Analytical method and comparison for static and dynamic headspace gas chromatography of anisole in water / 中华预防医学杂志

<p><b>OBJECTIVE</b>To establish and compare the method of static headspace gas chromatography hydrogen flame detector (static headspace method) and purge and trap gas chromatography-mass spectrometry (dynamic headspace method) of anisole in water.</p><p><b>METHODS</b>Nitrogen gas was used as carrier gas in the static headspace method, 5 g NaCl as matrix modifier was added into 10 ml water. The sample was balanced with high speed vibration at 75°C for 30 min, and anisole was detected by gas chromatography and quantified with external standard. Helium was used as carrier gas in dynamic headspace method, 5.0 ml water and 0.004 mg/L internal standard fluorobenzene was purged into the purge and trap apparatus. After purging, trapping and desorption, anisole was detected by the gas chromatography-mass spectrograph, confirmed by the retention time and comparison of mass-spectrogram in spectrum library and quantified with internal standard. The repeatability and sensitivity of assay were evaluated.</p><p><b>RESULTS</b>A good linear range for anisole was observed in static headspace gas chromatography and dynamic headspace gas chromatography-mass spectrometry, within the range of 10 - 500 µg/L and 0.5 - 60.0 µg/L respectively. The linear regression equation was Y = 782.150X + 1.3446 and Y = 0.0358X - 0.0209 respectively, both the correlation coefficient ≥ 0.999. The detection limit (LOD) were 0.002 µg/L and 0.110 µg/L, the lower limit of quantitation (LOQ) were 0.006 µg/L and 0.350 µg/L, the relative standard deviation (RSD) were 1.8% - 2.3% and 2.0% - 3.4%, and the spiking recovery were 93% - 101% and 96% - 101% respectively.</p><p><b>CONCLUSION</b>The methods of static headspace gas chromatography and dynamic headspace gas chromatography-mass spectrometry are simple and can measure anisole in water quickly, sensitively and accurately.</p>

Ultrastructure of hepatocytes in Gilbert's syndrome patients and chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the pathological characteristics of inborn hyperbilirubinemia of patients with Gilbert's syndrome (GS).</p><p><b>METHODS</b>Patients with GS (n = 7) and patients with chronic hepatitis B (CHB; n = 8) were enrolled in the study. GS was diagnosed by peripheral blood analysis results showing glucuronyl transferase gene mutation. The histology and ultrastructure of biopsied liver tissues were evaluated by light microscopy and transmission electron microscopy.</p><p><b>RESULTS</b>The GS group showed normal structure in the hepatic portal area and lobule; however, bile pigment granules with high electron density were noted in the hepatocytes. The CHB group showed abnormal structure of the hepatic lobules, including infiltration of inflammatory cells, necrotic regions, degenerated hepatocytes, bile duct injury, and fibrosis in the portal tracts; a few bile pigment granules were observed. The GS group also showed greater quantity and size of bilirubin deposits than the CHB group.</p><p><b>CONCLUSION</b>The histological and ultrastructural features of GS include normal hepatic lobule and deposition of bile pigment granules in hepatocytes.</p>

Effect of functionalized multi-walled carbon nanotubes on L02 cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the cytotoxic effect of multi-walled carbon nanotubes (MWCNTs) on human liver L02 cells and its relevant mechanism.</p><p><b>METHODS</b>MWCNTs, carboxyl modification MWCNTs (MWCNTs-COOH), and hydroxyl modification MWCNTs (MWCNTs-OH) were characterized by transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The carbon nanotubes at concentrations of 12.5, 25, 50, 100, and 200 μg/ml were incubated with human liver L02 cells for 24, 48 and 72 hours, respectively. The cell viability was evaluated by water soluble tetrazolium salts assay and the intercellular reactive oxygen species induced by the carbon nanotubes were detected by 2', 7'-dichlorodihydrofluorescein diacetate method.</p><p><b>RESULTS</b>Transmission electron microscope showed that the average outside diameters (10 to 20 nm) and the average length (10 to 30 μm) of the three MWCNTs were similar. Scanning electron microscope indicated that the three MWCNTs had a similar surface topography. X-ray photoelectron spectroscopy demonstrated that the MWCNTs-COOH and MWCNTs-OH had relatively high peak areas at 289 and 286ev, respectively,indicating that they have been modified by carboxyl and hydroxyl groups,respectively. Water soluble tetrazolium salts assay showed that the MWCNTs-COOH was less cytotoxic when compared to MWCNTs which demonstrated to be slightly more cytotoxic than MWCNTs-OH. The capability to induce increase in intracellular reactive oxygen species was in the following order: MWCNTs > MWCNTs-COOH > MWCNTs-OH.</p><p><b>CONCLUSIONS</b>Modification of MWCNTs with carboxyl group and hydroxyl group improves the biocompatibility of MWCNTs to some extents. MWCNTs-COOH has better compatibility than MWCNTs at the low concentration,and MWCNTs-OH showed better compatibility than MWCNTs after 48 hours. Different mechanisms may be involved in the interaction between cells and the MWCNTs with different chemical surfaces.</p>

Effect of chitosan gene nanoparticles on L02 cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of the gene nanoparticles using chitosan (CNP), arginine modified chitosan (ANP), or hexadecylated chitosan (HNP) as carriers on the human normal liver cell line L02.</p><p><b>METHODS</b>CNPs, ANPs, and HNPs were prepared using complex coacervation method. The size and zeta potential of the gene nanoparticles were measured using Zetasizer nanoZS. The nanoparticles at concentrations of 5, 10, 30, and 50 microg/ml (based on the content of DNA) were incubated with L02 cells, respectively. The cell viability was evaluated by MTT assay, and the effect of the gene nanoparticles on the cell apoptosis was analyzed by flow cytometry.</p><p><b>RESULTS</b>The zeta potential of the gene nanoparticles ranged from 12.10 to 14.63 mV, and their diameters ranged from 148.07 to 179.47 nm. MTT assay showed that the viability of L02 cells began to decrease when the concentration of CNPs reached 30 microg/ml and higher. Furthermore, the CNPs could induce cell apoptosis as the concentration of CNPs reached 30 microg/ml and higher.</p><p><b>CONCLUSION</b>CNPs can induce L02 cell apoptosis at relatively higher concentrations.</p>

Expression and purification of tissue factor pathway inhibitor in Pichia pastoris / 中国医学科学院学报

<p><b>OBJECTIVE</b>To generate recombinant human tissue factor pathway inhibitor (TFPI) in Pichia pastoris.</p><p><b>METHODS</b>To improve the expression of TFPI, a silent mutation was generated at the specific site of TFPI cDNA. Both wild-type TFPI cDNA and mutated TFPI cDNA were cloned into the expression vector pPic9. The constructed plasmids were subsequently transformed into Pichia pastoris cells GS115 and KM71, and the transformants were confirmed by polymerase chain reaction and DNA sequencing. The expression of recombinant protein was induced by addition of 0.5% methanol in the culture medium. The cell culture medium after induction was concentrated through ultra filtration. The recombinant protein was further purified by a three-step process (Heparin-sepharose CL-6B affinity chromatography, DEAE-Sepharose Fast Flow affinity chromatography, and Sephadex G75-gel filtration). The amount of the recombinant protein was quantified with gel imaging system. The activity of the recombinant protein was analyzed by the chromogenic substrate assay.</p><p><b>RESULTS</b>The amount of TFPI expressed in the mutated clone (1 mg/L) was much higher than that in the wild type clone (0.1 mg/L). The TFPI activity in the recombinant GS115 cells could be detected 12 hours after induction and reached the peak at 36 hours, while the TFPI activity in the recombinant KM71 cells started to show up at 24 hours after induction and reached the peak at 72 hours. The expression of recombinant protein in the silent mutant was significantly higher than those of wild type clone in both GS115 and KM71 host cells. The relative molecular mass of recombinant TFPI was approximately 42 000.</p><p><b>CONCLUSION</b>Introduction of the silent mutation at the specific site of TFPI cDNA can increase the recombinant protein expression in Pichia pastoris, which is much higher than that in insect cells or saccharomyces cerevisiae.</p>

A novel anti-DNA antibody modified coronary stent for site-specific plasmid DNA delivery / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the feasibility of using an endovascular metal stent as a highly efficient and site-specific gene delivery system.</p><p><b>METHODS</b>Stents were formulated with a collagen coating. Anti-DNA monoclonal antibodies were covalently bound to the collagen surface by a cross linking reagent of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Binding capacity and stability of antibody and plasmid DNA on stents were quantified by radioactive labeling. The gene transduction efficiency was evaluated in cell culture and in rabbits.</p><p><b>RESULTS</b>The amount of antibodies binding on collagen matrix through SPDP reaction was 15 times higher than that of through physical absorption (P < 0.005). The binding stability of plasmid was significantly better than the control groups (P < 0.01). There was no harmful effect on cell growth with the anti-DNA antibody modified stents. The stents retrieved from cell culture after 72 hours of incubation in A10 cells showed numerous transducted cells only infiltrating the surface coating indicating a highly localized and efficient gene delivery pattern. Results of in vivo gene transfer by this modified stent revealed (2.8 +/- 0.7)% of total cells transduction and the higher transduction location was neointimal layer (about 7%). No distal spread of vector was detectable in the anti-DNA antibody modified stent implantation animals.</p><p><b>CONCLUSIONS</b>Anti-DNA antibody modified stents represent a novel highly efficient and site-specific gene delivery system which can deliver various kinds of plasmid vectors. The release of plasmid DNA tethered on the stents could be controlled in some conditions. This novel system provided a novel platform for cardiovascular site-specific gene therapy.</p>

Chemical coupling of anti-dNA antibody on collagen coating / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the feasibility and stability of chemically conjugating IgM on collagen films.</p><p><b>METHODS</b>IgM was labeled with 125I using the chloramine-T method. Six collagen films were randomly divided into two groups. In chemical coupling group 125I-labeled IgM was chemically coupled with the films through N-succinmiclyl-3- (2-pyridyl-dithio) propionate reaction. In control group 125I-labeled IgM was absorbed onto collagen films. The amount of IgM on the collagen films and the amount of IgM remained on the films after extensive rinsing with phosphate buffered saline were monitored by counting the radioactivity of 125I.</p><p><b>RESULTS</b>The amount of antibodies loaded onto collagen films in the chemical coupling group was 15 times higher than that on the control films, showing significant statistical difference (P < 0.01). And the stability of conjugation antibodies on collagen films was significantly better than the control films.</p><p><b>CONCLUSION</b>Chemical coupling is an effective approach to immobilize antibodies on collagen for further plasmid DNA tethering.</p>