Pengaruh Glukosa Tinggi terhadap Proliferasi, Migrasi dan Ekspresi Gen OCT-4 pada Kultur Sel Dermal Fibroblast Manusia

Human dermal fibroblasts (HDF) termasuk sel mesenchymal yang diisolasi dari lapisan dermis kulit. HDF berpotensi digunakan untuk pengobatan penyembuhan luka berdasarkan pengobatan regeneratif. Peningkatan konsentrasi glukosa dapat merusak fungsi sel dan menghambat terapi penyembuhan luka. Penelitian ini dilakukan untuk mengkaji pengaruh glukosa tinggi pada proliferasi, migrasi dan ekspresi gen OCT-4 sel HDF sebagai model penyembuhan luka diabetes in vitro. Pada penelitian eksperimental ini, fibroblast diisolasi dari kulit setelah sirkumsisi, kemudian ditumbuhkan dalam medium Minimal Essential Medium (DMEM) Dulbecco lengkap dengan serum 10%. Untuk menguji efek glukosa tinggi pada proliferasi sel HDF dilakukan dengan uji CCK-8. Migrasi sel HDF dievaluasi menggunakan uji scratch-assay. RT-PCR digunakan untuk menentukan ekspresi gen OCT-4. Hasilnya menunjukkan bahwa glukosa tinggi (25 mM-50 mM) meningkatkan kemampuan proliferasi dan migrasi sel HDF. Efek glukosa tinggi tergantung pada dosis. Perlakuan glukosa pada dosis 75 mM akan menghambat kemampuan pertumbuhan HDF. Ekspresi gen OCT-4 meningkat secara bermakna pada pemberian glukosa dosis 25-50 mM. Hasil penelitian ini memberikan dasar untuk pengembangan terapi dalam kondisi diabetes bahwa terapi sel HDF dapat meningkatkan penyembuhan luka meskipun dalam kondisi glukosa tinggi.

Effects of sodium alginate capsules as 3D scaffolds on hormones and genes expression in preantral follicles of mice compared to 2D medium: An experimental study

Background: The improvement of in vitro maturation methods, which can activate the preantral follicle growth, plays a crucial role in the production of mature oocytes in reproductive technology. Objective: To evaluate the different concentrations of 3D scaffolds of sodium alginate on hormones and gene expression in mice preantral follicles. Materials and Methods: Immature female BALB/c mice (12-14 days) were sacrificed. The follicles were removed mechanically and transferred into α minimal essential medium with 5% fetal bovine serum. The preantral follicles were incubated with different concentrations of sodium alginate (0.25%, 0.5%, and 1%) and 2D medium for 12 days. The follicles were examined for antral formation following the 10th day and the diameter on days 6th and 12th. The levels of hormones (AMH, androstenedione, 17

Influence of follicle-stimulating hormone concentrations on the integrity and development of bovine follicles cultured in vitro

SummaryThis study investigated the in vitro culture of bovine follicles included in ovarian tissue for 2 or 6 days (D2 or D6), with the addition of different concentrations of follicle-stimulating hormone (FSH) (0, 10, 50, 100 or 200 ng/ml). Data were compared for follicular development, morphological integrity and diameter of follicles and oocytes. Ovaries (n = 10) from Nelore cows (n = 5) were divided into fragments (n = 11 per ovary) and were immediately fixed in Bouin’s solution (D0) or were individually cultured for 2 or 6 days in one of the described concentrations of FSH and then processed for histology. Compared with the rates of follicular development at D2 for minimal essential medium (MEM) (75.0%) and 50 ng/ml of FSH (71.1%), the best rates of follicular development at D2 were obtained with 10 (84.7%), 100 (87.5%) and 200 ng/ml of FSH (85.0%; P<0.05). After 6 days of cultivation, there were no differences among treatments regarding follicular growth. The morphological integrity of preantral follicles was better maintained by 100 ng/ml FSH for 2 and 6 days of cultivation (51.2 and 40.4%, respectively; P<0.05) than that for MEM (D2: 30.9%, D6: 20.8%), 10 (D2: 39.2%, D6: 22.8%), 50 (D2: 30.4%, D6: 28.8%) and 200 ng/ml FSH (D2: 45.2%, D6: 36.8%). FSH at 100 ng/ml provided the highest mean diameter averages: 34.5±10.8 µm at D2 and 33.2±12.5 µm at D6 (P<0.05). We concluded that the medium supplemented with 100 ng/ml FSH during in vitro culture provided appropriate conditions for the development and morphological integrity of preantral follicles in cattle.

In vitro culture of L3 larvae of nematodes obtained from the African giant snail Lissachatina fulica (Mollusca: Gastropoda) in Santa Fe de Antioquia

Introducción. Más de 170 municipios colombianos están invadidos por Lissachatina fulica, caracol africano que puede portar larvas de nematodos de interés en salud humana y veterinaria. Los parásitos entran al caracol huésped intermediario en el estadio de larva L1, y allí cambian a L2 y L3, formas estas capaces de infectar a vertebrados.Objetivo. Estandarizar el cultivo in vitro de las L3 portadas por especímenes de L. fulica recolectados en Santa Fe de Antioquia.Materiales y métodos. Entre julio y noviembre de 2014 se recolectaron 10 caracoles, se sacrificaron y se digirieron con ácido clorhídrico al 0,7 %. Las larvas se recuperaron mediante la técnica de Baermann; se cultivaron 36 días en los medios Schneider, mínimo esencial de Eagle modificado por Dulbecco (Dulbecco’s Modified Eagles Minimal Essential Medium, DMEM), y Roswell Park Memorial Institute (RPMI), con suero fetal bovino (SFB) al 20 % y sin este, y agua destilada con SFB al 20 %. Los medios de cultivo se cambiaron cada 36 horas. Las larvas se midieron con el microscopio utilizando reglilla ocular, y se evaluaron la supervivencia, la longitud y el ancho. Se calcularon datos estadísticos de resumen y se hicieron gráficos de cajas y bigotes, así como la prueba t de Student. El nivel de significación (p) se estableció como menor de 0,05.Resultados. El 50 % de las larvas sobrevivió, 85 % en DMEM con SFB al 20 %, el 70 % con RPMI más SFB al 20 %, el 60 % en RPMI, el 50 % en Schneider más SFB al 20 %, el 45 % en Schneider y el 40 % en DMEM. El control sobrevivió diez días. Hubo diferencias significativas entre la longitud inicial promedio de las larvas y la longitud final promedio en los medios con suplementos: inicial, 645,83 μm; final en DMEM más SFB al 20 %, 732,65 μm (p<0,001); en RPMI más SFB al 20 %, 718,79 μm (p<0,001), y en Schneider más SFB al 20 %, 696,12 μm (p<0,01). No hubo diferencias significativas entre la anchura inicial promedio, de 24,99 μm, y la final.Conclusiones. El mejor medio para cultivar las L3 de L. fulica fue el DMEM más SFB al 20 %. En la evaluación del crecimiento larval, la longitud fue más informativa que la anchura. Las larvas estudiadas no correspondieron a Angiostrongylus cantonensis, A. costaricensis ni Aelurostrongylus abstrusus.

Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles

This study demonstrated the effect of Morus nigra leaf extract during ovine ovarian tissue transportation on the survival and apoptosis of preantral follicles in vitro. High Performance Liquid Chromatography (HPLC) was used to determine the fingerprint chromatogram of the crude ethanolic extract. Four pairs of ovaries from four sheep were collected. The ovarian cortex was fragmented and one fragment was fixed in 10% buffered formaldehyde and processed for histological and TUNEL analysis (fresh control). The other fragments were placed in Minimal Essential Medium (MEM – control medium) or M. nigra extract (0.025; 0.05 or 0.1 mg/mL) and stored (simulating transport) at 4ºC for 6, 12 or 24 h. Preserved fragments (6 h) were also destined to histological and TUNEL analysis. HPLC analysis confirmed the presence of antioxidant compounds (rutin, isoquercetin e kaempferitrin) in the extract. There was a decrease (P < 0.05) in the percentage of morphologically normal preantral follicles after preservation in all treatments compared to the fresh control. The percentage of normal preantral follicles after preservation in M. nigra at 0.05 mg/mL for 6 h was higher (P < 0.05) than in MEM or 0.025 mg/mL M. nigra and similar (P > 0.05) to 0.1 mg/mL of the extract. Apoptosis increased (P < 0.05) after conservation for 6 h in all treatments compared to the fresh control. Moreover, TUNEL positive cells decreased (P < 0.05) after preservation in 0.05 or 0.1 mg/mL M. nigra compared to MEM or 0.025 mg/mL M. nigra. In conclusion, 0.05 mg/mL M. nigra extract can be used as a preservation medium for ovine ovarian tissue at 4°C for up to 6 h.

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

136 COMPARISON OF NCSU-23 AND ALPHA-MINIMAL ESSENTIAL MEDIA IN THE DEVELOPMENT OF ISOLATED PORCINE PREANTRAL FOLLICLES IN VITRO

To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) maintained at 30–35°C. The ovaries were cut into small pieces (1–3 mm), and preantral follicles were isolated mechanically. Preantral follicles from 280 to 300 μm in diameter were collected into a small dish containing medium TCM199 (Lonza 12–117F) supplemented with 5% fetal bovine serum. The follicles were transferred from the collecting medium to the culture medium that consisted of base medium (NCSU23 or α-minimal essential medium) supplemented with 3.5 μg mL–1 of insulin, 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, 7.5% porcine serum, and 1.5 ng mL–1 of FSH. The follicles were randomly distributed to the different experimental treatments and cultured for 6 days in 24-well cell culture plates, with 3 follicles per well in 280 μL of culture medium. The culture was carried out at 38.5°C in 5% CO2 in air. Culture medium was changed every 2 days with freshly prepared medium. The diameters of follicles were measured every 2 days, and each follicle was photographed and evaluated at 20× magnification. Forty-two follicles per group were analysed and collected in 4 replicates. Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SPSS, version 18, SPSS Inc., Chicago, IL, USA), where the independent variable was the sample (group and day of culture). Tukey's post hoc test was used to perform multiple comparisons; the α level was set at 0.05. All data were expressed as quadratic means with standard error of the means. Only the antrum formation was evaluated by chi-square test. The results, reported in Table 1, show that there was no statistical differences between follicle size between NCSU23 or α-minimal essential media, but at Day 6 there was a positive trend (P = 0.08). Otherwise, when we compared the size inside the groups, we observed that the preantral follicles grew more in α-minimal essential media than in NCSU23. The percentages of antrum formation were 65 v. 76% (NCSU23 and α-minimal essential media, respectively). These results support the use of α-minimal essential media because it had a positive effect on the antrum formation, and that after Day 4 some follicles could undergo a regression phase. Future studies will be necessary to evaluate the molecular status and the hormone production. Table 1.Follicle size (μm) from Day 0 to Day 6

Antiviral Activity of Silver Nanoparticles Immobilized onto Textile Fabrics Synthesized by Radiochemical Process

ABSTRACTAntiviral activity of metallic Ag nanoparticles immobilized on textile fabrics were investigated. The Ag nanoparticles synthesized by radiochemical process are firmly immobilized on the surface of support textile fabrics of cotton. Small Ag particles of about 2–4 nm were observed together with relatively large particles of more than 10 nm. The Ag nanoparticles showed antiviral activity against Influenza A and Feline Calicivirus. The antiviral activity significantly depended on the concentration of the Eagle’s minimal essential medium. It was implied that the surface passivation by inhibitory agent lead to the deactivation of metallic Ag nanoparticles.

Kit ligand promotes the transition from primordial to primary follicles afterin vitroculture of ovine ovarian tissue

SummaryThis study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days ofin vitroculture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) afterin vitroculture of ovine ovarian tissue.