An ectopic study of tissue-engineered bone with Nell-1 gene modified rat bone marrow stromal cells in nude mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique.</p><p><b>METHODS</b>bMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or Ad-beta-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with beta-tricalcium phosphate (beta-TCP) at a concentration of 2 x 10(7) cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.</p><p><b>RESULTS</b>Under current transduction conditions, gene transfer efficiency reached (57.9 +/- 6.8)%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was (18.1 +/- 5.0)%, significantly higher than those of untransduced group (11.3 +/- 3.2)% and LacZ group (12.3 +/- 3.1)% (P < 0.05).</p><p><b>CONCLUSIONS</b>This study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.</p>

Effects of clenbuterol on the metabolism of nitrogen and IGF-I level in isolated perfused rat liver / 中国应用生理学杂志

<p><b>AIM</b>To examine the liver mechanism with which clenbuterol is explained how to affect growth metabolism.</p><p><b>METHODS</b>Twenty-four adult SD rats were randomly divided into three groups, a control and two treatment groups. The technique of isolated perfused rat liver in vitro was used to study the effects of clenbuterol on urea nitrogen concentration of perfused medium, GPT activity and synthesis and secretion of IGF-I in isolated perfused rat liver.</p><p><b>RESULTS</b>Urea-nitrogen concentration of perfused medium was significantly affected by clenbuterol in a dose-dependent and time-dependent manner. The urea-nitrogen level was decreased by 15.02% (P > 0.05),17.97% (P > 0.05), 26.76% (P < 0.05) and 30.08% (P < 0.01) for 1 h, 2 h, 3 h, 4 h after administering clenbuterol at the dose of 1 x 10(-6) mol/L, respectively, compared to that of control. 1 x 10(-8) mol/L CL had the similar effect on urea-nitrogen level. GTP activity of isolated perfused rat liver was inhibited by clenbuterol. The enzyme activity was decreased by 24.65% (P < 0.05) at the dose of 1 10(-6) mol/L CL in clenbuterol-treated 4h. The production and secretion of IGF-I were also influenced by clenbuterol in isolated perfused rat liver. IGF-I concentration of rat liver was increased by 19.77% (P < 0.05) in 4 h clenbuterol treatment (1 x 10(-6) mol/L). Meanwhile, IGF-I concentration of perfusion medium was also elevated though the difference was not significant compared with control.</p><p><b>CONCLUSION</b>It is suggested that clenbuterol may improve growth metabolism by means of increasing nitrogen retention and enhancing IGF-I production and secretion of rat liver.</p>

Effects of clenbuterol on nitrogen metabolism and G6PDH activity of rat hepatocyte / 药学学报

<p><b>AIM</b>To study the effects of beta 2-adrenergic receptor-selective agonist clenbuterol on nitrogen metabolism and glucose-6-phosphate dehydrogenase activity of rat hepatocyte and its pharmacological mechanism.</p><p><b>METHODS</b>Biochemical methods were used to study the influence of clenbuterol on urea-nitrogen concentration of hepatocyte culture medium, 3H-leucine incorporation into hepatocyte, insulin-like growth factor I (IGF-I) production and glucose-6-phosphate dehydrogenase (G6PDH) activity of rat hepatocyte.</p><p><b>RESULTS</b>The results showed that urea-nitrogen production by cultured rat hepatocytes was markedly affected with clenbuterol treatment (1 x 10(-6) mol.L-1), urea-nitrogen concentration of culture medium was decreased by 25.51% (P < 0.05) compared with control. The inhibitory effect of hepatocyte urea-nitrogen production of clenbuterol was blocked by propranolol, a beta-adrenoreceptor antagonist (1 x 10(-6) mol.L-1), but hepatocyte urea-nitrogen level was not affected with propranolol treatment only (P > 0.05). The content of 3H-leucine incorporation in rat hepatocyte was significantly increased by 23.35% (P < 0.05) with clenbuterol-treatment (1 x 10(-6) mol.L-1), and the enhanced effect of 3H-leucine incorporation into hepatocyte was antagonized by propranolol (1 x 10(-6) mol.L-1. The level of 3H-leucine incorporation of rat hepatocyte was not influenced by propranolol alone. IGF-I production of rat hepatocyte might be affected by clenbuterol. IGF-I concentration of culture medium was increased by 39.46% with clenbuterol (1 x 10(-6) mol.L-1), but no significant difference was found compared with the control (P > 0.05). Moreover, G6PDH activity of rat hepatocyte was significantly decreased by 43.36% (P < 0.05) with clenbuterol treatment (1 x 10(-6) mol.L-1), and the declined effect of clenbuterol was antagonized by propranolol. G6PDH activity of rat hepatocyte was not affected on condition that propranolol was administered alone (P > 0.05).</p><p><b>CONCLUSION</b>It is suggested that clenbuterol may regulate nitrogen and fat metabolism by means of increasing nitrogen retention and protein synthesis, and decreasing G6PDH activity of rat hepatocyte for pharmacological effects.</p>