ABSTRACT
Objective To investigate the relationship between the content of flavonoids and the key enzyme genes expression in different tissues of Bupleurum chinense and B. scorzonerifolium. Methods The roots, stems, leaves, and fruits of B. chinense and B. scorzonerifolium were used as test materials, determination of flavonoids (rutin, quercetin, kaempferol, and isorhamnetin) in different tissues by HPLC, determination of total flavonoids by UV spectrophotometry, the tissues expression of key enzyme genes (IFS, F3H, and DFR) in flavonoids synthesis was determined by real-time quantitative PCR, correlation analysis was performed with SPSS. Results The content of flavonoids in the aerial parts (stems, leaves, and fruits) of B. chinense and B. scorzonerifolium was significantly higher than that in roots, the content of flavonoids was mainly rutin, and the content of rutin in the leaves of B. chinense leaves was up to 106.961 mg/g; The distribution of total flavonoids in B. chinense and B. scorzonerifolium was obviously different, the content was from high to low: leaves ≥ fruit > stem > root; The expression of B. chinense IFS, F3H, and DFR gene in the aerial parts was much higher than that in roots, IFS gene was significantly positive correlated with rutin (P < 0.05), F3H gene was significantly positive correlated with DFR gene (P < 0.05), but the expression of IFS, F3H, and DFR gene in each tissues of B. scorzonerifolium was at lower level. Conclusion The content of flavonoids in different parts of B. chinense and B. scorzonerifolium was consistent with the expression of flavonoids synthesis key enzyme genes, the differential expression of key enzyme genes regulates the synthesis and accumulation of flavonoids in different tissues.
ABSTRACT
Oviductal molecules have the potential to improve the reproductive biotechnologies. In camelids, knowledge and assessment of the oviductal environment are necessary to successfully develop species-specific reproductive technologies, especially because of the camelids reproductive particularities. Among the oviductal factors, the matrix metalloproteinases/tissue inhibitor of matrix metalloproteinases system (MMPs/TIMPs) should be investigated more thoroughly due to their participation in reproductive processes. Consequently, the current study assayed gene and protein expression of MMPs throughout the llama oviduct. MMPs zymogen and active forms in the oviductal fluid were also characterized. MMP2 and MMP9 transcripts were detected in ampulla, isthmus, utero-tubal junction and papilla, being MMP2 and MMP9 2.15 and 1.10 folds higher in papilla than in ampulla, respectively. In addition, differences in immunolocalization of MMP2 and MMP9 between the epithelial mucosa layers of the oviductal segments were observed. The presence of MMPs in the epithelium suggests their secretion into the oviductal lumen. Coincidently, bands of 62 and 94 kDa, corresponding to MMP2 and MMP9 were detected by zymography in the oviductal fluid. Treatment with an exogenous activator (APMA) suggests that they are present as proMMPs. TIMP2 and TIMP1, the specific inhibitors of MMP2 and MMP9, respectively, were expressed in each oviductal segment, indicating a well-regulated control of MMP proteolytic activity in the oviduct. These findings prove that the llama oviduct produces and secretes MMPs into the oviductal lumen, suggesting that these enzymes may have an unknown role in the preparation of the oviductal environment for gametes, fertilization and early embryo development in camelids.
Las moléculas oviductales tienen el potencial para mejorar las biotecnologías reproductivas. En los camélidos, debido a sus peculiares características reproductivas, el conocimiento del ambiente oviductal constituye una herramienta útil para el desarrollo de tecnologías reproductivas específicas para estas especies. Entre los factores oviductales de interés se encuentran las metaloproteasas de matriz (MMPs) y sus inhibidores específicos (TIMPs), los cuales han sido involucrados en diferentes procesos reproductivos. Por estas razones, en este trabajo se caracterizó la expresión génica y proteica de MMP2 y MMP9 en el oviducto de llama. Además, se analizó la presencia de las formas activas e inactivas (zimógenos) de estas enzimas en el fluido oviductal. Se observó que todos los segmentos oviductales, ámpula, istmo, unión útero-tubal y papila, expresan MMP2 y MMP9, siendo los niveles de expresión de MMP2 y MMP9 más elevados en papila respecto a ámpula; 2,15 y 1,10 veces respectivamente. Asimismo, se observaron diferencias en la distribución de las MMPs a nivel de la mucosa entre los segmentos oviductales. Consecuentemente, bandas con actividad gelatinolítica de 62 y 94 kDa, se detectaron en el fluido oviductal, las cuales corresponderían a las formas inactivas de la MMP2 y la MMP9, respectivamente. Los inhibidores específicos de MMP2 y MMP9; TIMP2 y TIMP1, también se detectaron en los segmentos oviductales, indicando su probable participación en la regulación de la actividad proteolítica de las MMPs en el oviducto de llama. En conjunto, los datos de este trabajo demuestran que el oviducto de la llama produce y secreta MMPs al lumen oviductal; sugiriendo que estas enzimas pueden participar en la preparación del ambiente oviductal para la recepción de los gametos, la fecundación y el desarrollo embrionario temprano en camélidos.
Subject(s)
Animals , Female , Camelids, New World , Tissue Inhibitor of Metalloproteinases/metabolism , Matrix Metalloproteinases/metabolism , Fallopian Tubes/metabolism , Immunohistochemistry , Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinases/genetics , Matrix Metalloproteinases/geneticsABSTRACT
Mesenchymal stem cells (MSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no gene expression follow up analysis has been undertaken to characterize the in vitro adipogenic differentiated MSCs. The main goal of this study was to focus on MSCs and to analyze their differentiation capacity. To achieve this aim, bone marrow MSCs from sprague dawely rats were isolated, expanded in monolayer culture and characterized with respect to their cluster of differentiation (CD) and ability for adipogenic differentiation capacity. The expression of CD44, CD45, CD29, CD34, and CD90 on bone marrow derived MSCs was characterized using flow cytometry. Adipogenesis was determined by staining with oil-red O and reverse transcription polymerase chain reaction assessments of lipoprotein lipase, leptin, adiponectin and adipocyte genes at different time intervals, after 4, 7, 14, and 21 days. Our results revealed that the pattern of CD marker expression was highly positive significant with CD29, CD44, and CD90 when compared with CD34 and CD45. MSCs showed proliferative potential and were capable of adipogenic differentiation characterized by reddish brown-droplets following staining with oil-red O and expression of molecular bands of genes. These results demonstrate, at the morphological, immunophenotyping and gene expression levels, the multipotency of MSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.
Subject(s)
Animals , Rats , Adipocytes , Adipogenesis , Adiponectin , Bone Marrow , Flow Cytometry , Follow-Up Studies , Gene Expression , Immunophenotyping , Leptin , Lipoprotein Lipase , Mesenchymal Stem Cells , Polymerase Chain Reaction , Reverse Transcription , Tissue Engineering , TransplantsABSTRACT
Mesenchymal stem cells (MSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no gene expression follow up analysis has been undertaken to characterize the in vitro adipogenic differentiated MSCs. The main goal of this study was to focus on MSCs and to analyze their differentiation capacity. To achieve this aim, bone marrow MSCs from sprague dawely rats were isolated, expanded in monolayer culture and characterized with respect to their cluster of differentiation (CD) and ability for adipogenic differentiation capacity. The expression of CD44, CD45, CD29, CD34, and CD90 on bone marrow derived MSCs was characterized using flow cytometry. Adipogenesis was determined by staining with oil-red O and reverse transcription polymerase chain reaction assessments of lipoprotein lipase, leptin, adiponectin and adipocyte genes at different time intervals, after 4, 7, 14, and 21 days. Our results revealed that the pattern of CD marker expression was highly positive significant with CD29, CD44, and CD90 when compared with CD34 and CD45. MSCs showed proliferative potential and were capable of adipogenic differentiation characterized by reddish brown-droplets following staining with oil-red O and expression of molecular bands of genes. These results demonstrate, at the morphological, immunophenotyping and gene expression levels, the multipotency of MSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.