Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector.</p><p><b>METHODS</b>First, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells.</p><p><b>RESULTS</b>The construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69.</p><p><b>CONCLUSION</b>The construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.</p>

Transport mechanism of danshensu across Caco-2 monolayer model / 中国中药杂志

<p><b>OBJECTIVE</b>To study the absorption mechanism of Danshensu by using Caco-2 monolayer model .</p><p><b>METHOD</b>Caco-2 cell monolayer model was used to study the bi-direction transport of Danshensu. An LC-MS method was developed to measure the concentration of Danshensu in cell culture medium and calculate the apparent permeability coefficients (P(app)). The effects of time, drug concentration and inhibitor on the absorption of Danshensu were studied.</p><p><b>RESULT</b>Transport of Danshensu was time and concentration dependent, and it was also effected by P-glycoprotein inhibitor. P(app) increased with time increase and tended to become saturated at some point. It, however,decreased while concentration of Danshensu increased. P(ratio) is larger than 1.5 . Verapamil can cause significantly effect on transport of Danshensu: P(app,A-B) increased and P(app,B-A) decreased .</p><p><b>CONCLUSION</b>The absorption of Danshensu in Caco-2 cell model may be an active transportation mediated by P-glycoprotein transporter.</p>

Effects of Gardenia-aweto compound by different extraction method on antagonizing acute respiratory distress syndrome / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of Gardenia-Aweto compound (GAC) and two component on preventing acute respiratory distress syndrome (ARDS) by the rabbit model of ARDS induced by intravenous injection of oleic acid. To detect the efficiency component of GAC in preventing ARDS.</p><p><b>METHOD</b>GAC was divided into two compounts, ethanol-soluble components (ESC) and ethanol-deposition components (EDC), based on polarity. Forty-three new zealand rabbits were randomly divided into five groups, the blank control group, the model group, the GAC groups, the ESC group, and the EDC group. The ARDS model was induced by intravenous injection of oleic acid. Dynamic changes of arterial blood gas, lung index, albumin in bronchoalveolar lavage fluid (BALF) in different groups and lung histological changes were observed and compared.</p><p><b>RESULT</b>As compared with the blank group, in the model group, GAC group, ESC group, EDC group the arterial PO2 and oxygen saturation deprived continuously. While SO2 in GAC group at time points 30, 60, 90, 120 min (P < 0.05 or 0.01) and SO2 in ESC group at time points 30, 60, 90 min were higher than those in ARDS group. PO2 in ESC group at time points 30, 60 min (P < 0.05) were higher than those in ARDS group. The value of LI and W/D were higher in ARDS group than in sham group (P < 0.01), they were much lower in HD group than in ARDS group (P < 0.01). Concentration of BALF-albumin increased markedly in ARDS group and pre-treatment groups compared with sham group, but it was much lower in GAC group and ESC group, there was a significant difference between GAC group (P < 0.01), ESC group (P < 0.05) and ARDS group. The lung histological changes had been improved in GAC group and ESC group. But no significantly difference between above-mentioned parameters was found in comparison in the model group and in the EDC group.</p><p><b>CONCLUSION</b>Preventive administration of GAC or ESC an protect the damaged lung function in ARDS rabbits induced by oleic acid. The efficiency component of GAC in preventing ARDS is ESC. GAC antagonizing ARDS may relate to its anti-inflammatory, immuno-modulatory, anti-oxidant and antithrombotic effects.</p>