Screening for potential bioactive components of Yin-zhi-huang using high bilirubin HepaRG cells incubating with serum from animals / 药学学报

A hyper-bilirubin cell model was established for its relevance to the pathological state of jaundice in human. This model was used to screen for the pharmacological components of Yin-Zhi-huang (YZH). Total bilirubin, indirect bilirubin in cells, and direct bilirubin in extracellular fluid were quantified after HepaRG cells were incubated with serum from rats injected with multiple components of YZH. Cellular uptake was determined by dynamic multiple reaction monitoring (DMRM) using LC-MS/MS. We found that the stable hyper-bilirubin HepaRG cell model could be established by incubating cells with 40 μg·mL-1 bilirubin and 50 μg·mL-1 probenecid. When the hyper-bilirubin cell model was incubated with serum from rats of YZH injection, there were 52.4% and 60.1% decrease in intercellular total bilirubin and indirect bilirubin, respectively, and 52.5% increase in extracellular direct bilirubin. Using DMRM mode, 53 components could be determined, and 8 potential bioactive candidates were identified from the serum. This method could be used to screen for bioactive metabolites of YZH. This strategy is simple, highly active, sensitive and specific, providing a new method for high throughput screening of therapeutic or toxic metabolites from traditional Chinese medicine. The regulations of Ethics Committee in the First Hospital of Lanzhou University were abided in the rat experiment of this study.

Study of pharmacokinetics of digoxin in ovariectomized rats model / 药学学报

This study aims to investigate the change of plasma concentration of digoxin (DIG) in rats with ovariectomy. Twelve female SD rats were randomly assigned into ovariectomized group and sham group (n = 6). All rats plasma was collected after a single dose of 2 mg x kg(-1) DIG administrated orally, serum DIG concentration was determined by LC-MS/MS. The level of P-gp in the intestinal was analyzed by Western blotting. Pharmacokinetic calculations were performed on each individual using DAS 2.0 practical pharmacokinetic software. Compared with the sham group, C(max) of ovariectomized group decreased significantly (P < 0.01). There was no significant difference of AUC(0-t), and the level of P-gp was elevated in ovariectomized group. It was found that C(max) of DIG was significantly reduced after ovariectomy, and the change was associated with the decreased level of estrogen, which contributes to the increased level of P-gp.

A new targeting approach for breast cancer gene therapy using the human fatty acid synthase promoter.

Gene therapy with adenoviral vectors is a promising new approach for the treatment of refractory advanced breast cancer. Strategies to restrict adenoviral-mediated therapeutic gene expression are important to avoid harming normal cells. Fatty acid synthase (FAS) is overexpressed in several human cancers. FAS is highly expressed in infiltrating breast cancer tissue, and always associated with malignant phenotypes and poor prognosis. In this study, expression of the FAS was evaluated in three breast cancer cell lines. A 680 bp-FAS promoter was cloned and its transcriptional activity was analyzed in breast cancer cell lines. We made a recombinant adenovirus construct carrying herpes simplex virus thymidine kinase (HSV-TK) driven by human FAS promoter (Ad-FAS-TK) and analyzed its target cytotoxicity in vitro and in vivo against human breast cancer cells combined with prodrug ganciclovir (GCV). The results show that the expression of FAS varies in the three breast cancer cell lines examined (respectively, SK-Br3>MCF-7>MDA-MB-231), but FAS promoter can initiate relative high transcriptional activities in all three kinds of cancer cells while little in normal fibroblast cells. Furthermore, FAS promoter can drive the therapeutic gene in a wider range of human breast cancers than cerbB2 promoter and exhibit a stronger activity than midkine (MK) promoter. Combination of Ad-FAS-TK and GCV treatment exhibited strong-targeted cytotoxic effect on breast cancer cells but showed little activity in normal fibroblast cells. The tumorigenic capability of breast cancer cells treated with Ad-FAS-TK/GCV was completely inhibited in vitro and in vivo assays. In conclusion, adenoviral-mediated suicide gene therapy controlled by tumor associated-FAS promoter can induce specific cytotoxic effect on human breast cancer cells in vitro and in vivo. So it is a promising target for the development of gene therapy against breast cancers.

Isolation and differentiation of embryonic stem cells from BALB/c mouse / 神经科学通报·英文版

Objective To investigate the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula of 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.

Construction of eukaryotic expression vector for KGPcd gene from Porphyromonas gingivalis and expression in mammalian cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.</p><p><b>METHODS</b>Eukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.</p><p><b>RESULTS</b>It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.</p>