ABSTRACT
Objective To investigate the effects of parathyroid hormone (PTH) on the synthesis and secretion of collagen Ⅲ and fibronectin (FN), and the expressions of plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA in cultured human renal tubular epithelial cells (HK-2).Methods HK-2 cells were cultured in DMEM-F12 medium supplemented with 5% FBS. Cells were exposed to different concentrations of PTH (0, 10-12, 10-11, 10-10, 10-9, 10-8 mol/L) for 48 h, or 10-8 mol/L PTH at different time (0, 12, 24, 48, 72 h). The gene expressions of collagen Ⅲ,FN, PAI-1, MMP-1, and TIMP-1 were detected by semi-quantitative RT-PCR. The protein expression of collagen Ⅲ was detected by Western blotting. The level of FN in the supernatant was assayed by enzyme linked immunosorbent assay (ELISA). Results PTH increased gene expressions of collagen Ⅲ, FN, PAI-1 and TIMP-1 in a dose- and time-dependent manner, but decreased MMP-1 gene expression. Then the ratio of MMP-1/TIMP-1 was decreased. PTH increased the collagen Ⅲ protein expression in cultured HK-2 cells and the level of FN in the supernatant of cultured HK-2 cells in a dose- and time-dependent manner. Conclusion PTH can up-regulate PAI-1, TIMP-1 gene expressions, and down-regulate MMP-1 gene expression,resulting in elevation of extracellular matrix (ECM) synthesis and reductim of degradation.
ABSTRACT
A squalene synthase gene cloned (GuSQS1, accession number in GenBank database: AM182329) from Glycyrrhiza uralensis was transferred into G. uralensis via Agrobacterium rhizogenes A4 for investigating biosynthesis pathway and enhancing synthesis of glycyrrhizic acid (GA). Hypocotyl explants from G. uralensis were infected with A. rhizogenes A4 containing GuSQS1 gene to induce the hairy roots. The hairy root lines established were selected in medium containing 0.8 mg x L(-1) phosphinothricin (PPT) and analyzed by PCR and southern blotting. The transgenic hairy roots were cultured in liquid MS medium. GA contents in transgenic hairy roots were detected by HPLC. Results showed that maximal GA content in transgenic hairy root lines was 3.6 times as high as in wild type hairy roots.
Subject(s)
Cells, Cultured , Farnesyl-Diphosphate Farnesyltransferase , Genetics , Metabolism , Glycyrrhiza uralensis , Genetics , Metabolism , Glycyrrhizic Acid , Metabolism , Molecular Sequence Data , Plant Proteins , Genetics , Metabolism , Plant Roots , Genetics , Metabolism , Plants, Genetically Modified , Genetics , MetabolismABSTRACT
Object To establish and optimize the technology of clonig rapid propagation by tissue culture in Scutellaria baicalensis Georgi for the protection and development of nature resource as well as seeking for new breeding way of S. baicalensis by biotechnology. Methods Research on the optimized technology of tissue culture including callus inducing, differentiation, plantlet propagation, and rooting were designed. Results Callus could be induced from nodes and internodes easily with 100% inducing rate compared with that from leaves. PP 333 has significant stocky effect to the plantlets. Conclusion The nodes are good explant source in inducing callus. PP 333 in certain concentration which is added to culture media could improve and regulate the differentiation of callus and growth of plantlets as well as increase the survival rate of cultivated seeding.
ABSTRACT
Object To determine the baicalin content in skullcap polyploid by MEKC and HPLC, and to provide the reliable method used to determine a large number of samples. Methods On the basis of methodology of MEKC and HPLC, the baicalin in Scutellaria baicalensis Georgi. was determined, the data and relevant analysis were compared. Results The data of the content determined by HPLC was similar to that by MEKC, but the former showed slightly higher 1%-3%. There was no significant variation between the two methods with higher correlation coefficient. Conclusion Both the above two methods could be accurately used to determine the baicalin in S. baicalensis. The MEKC has some advantages, such as quick, save time with lower solvent cost, and is suitable for the large number of samples in the determination of baicalin of S. baicalensis.