AIM: To investigate the effect of 7-(4-methoxyphenyl)-5, 8a-diphenyl-1,2, 3, 7, 8, 8a-hexahydroimidazo[1,2-a] pyridine (TIP-6) on cell proliferation in human hepatoma cell line HepG2 and human normal hepatocyte cell line L02. METHODS: Typan blue assay was used to check the effect of TIP-6 on cell proliferation. The changes of cell morphology were observed by the phase contrast microscope. Flow cytometry (FCM) was used to check cell cycle. Autophagy and autophagic cell death were detected after acridine orange (AO) staining under fluorescent microscopy. Apoptosis was analyzed by Annexin V/7-AAD, DAPI staining and DNA ladder. NF-κB expression was detected with cellular immunochemistry. RESULTS: Cell proliferation inhibiting effect was appeared when treated with TIP6 from 60 μmol/L to 200 μmol/L, which was correlated with treated concentrations and time. The proliferation rates were just 12.10% and 18.75% (vs control) under 200 μmol/L 72 h in HepG2 and L02 respectively. Vacuolization were found more and more frequently with the increasing of TIP-6 concentrations and treated time prolonged. FCM results indicated that cells were blocked in G2/M phase, and more sensitive were found in HepG2 than L02. AO staining results indicated that the phenomenon of autophagy and autophagic cell death were occurred and appeared more potent with more TIP-6 and longer time treated. No apoptosis markers were found with Annexin V/7-AAD and DAPI staining, and no DNA ladders were found either, these indicated that TIP6 didnt induce apoptosis in these cells. NF-κB was found increased after treated with TIP-6, and the autophagic vacuole became more and more with the increasing of NF-κB protein, but the proliferation rates decreased at the same time. CONCLUSION: TIP-6 inhibited cell proliferation and induced autophagy and autophagic cell death in HepG2 and L02 cells. NF-κB activation may be involved in these effects.

The NSP2 gene of porcine reproductive and respiratory syndrome virus (PRRSV)S1 strain was partly amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a fusion protein GST-tNSP2 with molecular weight of 50 kDa was expressed in E.coli. The purified GST-tNSP2 protein showed a strong reaction with the PRRSV-positive sera in Western blot assay. Balb/c mice were immunized with the purified protein, and the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0. After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5. They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA, but not with the PRRSV SY0608 strain. Both of the McAbs belong to IgG1 isotype, and their light chains belong ? type. The expressed GST-tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.

Adult ; Carcinoma ; complications ; Female ; Humans ; Kidney Neoplasms ; complications ; Solitary Fibrous Tumors ; complications ; Thyroid Neoplasms ; complications

·AIM:To present a case of late onset bilateral keratectasis after laser in situ keratomileusis (LASIK) for myopia with rigid gas-permeable contact lenses with a brief review of literature on this subject.·METHODS:A 27-year-old woman underwent bilateral uneventful LASIK for moderate myopia. Preoperative cycloplegic refractions were -5.50/-0.50×50° right eye (OD) and - 4.50/-1.00×15° left eye (OS).Corneal pachymetry was 526μm OD and 541μm OS, Preoperative corneal topography was normal and did not reveal any keratoconus or forme fruste keratoconus.Following the creation of flaps with 160μm plates,ablations of 102μm OD and 86μm OS were performed,estimated to leave residual stromal beds of 264μm OD and 295μm OS.·RESULTS:Twenty-nine months postoperatively,the patient developed bilateral inferior keratectasia of -12.50/-4.00×160° OD and -6.00/- 4.25×125° OS.Visual acuity was reduced in both eyes;the central cornea had steepened; and pachymetry showed central corneal thinning.Keratectasia was diagnosed,and rigid contact lenses were fitted.Three years later,the patient achieved satisfactory visual acuity and all-day lens wear with minimal complications.·CONCLUSION:Late keratectasia may follow LASIK for low to moderate myopia despite a thorough preoperative work-up.Rigid contact lenses can offer a safe,reversible option for improving visual acuity in such patients by delaying or avoiding the need for intracorneal ring segments implanting or penetrating keratoplasty.

The protoplasts of original Aspergillus niger strain Uco-3 were treated with the cooperation of UV and ?-ray to obtain the high-yielding strain producing the thermostable ?-galactosidase. Under the optimum conditions of formation and regeneration protoplasts were prepared. According to the interaction of positive mutation rate and radiation dose,the optimum condition was determined. The optimum dose of UV was 4 minutes and the optimum dose of ?-ray was 500 Gy. After mutagenetic treatment of protoplasts and selection from a lot of mutants,a mutant DL116 producing the thermostable ?-galactosidase was obtained. The ?-galactosidase activity of DL116 was increased from 16.27 U/mL to 44.37 U/mL,which was higher than that of strain Uco-3.

Objective To study the therapeutic effect of thalidomide(Tha) on murine hepatocellular carcinoma. Methods In murine transplanted hepatoma model, thalidomide was administered intragastrically alone (200 mg/kg daily for 10 days) or in combination with doxorubicin. The antitumor activity of Tha was observed in solid and ascitic tumor models. Results Tha induced significant growth inhibition of solid hepatoma without obvious toxicity on peripheral blood cells and lymphocyte proliferation. Although Tha alone had no effect on the survival of mice with ascitic tumor, it showed a synergistic antitumor activity in combination with doxorubicin (Dox) in both solid and ascitic tumor models. Moreover, Tha reduced Dox-induced cytopenia and immunosuppression. Histological analysis of Tha-treated tumors revealed remarkably enhanced tumor necrosis and lymphocyte infiltration on the edge of tumor tissues. Conclusion Tha has definite therapeutic effect on murine hepatoma,and the combination with Dox shows an enhanced therapeutic potential.

Based on the analysis of metabolic pathway Streptococcous zooepidemicus for hyaluronic acid (HA) synthesis, nucleotide,especially uracil, was considered to be important to cell growth and metabolism. When 0.005 g·L-1 uracil added in the media in which yeast extract as complex nitrogen source, cell growth and HA production were increased by 32 % and 34 % respectively. From analysis of amino acid in fermentation process, it was show that arginine(Arg) was needed for cell metabolism,and concentration of free Arg maintained at 0 g·L-1 in fermentation process, which was proposed to limit cell growth and HA production. By shake-flask experiment HA concentration reached 0.510 g·L-1 whene 0.06 g·L-1 Arg added,in the fermentation with 2.5 L fermentor, when uracil 0.005 g·L-1 and Arg 0.06 g·L-1 were added, the rate of cell growth increased, maximum of specific growth rate, concentration of HA and HA molecular weight reached 0.67 h-1 ,5.2 g·L-1 and 2.15×106 Da from 0.54 h-1,4.2 g·L-1,2.0×106 Da,respectively.

Objective To study the therapeutic effect of thalidomide(Tha) on murine hepatocellular carcinoma. Methods In murine transplanted hepatoma model, thalidomide was administered intragastrically alone (200 mg/kg daily for 10 days) or in combination with doxorubicin. The antitumor activity of Tha was observed in solid and ascitic tumor models. Results Tha induced significant growth inhibition of solid hepatoma without obvious toxicity on peripheral blood cells and lymphocyte proliferation. Although Tha alone had no effect on the survival of mice with ascitic tumor, it showed a synergistic antitumor activity in combination with doxorubicin (Dox) in both solid and ascitic tumor models. Moreover, Tha reduced Dox-induced cytopenia and immunosuppression. Histological analysis of Tha-treated tumors revealed remarkably enhanced tumor necrosis and lymphocyte infiltration on the edge of tumor tissues. Conclusion Tha has definite therapeutic effect on murine hepatoma,and the combination with Dox shows an enhanced therapeutic potential.

To explore the possibility of prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a expression vector and recombinant GST-hHSS fusion protein was expressed under IPTG induction in BL-21(DE3) cells. The recombinant HSS was purified with His.Tag affinity chromatography, and its bioactivity was analyzed. The results showed that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol. The soluble GST-hHSS expression reached up to 30% of the whole soluble protein of bacteria as determined by densitometry. The cleavage of GST-hHSS fusion protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively. The molecular weight of recombinant HSS protein was identical to theoretical deduction based on the DNA sequences. The protein homology of 15 kD hHSS could be efficiently eluted out after Factor Xa cleavage. It is further indicated that the recombinant hHSS is able to proliferate hepatoma cells of BEL-7402 in the preliminary experiments.
Cell Division ; drug effects ; Gene Expression ; Glutathione Transferase ; genetics ; Growth Substances ; genetics ; isolation & purification ; pharmacology ; Humans ; Peptides ; genetics ; isolation & purification ; pharmacology ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; pharmacology ; Tumor Cells, Cultured

The heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme metabolism, has been recently defined as a novel stress-stimulated protein, since the intracellular expression of HO-1 in response to various stimuli as oxidation, ischemia and endotoxin injury has been proved to be able to protect the cells from damage. In this study, a retroviral vector containing human HO-1 gene was constructed and transfected to rat vascular smooth muscle cells (VSMCs). Using Southern and Northern blot analyses, the integration and mRNA expression of HO-1 gene in the transfected cells were confirmed. The profound protein expression of HO-1 as well as HO enzyme activity in the transfected cells increased by 1.8-fold and 2.0-fold respectively as compared with the non-transfected cells. It was found that the HO-1 transfected-VSMCs presented dominant resistance to toxicity produced by exposure to H2O2, as a significant protective effect of HO-1 marked by cell survival and LDH leakage was observed when 200, 400 and 600 micromol/L of H2O2 were used. The protection of HO-1 rapidly declined after the transfected-VSMCs were pretreated 24 h with an HO-1 specific inhibitor (ZnPP-IX). The results of this investigation suggest that the functional expression of HO-1 gene within VSMCs raises an alternative ability to protect the vascular cells against active oxygen injury.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Hydrogen Peroxide ; toxicity ; Muscle, Smooth, Vascular ; enzymology ; pathology ; physiology ; Oxidants ; toxicity ; Rats ; Rats, Inbred WKY ; Retroviridae ; genetics ; Transfection