferation ; drug effects ; Chitosan ; analogs & derivatives ; pharmacology ; Female ; Fibroblasts ; cytology ; metabolism ; Filtering Surgery ; Male ; Membranes, Artificial ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Rabbits

OBJECTIVETo study the effect of matrix metalloproteinases inhibitor GM6001 in suppressing scar tissue formation in the filtering passage after glaucoma filtration surgery.

METHODSTwenty-four pigmented rabbits (48 eyes) underwent trabeculectomy followed by subconjunctival injection of GM6001 in the right eye (treated eyes) and injection of PBS in the left eye (control) once a day. The intraocular pressure was monitored postoperatively and proliferating cell nuclear antigen (PCNA)- and α-smooth muscle actin (α-SMA)-positive cells in the filtering pathway were detected using immunohistochemistry.

RESULTSOn postoperative days 7, 14, 21, and 28, the intraocular pressure was significantly lower in the treated eyes (GM6001) than in the control eyes (P<0.01). The counts of PCNA- and α-SMA-positive cells were also significantly lowered in the treated than in the control eyes (P<0.01).

CONCLUSIONGM6001 can inhibit excessive proliferation of the fibroblasts in the filtering pathway to suppress scar tissue formation and prolong the existence of the functional filtration bleb in rabbits.

Actins ; metabolism ; Animals ; Cicatrix ; pathology ; prevention & control ; Dipeptides ; pharmacology ; Filtering Surgery ; adverse effects ; Glaucoma ; surgery ; Intraocular Pressure ; Postoperative Complications ; Proliferating Cell Nuclear Antigen ; metabolism ; Rabbits

OBJECTIVE: To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer. METHODS: Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay. RESULTS: Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G₁/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05). CONCLUSION Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.
Humans ; Animals ; Mice ; Oldenlandia/metabolism* ; Proliferating Cell Nuclear Antigen ; Stomach Neoplasms ; Flavonoids/pharmacology* ; Reactive Oxygen Species/metabolism* ; Mice, Nude ; Ki-67 Antigen ; Cell Line, Tumor ; Apoptosis ; Plant Extracts/pharmacology* ; Caspases ; Cell Proliferation