[Study on ultrasonic extraction and macroporous resin purification techniques of total flavones from Sedum lineare].

OBJECTIVE: To optimize the ultrasonic extraction condition and purification process of total flavonoids in Sedum lineare by macroporous resin. METHODS: Optimum ultrasonic extraction conditions (ethanol concentration, solid/liquid, ultrasonic time, working temperature) were obtained through orthogonal design. The adsorption ratio and eluting ratio of total flavones were selected as indexes. RESULTS: The optimum ultrasonic extraction condition was 80% ethanol, solid/liquid 1:40, ultrasonic 20 min at 30 degrees C. The adsorbed AB-8 macroporous resin column was eluted by 80% ethanol at the eluting velocity of 2 mL/min, the eluting ratio and purity of total flavones was 94.72% and 43.51%, respectively. CONCLUSION: The method is simple and low cost.

[Preparation of anti-P21-activated kinase 5 polyclonal antibody and its application in dental germ cells].

OBJECTIVE: To clone PAK5-N terminal sequence for expression in E. coli to prepare its polyclonal antibody, and examine the role of PAK5 in dental germ cells. METHODS: Based on human PAK5 cDNA sequence, PCR primers were designed to amplify PAK5-N terminal sequence. The PCR product was cloned into the expression vector pGEX-4T-1 EcoRI/XhoI sites, and the recombinant plasmids were identified by agarose gel electrophoresis followed by DNA sequence analysis. The recombinant plasmids were transformed into E. coli BL21 and the expression of GST-fusion protein was induced by IPTG. Glutathione-Sepharose beads were used to purify GST-fusion PAK5-N-terminal fragment. Anti-PAK5 polyclonal antibody was obtained in immunizing rabbits with purified GST-PAK5 N-terminal fusion protein, and the antibodies were purified by protein A beads and used for detection of PAK5 expression in dental germ cells. RESULTS AND CONCLUSIONS: We successfully cloned PAK5-N terminal gene fragment, and achieved protein expression, purification and production of PAK5-NT polyclonal antibody. The results of Western blotting indicated that PAK5 can be highly expressed in the dental germ cells, suggesting that PAK5 may play an important role in biological function of dental germ cells.

[Effects of cyclosporin A on gene expression profiles of NIT-1 pancreatic beta cell line].

OBJECTIVE: To observe the effects of cyclosporin A (CsA) on gene expression profiles of NIT-1 pancreatic beta cells cell line using microarray technique. METHODS: NIT-1 cells were exposed to cyclosporin A treatment (10 micromol/L) for 24 h and the differential gene expressions were assessed using microarray technique. RESULTS: After 24h of CsA treatment, 38 of the 4096 genes tested were up-regulated, including 13 genes with known functions involving stress response, cell growth and protein synthesis. Meanwhile 46 genes were down-regulated, including 25 genes with known functions involving cell growth and maturation, oxidative phosphorylation and protein synthesis. Changes of gene expression of Zfr,Tpi and Pax6 were confirmed by semi-quantitative reverse transcription polymerase chain reaction. CONCLUSIONS: CsA treatment for 24 h induces changes in the gene expression profiles of NIT-1 pancreatic beta cells. Down-regulation of the genes related to cell growth and maturation, oxidative phosphorylation and protein synthesis may partly explain the mechanisms that CsA inhibits the release of insulin from pancreatic beta cells.