RESUMEN
Mesenchymal stem cells (MSC) are modern tools in regenerative therapies of humans and animals owed to their immunomodulatory properties, which are activated in a pro-inflammatory environment. Different preconditioning strategies had been devised to enhance the immunomodulatory properties of MSC. In this research, we evaluated the immunological attributes of equine adipose MSC (eAMSC) before and after preconditioning in vitro with prostaglandin E2 (PGE2), substance P (SP), their combination and IFNγ. PGE2/SP was the best combination to keep or enhance the mesodermal lineage differentiation of eAMSC. Alongside with this, preconditioning of eMSC with PGE2 and SP did not affect expression of stemness MSC surface phenotype: CD90+, CD44+, MHC class I+, MHC class II- and CD45-, assessed by cytometry. Both naïve and preconditioned eAMSC expressed genes related with immune properties, such as MHC-I, PTGES, IL6, IL1A, TNFα and IL8 assessed by qPCR. Only TNFα was under expressed in treated cells, while the other markers were either overexpressed or not changed. In no cases MHC-II expression was detected. The antiproliferative effect of preconditioned eAMSC exposed to activated peripheral blood mononuclear cells (PBMC) showed that SP treatment significantly inhibited proliferation of LPS stimulated PBMC. When eAMSC were stimulated with Poly I:C, all the treatments significantly inhibited proliferation of stimulated PBMC (p < 0.05). Direct contact (coculture) between the preconditioned eAMSC and PBMC, induced a shift of significantly more (CD4/CD25/FOXP3)+ T-regulatory PBMC than naïve eAMSC. In the experiments of this research, we investigated the secreted proteomic profile of naïve and preconditioned eAMSC, 42 up-regulated and 40 down-regulated proteins were found in the proteomic assay. Our proteomic data revealed profound changes in the secretory pattern of MSC exposed to different treatments, compared to naïve eAMSC as well as among treatments. In overall, compared to naïve cells, the protein profile of preconditioned cells resembled the mesenchymal-epithelial transition (MET). Here we showed that the combined use of PGE2 and SP provoked in overall the highest expression of anti-inflammatory markers as well as lead to an increased acquisition of a T-regulatory phenotype in preconditioned eAMSC without affecting their "stemness".
Asunto(s)
Dinoprostona/inmunología , Caballos/inmunología , Células Madre Mesenquimatosas/inmunología , Proteínas/metabolismo , Sustancia P/inmunología , Animales , Biomarcadores/metabolismo , Diferenciación Celular/inmunología , Proliferación Celular , Citometría de Flujo/veterinaria , Interferón gamma/inmunología , Masculino , Células Madre Mesenquimatosas/metabolismo , Mesodermo/citología , Proteoma , Vías Secretoras/inmunologíaRESUMEN
The objective of this study was to evaluate, in the domestic cat, the effect of ovarian stimulation with eCG prior to oocyte in vitro maturation (priming) on in vitro and in vivo development after in vitro fertilization (IVF). For this purpose, oocyte donors were either 1) treated with a single dose of 200 IU eCG four days before oocyte recovery (eCG group), or, 2) given no treatment before oocyte recovery (control group). Ovaries of both groups were collected by ovariohysterectomy (OVH) and cumulus-oocyte complexes (COCs) were recovered by slicing. Immature COCs from both groups were matured in vitro (IVM) for 26-28 h. IVF was done with refrigerated epididymal sperm. After 24 h co-incubation, presumptive zygotes were cultured in vitro for eight days. The rates of cleavage, morulae, blastocyst development and hatching were estimated. Some blastocysts were stained for total cell counting and others were used for gene expression analysis of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6). Additionally, to evaluate in vivo development, embryos from the eCG group were transferred at Day 5 and Days 7 or 8 of IVC to synchronized cat recipients. The results showed that, eCG priming increased significantly the rate of blastocyst development as compared to the control group (37.9 and 25.6%, respectively) (P < 0.05). No differences were observed in total cell number of blastocysts and hatching blastocysts (mean ± SD) between the eCG and control groups (420.6 ± 193.6 and 347.0 ± 237.1, respectively) (P > 0.05). In the gene expression analysis, blastocysts generated in the eCG group had higher expression of OCT4 than blastocysts from the control group (P < 0.05). However, no significant differences were observed in the relative expression of SOX2, NANOG, CDX2 and GATA6 (P > 0.05). Additionally, six embryo transfer (ET) procedures were done, three with Day 5 embryos and three with Day 7 or 8 embryos. Recipients from both ET groups delivered live kittens. The total pregnancy rate was 4/6 (67%), meanwhile the live birth rate was 2/6 (33%). In conclusion, eCG priming improved the rate of blastocyst development in vitro and increased relative expression of OCT4. These results demonstrate that eCG priming of oocytes donors before IVM improves oocyte competence, enhance in vitro embryo development and allows live births of healthy offspring after ET.
Asunto(s)
Gonadotropina Coriónica/farmacología , Embrión de Mamíferos/fisiología , Desarrollo Embrionario/fisiología , Fertilización In Vitro/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/fisiología , Animales , Gatos , Transferencia de Embrión , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Histerectomía/veterinaria , Ovariectomía/veterinaria , EmbarazoRESUMEN
Quality of in vitro-produced embryos is influenced by changes in gene expression in response to adverse conditions. Gene markers for predicting 'good embryos' do not exist at present. We propose that the expression of pluripotency markers OCT4-SOX2-NANOG in D9 (day 9) bovine demi-embryos correlated with development at D13 (day 13). Day 8 in vitro-produced blastocysts were split in two cloned halves, one half (D9) was subjected to analysis of pluripotency markers and the other was kept in culture until D13 of development. Embryo development was scored and correlated with its own status at D9 and assigned to one of two categories: G1, arrested/dead; or G2, development up to D13. SOX2 and NANOG expression levels were significantly higher in embryos from G1 and there was also negative correlation between SOX2 and embryo survival to D13 (G3; r = -0.37; P = 0.03). We observed a significant reduction in the expression of the three studied genes from D9 to D13. Furthermore, there was a correlation between the expression of pluripotency markers at D9 and embryo diameter and the expression of trophoblastic markers at D13 (TP1-EOMES-FGF4-CDX2-TKDP1). Finally, the quotient between the relative expression of SOX2 and OCT4 in the D9 blastocysts from G1 and G2 showed that embryos that were considered as competent (G2) had a quotient close to one, while the other group had a quotient of 2.3 due to a higher expression of SOX2. These results might indicate that overexpression of SOX2 at the blastocyst stage had a negative effect on the control of embryonic developmental potential.
Asunto(s)
Blastocisto/metabolismo , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción SOXB1/genética , Animales , Blastocisto/citología , Bovinos , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Factores de TiempoRESUMEN
Adult stromal mesenchymal stem cells (MSCs) have been postulated as responsible for cell renewal in highly and continuously regenerative tissues such as the endometrium. MSCs have been identified in the endometrium of many species including humans, rodents, pets and some farm animals, but not in horses. The objective of this work was to isolate such cells from the endometrium of mares and to compare their main biological attributes with horse adipose-derived MSCs. Here we successfully isolated and characterized endometrial MSCs (eMSCs) from mares. Said cells showed fibroblast-like morphology, grew on plastic, had doubling population times of 46.4 ± 3.38 h, underwent tri-lineage (osteo, chondro and adipogenic) differentiation after appropriate inductions, migrated toward the attraction of fetal calf serum and displayed a pattern of surface markers commonly accepted for horse MSCs. All these are properties of MSCs. Some of these attributes were shared with equine adipose-derived MSCs, but the migration pattern of eMSC at 12 and 24 h after stimulation was reduced in comparison with adipose MSCs. Also, expression of CD44, CD90 and MHCI surface markers were dramatically down-regulated in eMSCs. In conclusion, equine-derived endometrial MSC share biological attributes with adipose MSC of this species, but displayed a different surface marker phenotype and an impaired migration ability. Conceivably, this phenotype is distinctive for MSC of this origin.
Asunto(s)
Tejido Adiposo/citología , Endometrio/citología , Caballos/fisiología , Proteínas de la Membrana/metabolismo , Células Madre Mesenquimatosas/fisiología , Animales , Biomarcadores , Movimiento Celular , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Proteínas de la Membrana/genéticaRESUMEN
The kodkod population is in constant decrease and the somatic cell nuclear transfer (SCNT) might help to preserve the genetic pool of this species. The cell cycle synchronization of donor cells plays a crucial role in SCNT. The objective of this research was to evaluate two different methods for quiescence induction, serum starvation (SS) and contact inhibition (CI), both for 1, 3 and 5 days, on skin fibroblast from domestic cat and kodkod. Flow cytometry analysis revealed that in domestic cat, SS and CI, both at 3 and 5 days, increased the percentage of fibroblasts in G0/G1 compared to growing cells (GC) (p < .05). In kodkod, only SS for 3 and 5 days and CI for 1 and 3 days increased the percentage of fibroblasts in G0/G1 compared to GC (p < .05). Viability analysis by differential staining revealed that SS for 5 days decreased the proportion of live fibroblasts in domestic cat and kodkod (p < .05). Regarding gene expression analysis, in domestic cat fibroblasts, no differences were found in the BAX/BCL2 ratio in SS and CI (both at 1, 3 and 5 days) compared to GC. In kodkod fibroblasts, BAX/BCL2 ratio was increased in CI at 3 and 5 days compared to SS at 3 and 5 days (p < .05). In conclusion, in kodkod fibroblasts SS for 5 days and CI after 3 days might have a negative impact on cellular viability. According to these results, we suggest SS for 3 days for cell cycle synchronization in kodkod fibroblasts.
Asunto(s)
Ciclo Celular/fisiología , Felidae/fisiología , Fibroblastos/citología , Técnicas de Transferencia Nuclear/veterinaria , Animales , Apoptosis/genética , Supervivencia Celular , Clonación de Organismos/veterinaria , Inhibición de Contacto , Medio de Cultivo Libre de Suero , Perfilación de la Expresión Génica , Fase de Descanso del Ciclo CelularRESUMEN
Stem cells have been postulated as responsible for cell regeneration in highly and continuously regenerative tissues such as the endometrium. Few studies in cattle have identified and specified the presence of stem cells in the endometrium during the oestrous cycle. The aim of this study was to investigate the presence of mesenchymal stem cells (MSCs) in the bovine endometrium during the follicular phase (FP) of the oestrous cycle. Uterine tissue was collected in the time-frame comprising day 18 of the cycle and ovulation (day 0). We isolated, cultured and expanded four primary cell lines from endometrium and identified byRT-qPCR the expression of OCT4, SOX2 but not NANOG (undifferentiated/embryonic markers), CD44 (MSCs marker) and c-KIT (stem cell marker) genes; and the encoded Oct4, Sox2 and Cd44 proteins by Western blot or immunostaining of paraffin-embedded tissue in endometrium. We demonstrated that cells isolated from bovine endometrium displayed essentially the same gene expression pattern; however, at the protein level, Oct4 and Cd44 were not detected. Besides, they showed typical functional characteristics of MSCs such as fibroblast-like morphology, plastic adherence, high proliferative capacity, clone formation in vitro and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. We obtained for the first time an extensive characterization of undifferentiated cells populations contained in the bovine endometrium during the FP of the oestrous cycle.
Asunto(s)
Endometrio/citología , Fase Folicular , Células Madre Mesenquimatosas/citología , Animales , Bovinos , Diferenciación Celular , Células Cultivadas , Ciclo Estral , Femenino , Expresión GénicaRESUMEN
Embryo splitting has been used for the production of identical twins and to increase the pregnancy rate per available embryo. Split blastocysts can develop to term; however, little is known about the impact on gene expression of split embryos, especially at the whole transcriptome level. This work was aimed to evaluate the effect of blastocyst splitting on global gene expression profile at the elongation stage. For that, split and time-matched nonsplit (control group) bovine blastocysts were transferred to a bovine recipient and recovered at Day 17 of development. The number of collected embryos, their size, and global gene expression was compared between both groups. From 16 transferred split embryos, six (37.5%) were collected, whereas nine elongated were recovered from 17 nonsplit (52.9%). Neither the recovery rate nor the average length of the elongated embryos was significantly different between both groups. However more than 50% of embryos from the control group had a length surpassing 100 mm, whereas only 33% of the split embryos reached that size. Global gene expression was performed in individual elongated embryos from both groups using Two-Color Microarray-Based Gene Expression Analysis. From detected genes, 383 (1.31%) were differentially expressed between both groups, among them, 185 (0.63%) were downregulated and 198 (0.67%) genes were upregulated in split embryos. Bioinformatic analysis of differentially expressed genes revealed that embryo splitting affects transcriptomes of resulting elongated embryos, mainly downregulating genes involved in matrix remodelation, control of growth, detoxification, and transport of metabolites. These in turns might have a detrimental impact on the developmental potential of produced embryos.
Asunto(s)
Blastocisto/citología , Bovinos/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Transcriptoma/fisiología , Animales , Blastocisto/fisiología , Desarrollo Embrionario , Femenino , Fertilización In Vitro , EmbarazoRESUMEN
Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1-5) and late luteal phases (days 13-18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD-117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin-embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c-KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.
Asunto(s)
Bovinos , Endometrio/citología , Ciclo Estral , Células Madre Mesenquimatosas/citología , Factor 3 de Transcripción de Unión a Octámeros/genética , Animales , Biomarcadores/análisis , Diferenciación Celular , Células Cultivadas , Femenino , Expresión Génica , Receptores de Hialuranos/análisis , Receptores de Hialuranos/genética , Factor 3 de Transcripción de Unión a Octámeros/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción SOXB1/análisis , Factores de Transcripción SOXB1/genéticaRESUMEN
Repair of injured soft and hard tissues in horses can benefit greatly from the use of regenerative therapies with mesenchymal stem cells (MSC). Vitamin-C and platelet-rich-plasma had been used for in vitro differentiation of MSC. This study was aimed to evaluate the effect of vitamin-C, platelet-rich-plasma and their combination on the in vitro differentiation of adipose horse MSC. We isolated MSC from horse fat and differentiated them in vitro into osteogenic and chondrogenic lineages, as demonstrated by specific staining and RT-qPCR of selected genes. Combining vitamin-C and plasma-rich-platelet positively affected the ability of MSC to differentiate in vitro into mesodermal lineages during 14 days of culture; this effect was not as marked when differentiation was attempted for 21 days. This provides valuable information on the effect of combined use of these molecules in regenerative therapies and their potential application along stem cells for lesions of musculoskeletal tissue in sport horses.
Asunto(s)
Tejido Adiposo/metabolismo , Ácido Ascórbico/metabolismo , Diferenciación Celular/fisiología , Caballos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Plasma Rico en Plaquetas/metabolismo , Tejido Adiposo/citología , Animales , Diferenciación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Histocitoquímica/veterinaria , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Osteonectina/genética , Osteonectina/metabolismo , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Factor de Transcripción SOX9/genéticaRESUMEN
Viruses depend on cell metabolism for their own propagation. The need to foster an intimate relationship with the host has resulted in the development of various strategies designed to help virus escape from the defense mechanisms present in the host. Over millions of years, the unremitting battle between pathogens and their hosts has led to changes in evolution of the immune system. Snake venoms are biological resources that have antiviral activity, hence substances of significant pharmacological value. The biodiversity in Brazil with respect to snakes is one of the richest on the planet; nevertheless, studies on the antiviral activity of venom from Brazilian snakes are scarce. The antiviral properties of snake venom appear as new promising therapeutic alternative against the defense mechanisms developed by viruses. In the current study, scientific papers published in recent years on the antiviral activity of venom from various species of snakes were reviewed. The objective of this review is to discuss the mechanisms of resistance developed by viruses and the components of snake venoms that present antiviral activity, particularly, enzymes, amino acids, peptides and proteins.
Asunto(s)
Animales , Venenos de Crotálidos , Farmacorresistencia ViralRESUMEN
The objective of this study was to identify microRNAs (miRNAs) expressed in bovine (Bos Taurus) cloned embryos at Day 17 of development (Day 0=day of nucleus transfer or in vitro fertilization) during elongation. Day 7 bovine expanded blastocysts produced by hand made cloning (HMC) or in vitro fertilization were bulk-transferred to synchronized recipient cattle (48 HMC embryos to 10 recipients and 28 in vitro-produced embryos to four recipients). Elongated embryos were retrieved at Day 17; miRNAs were isolated and subjected to microarray screening using custom composite slides spotted with human, mouse, and rat and in silico-predicted miRNAs. An initial profile of expressed miRNAs was determined in cloned embryos and somatic donor cells; this profile changed after somatic cell nucleus transfer, identifying differentially expressed miRNAs between cloned and in vitro-produced bovine embryos. Furthermore, microarray data were validated using a miRNA-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) approach (miR-Q). There was an 83% correlation (P=0.01) between microarray and qPCR data. Based on qRT-PCR, correct reprogramming of some miRNAs from the donor cells was confirmed in cloned bovine embryos, whereas other somatic miRNAs were not appropriately reprogrammed. Some of the miRNAs that were equally reprogrammed clustered on the same chromosomal location in the bovine genome. In conclusion, reprogramming of miRNAs seemed to occur in cloned bovine embryos. This could have profound implications for elucidating nuclear reprogramming in somatic cloning, as well as for the role of miRNAs in preimplantation mammalian development.
Asunto(s)
Embrión de Mamíferos/metabolismo , MicroARNs/metabolismo , Animales , Bovinos , Clonación de Organismos , Técnicas de Cultivo de Embriones , Fertilización In Vitro , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg(-1) day(-1) intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3% for piplartine and 55.1 and 56.8% for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.
Asunto(s)
Alcaloides/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Benzodioxoles/uso terapéutico , Piper/química , Piperidinas/uso terapéutico , Piperidonas/uso terapéutico , Alcamidas Poliinsaturadas/uso terapéutico , Sarcoma 180/tratamiento farmacológico , Alcaloides/aislamiento & purificación , Alcaloides/toxicidad , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/toxicidad , Benzodioxoles/aislamiento & purificación , Benzodioxoles/toxicidad , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Riñón/efectos de los fármacos , Riñón/patología , Hígado/efectos de los fármacos , Hígado/patología , Ratones , Trasplante de Neoplasias , Piperidinas/aislamiento & purificación , Piperidinas/toxicidad , Piperidonas/aislamiento & purificación , Piperidonas/toxicidad , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Extractos Vegetales/toxicidad , Raíces de Plantas/química , Alcamidas Poliinsaturadas/aislamiento & purificación , Alcamidas Poliinsaturadas/toxicidad , Sarcoma 180/patología , Bazo/efectos de los fármacos , Bazo/patologíaRESUMEN
Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg-1 day-1 intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3 percent for piplartine and 55.1 and 56.8 percent for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.
Asunto(s)
Animales , Femenino , Ratones , Alcaloides/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Benzodioxoles/uso terapéutico , Piper/química , Piperidinas/uso terapéutico , Piperidonas/uso terapéutico , Alcamidas Poliinsaturadas/uso terapéutico , /tratamiento farmacológico , Alcaloides/aislamiento & purificación , Alcaloides/toxicidad , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/toxicidad , Benzodioxoles/aislamiento & purificación , Benzodioxoles/toxicidad , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Riñón/efectos de los fármacos , Riñón/patología , Hígado/efectos de los fármacos , Hígado/patología , Trasplante de Neoplasias , Piperidinas/aislamiento & purificación , Piperidinas/toxicidad , Piperidonas/aislamiento & purificación , Piperidonas/toxicidad , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Extractos Vegetales/toxicidad , Raíces de Plantas/química , Alcamidas Poliinsaturadas/aislamiento & purificación , Alcamidas Poliinsaturadas/toxicidad , /patología , Bazo/efectos de los fármacos , Bazo/patologíaRESUMEN
The cell cycle stage of donor cells is an important factor influencing developmental ability of nuclear transfer embryos. In the present experiment, cumulus and fibroblast cells of cattle were subjected to flow cytometric cell cycle analysis before being used in somatic cloning experiments. The following experimental groups were analyzed for each cell type: (1) actively dividing cells, (2) cells confluent for 4 days, (3) cells starved for 1, 2, 3 or 5 days. Using the propidium iodide flow cytometric assay, there were no significant differences (P > or = 0.05) in the percentage of cells in G0/G1 regardless of origin and type of cell, after confluency or serum starvation. Differences with the growing cells were found (P < or = 0.01). To determine what subset of cells in G0/G1 were in the G0 subphase of the cell cycle, an immunofluorescence analysis was conducted using monoclonal anti-PCNA antibodies in a FACS assay. There were not statistically significant differences in the percentage of cells that enter G0, between confluent and any starved group for either type of cells. Bovine fibroblast cells, confluent or serum starved for 3 days, were used in nuclear transfer experiments. A slight trend for a more desirable fusion rate in starved cells was detected, and embryo cleavage was greater in starved cells, however, in vitro development to blastocysts was similar between groups. Data indicate that prolonged culture of cells in the absence of serum does not imply a shift in the percentage of cells that enter G0/G1 or G0 alone, and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs, because there are negative effects such as apoptosis, associated with serum starvation.
Asunto(s)
Bovinos/fisiología , Fibroblastos/citología , Células de la Granulosa/citología , Animales , Ciclo Celular/fisiología , Medio de Cultivo Libre de Suero , ADN/fisiología , Transferencia de Embrión/veterinaria , Femenino , Citometría de FlujoRESUMEN
The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect of two protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesis is that freezing affects cell membranes in a way that interferes with the fusion process upon cloning but without hampering normal cell development in vitro. We found that freezing cell lines without controlling the cooling rate gives lower yields in the fusion step and in the final development into blastocysts, compared with cells frozen with a controlled cooling rate of approximately 1 degrees C/min. Transmission electron microscopy of the cells subjected to different freezing procedures showed major damage to the cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning is a critical step in the procedure and should be monitored carefully using a method that allows for a step-wise, controlled cooling rate.
Asunto(s)
Blastocisto/fisiología , Clonación de Organismos , Criopreservación , Fibroblastos , Células Híbridas/fisiología , Conservación de Tejido/métodos , Animales , Blastocisto/ultraestructura , Bovinos , Fusión Celular , Línea Celular , Femenino , Técnicas In Vitro , Micromanipulación , Técnicas de Transferencia Nuclear , Oocitos , Embarazo , Piel/citologíaRESUMEN
The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that a direct instillation of a recombinant adenoviral vector containing an expression cassette for the human growth hormone gene into the mammary gland of mice and goats allowed for the efficient secretion of human growth hormone in the milk. Through this approach we were able to express human growth hormone at maximal levels of 2.8 mg/ml in the milk of mice and up to 0.3 mg/ml in goat milk. We found that the expression levels were closely dependent on both the degree of differentiation of the secretory epithelium and on the adenoviral dose used. Here we demonstrated that the direct transduction of mammary epithelial cells by means of a recombinant adenovirus could be a suitable alternative to transgenic technology for the production of recombinant proteins of biopharmaceutical interest.
Asunto(s)
Adenoviridae/genética , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Cabras/genética , Hormona de Crecimiento Humana/biosíntesis , Hormona de Crecimiento Humana/genética , Leche/química , Ingeniería de Proteínas/métodos , Animales , Hormona de Crecimiento Humana/química , Humanos , Glándulas Mamarias Humanas/metabolismo , Ratones , Leche/metabolismo , Transformación Genética/genéticaRESUMEN
The use of live bioreactors for the expression of human genes in the mammary gland of transgenic animals is one of the most cost-effective ways for the production of valuable recombinant therapeutic proteins. Among the transgenic species used so far, rabbits are good candidates for the expression of tens to hundreds of grams of complex proteins in the milk during lactation. The lactating mammary gland of rabbits has proven to be effective in the processing of complex proteins. In this work. the potential use of rabbits as bioreactors is discussed based on our results and the published data.