[Influence of vitexin on the expression of inflammatory cytokines in dental pulp stem cells induced by lipopolysaccharide].

PURPOSE: To investigate the effect of vitexin (VTX) on the expression of inflammatory cytokines in human dental pulp stem cells(hDPSCs) induced by lipopolysaccharide(LPS), and to explore the underlying mechanism. METHODS: hDPSCs were isolated and cultured, and CCK-8 method was used to detect the effect of VTX on proliferation of hDPSCs. hDPSCs were randomly divided into 4 groups: blank group (without LPS and VTX)，LPS group (2 µg/mL LPS)，2 µg/mL LPS + 25 µmol/L VTX，2 µg/mL LPS + 50 µmol/L VTX. The cells of all groups were cultured for 48 h. The gene levels of IL-1ß， IL-6 and IL-8 in hDPSCs were detected by real time qPCR(RT-qPCR). The change of COX-2 and MAPKs signaling pathways were detected by Western blot. SPSS 16.0 software package was used for statistical analysis. RESULTS: When the VTX concentration was less than 200 µmol/L, the cell viability was not affected(P>0.05). VTX at 25 and 50 µmol/L significantly reduced LPS-induced expression of IL-1ß, IL-6 and IL-8 at gene levels and COX-2 at protein level (P<0.05). CONCLUSIONS: VTX significantly inhibited the activation of ERK and p38 signaling pathway. VTX can reduce LPS-induced inflammatory cytokine expression in hDPSCs via restraining the activation of ERK and p38 signaling pathway.

[Effects of lyophilized on the biological activities of lentiviral vector of bone morphogenetic protein 2].

PURPOSE: To investigate the influence of lyophilization on the biological activity of recombinant lentiviral vectors of bone morphogenetic protein 2(BMP-2). METHODS: Recombinant lenti-BMP-2 was constructed. lenti-BMP-2 was transfected with rat bone marrow stromal cells (BMSCs) by multiplicity infection (MOI) of 10, 25, 50, 100, 200. The infection efficiency was observed by X-gal staining. Under suitable conditions, the lenti-BMP-2 and 10% trehalose ratio of lyophilized protective agent was mixed into the lyophilization form. Before and after lyophilization, the effect of lenti-BMP-2 on the proliferation of BMSCs was evaluated by MTS assay. The expression of BMP-2 protein in the cells of lyophilized lenti-BMP-2 was detected by ELISA method. The expression of Runx-2, OCN, Col1 and OPN in BMSCs was detected by real-time PCR after transfection of lyophilized lenti-BMP-2. SPSS13.0 software package was used for statistical analysis. RESULTS: X-gal staining showed an MOI of 100 pfu/cell, and stable transfection efficiency. Before and after lyophilization, no significant change was observed in regard to the effect of lyophilized lenti-BMP-2 on BMSCs proliferation (P>0.05). ELISA method showed that BMSCs transfected by lyophilized lenti-BMP-2 could express BMP-2 protein continuously and stably at a high level. Before and after lyophilization, the result of real-time PCR showed that no significant difference in the expression of OPN,Col1,OCN and Runx-2 in BMSCs (P>0.05). CONCLUSIONS: Lyophilized lenti-BMP-2 with trehalose can maintain high activity for a long time as an effective and reliable storage method.

[Preparation of lyophilized lentiviral vector of bone morphogenetic protein 2].

PURPOSE: To construct recombinant lentiviral vectors of bone morphogenetic protein 2(BMP-2) gene and prepare a stable lyophilized state. METHODS: The BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and mixed with different stabilizers in an appropriate proportion and then lyophilized. The optimum stabilizer was selected according to the appearance and virus titer after being lyophilized. The quality of lyophilized product was measured by thermal stability of the virus, PCR and gene sequencing. SPSS13.0 software package was used for statistical analysis. RESULTS: Recombinant BMP-2 lentiviral vector was successfully constructed. 10% trehalose, 1% bovine serum albumin, 3%mannitol and 0.5% gelatin in group B showed good protection on BMP-2 gene lentiviral vector. The virus titer decreased 0.42 LgPFU/mL after being lyophilized, which was better than group A, C and the control group and the difference was statistically significant (P<0.05); Lyophilized protective agent made from group B still maintained a good appearance at 37 degrees centigrade after 28 days and the virus titer decreased 0.63 LgPFU/mL. The virus titer in liquid lentiviral infection control group decreased rapidly to 2.37 LgPFU/mL 1 week later and the difference between the two groups was statistically significant (P<0.05); PCR and gene sequencing showed that the target gene information after redissolve had no loss or mutation. CONCLUSIONS: Appropriate selection of lyoprotectants can effectively protect the biological stability of recombinant BMP-2 gene lentiviral vector.

[Expression and significance of DNA methyltransferase in sera of patients with lung cancer].

OBJECTIVE: To explore the protein expressions of DNMT1, DNMT3a and DNMT3b in sera of lung cancer patients. METHODS: Enzyme-linked immunosorbent assay (ELISA) method was used to measure the protein expressions of DNMT1, DNMT3a and DNMT3b in 136 lung cancer patients hospitalized at Department of Respiratory Diseases, First Affiliated Hospital, Zhengzhou University during September 2012 to June 2013. And 147 healthy controls were selected from a population of physical examination at Sixth People's Hospital of Zhengzhou. And the relationship was analyzed between protein expressions of DNMT1, DNMT3a and DNMT3b and clinic characteristics of lung cancer. RESULTS: The protein expressions of DNMT1, DNMT3a and DNMT3b in patients with lung cancer (15 ± 10, 997 ± 76 , 302 ± 25) were higher than those of the controls (13 ± 10, 344 ± 93, 108 ± 22). And there were statistical significance (t = 3.28, 62.51, 37.27; P = 0.021, 0.000, 0.000). The results of Logistic regression show that the protein expressions DNMT1, DNMT3a and DNMT3b increased morbidity for lung cancer (χ(2) = 14.811, 26.768, 12.057; P = 0.000, 0.000 0.001), especially so for DNMT1 (OR = 1.545, 95%CI: 1.238-1.928). No correlation existed between the protein expressions of DNMT1, DNMT3a, DNMT3b and histological types or stages (P > 0.05). CONCLUSIONS: The high protein expressions of serum DNMT1, DNMT3a and DNMT3b increase morbidity for lung cancer. And these markers may predict the early occurrence of lung cancer.

[Determination of cetirizine dihydrochloride by anti-fluorescence quenching on rhodamine B-sodium tetraphenylborate system].

The sodium tetraphenylboron has the fluorescence quenching effect on rhodamine B, making the fluorescence signal intensity weaken or even disappear. But cetirizine dihydrochloride has the anti-quenching effect on rhodamine B--the fluorescence signal is enhanced, and there was a linear relationship between the fluorescence signal enhancement degree and drug concentration. In the present paper, 491 and 610 nm were chosen as the excitation and emission wavelength for measurement of the fluorescence intensity difference deltaF = F1 - F0 of blank solution and test solution, and according to positive correlation between the value of deltaF and cetirizine hydrochloride concentration of test solution, a new method of anti-fluorescence quenching for the determination of cetirizine hydrochloride was established. The linear regression equation was deltaF = 0.727 5m - 2.357, correlation coefficient was 0.997 2, linear range was 3.50-129.3 mg x L(-1), the detection limit was 1.05 mg x L(-1) and RSD was 1.2%. The cetirizine hydrochloride tablets and cetirizine hydrochloride capsules from different manufacturers were determined by this method, the measured values were basically in line with the labeled amount of drugs, and the recovery rate was between 92% and 106%.

[A new immuno-nanosliver resonance scattering spectral probe for assay of IgG].

Using tri-sodium citrate as reducer, stable silver nanoparticles the size of about 20 nm were prepared by the microwave high pressure procedure with simplicity and rapidity. At pH 9.0, sliver nanoparticle was used to label goat anti-human IgG (GIgG) to obtain an immuno-nanosilver resonance scattering spectral probe (AgGIgG) for IgG. In pH 6.0 buffer solution and in the presence of polythylene glycol (PEG) and KCl, the immune reaction of IgG with AgGIgG took place, the silver nanoparticles released from AgGIgG produced aggregations, and the resonance scattering intensity at 485 nm (I(485 nm)) was enhanced greatly. The influence factors such as pH value, buffer solution volume, concentration of AgGIgG, KCl, PEG-4000, PEG-6000, PEG-10000 and PEG-20000, incubation temperature and time were considered, respectively. Under the conditions of 0.40 mL of pH 6.0 phosphate buffer solution, 1.20 mL of 9.8 microg x mL(-1) AgGIgG, 0.20 mL of 20% PEG-6000, 0.50 mL of 10% KCl, and ultrasonic irradiation for 25 min at room temperature, the increased intensity deltaI(485 nm), was proportional to the IgG concentration (c(IgG)) from 0.004 to 0.48 microg x mL(-1), with a detection limit of 2.4 ng x mL(-1). The regress equation was deltaI485 nm = 76.8c(IgG) + 4.7. The effect of foreign substances such as 20 microg x mL(-1) Ni2+, Fe2+, Pb2+ and BSA,60 microg x mL(-1) Cu2+, Ca2+ and HSA,60 microg x mL(-1) Mg2+ and Mn2+, 320 microg x mL(-1) Zn2+, glucose and urea on the deltaI(485 nm) was examined, respectively. Results showed that there was no interference. This assay showed high sensitivity and good selectivity for quantitative determination of IgG in human serum with satisfactory results. The analytical results were in agreement with that of the reference results.

Inhibition of hepatic interleukin-18 production by rosiglitazone in a rat model of nonalcoholic fatty liver disease.

AIM: To investigate the effects of rosiglitazone (RGZ) on expression of interleukin-18 (IL-18) and caspase-1 in liver of non-alcoholic fatty liver disease (NAFLD) rats. METHODS: Twenty-eight Sprague-Dawley (SD) rats were randomly divided into control, NAFLD, and RGZ treated NAFLD groups. A NAFLD rat model of NAFLD was established by feeding the animals with a high-fat diet for 12 wk. The NAFLD animals were treated with RGZ or vehicle for the last 4 wk (week 9-12) and then sacrificed to obtain liver tissues. Histological changes were analyzed with HE, oil red O and Masson's trichrome staining. Expressions of IL-18 and caspase-1 were detected using immunohistochemical staining and semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: The expression levels of both IL-18 and caspase-1 were higher in the liver of NAFLD group than in the control group. Steatosis, inflammation and fibrosis, found in the liver of NAFLD rats, were significantly improved 4 wk after RGZ treatment. The elevated hepatic IL-18 and caspase-1 expressions in NAFLD group were also significantly attenuated after RGZ treatment. CONCLUSION: RGZ treatment can ameliorate increased hepatic IL-18 production and histological changes in liver of NAFLD rats. The beneficial effects of RGZ on NAFLD may be partly due to its inhibitory effect on hepatic IL-18 production.

[Effect of boron and fluoride on the expression of enamelin in rat incisor].

OBJECTIVE: To investigate the effects of overdose fluoride, boron and two factors on the expression of enamelin in rat incisor. METHODS: 32 Wistar rats were randomly divided into 4 groups. Group I: The distilled water was given. Group II: 220 mg/L NaF were given. Group III: 382 mg/L Na2B4O2.10H2O were given. Group IV: 220 mg/L NaF and 382 mg/L Na2B4O2.10H2O were given. The rats were sacrificed in the eighth week. HE staining was used to observe the morphology of ameloblasts. Immunohistochemical staining was used for study the expression of enamelin in rat incisors. RESULTS: The results showed that the expression of enamelin was reduced in the group II (P<0.01). Compared with group I, the expression of enamelin in group IV had no significant difference. The expression of enamelin in group IV and group II had significant difference (P<0.01). CONCLUSION: The overdose fluoride can inhibit the expression of enamelin. The effection was weaken when boron added. Boron reduced the toxicity of fluoride on teeth.

[The effect of overdose fluoride on the expression of Cbfalpha1 in the ameloblasts of rat incisor].

PURPOSE: To evaluate the effect of overdose fluoride on the expression of Cbfalpha1 in the ameloblasts of rat incisor and its correlation with dental fluorosis at protein level. METHODS: Twenty Wistar rats were divided randomly into two groups, one was fed with distilled water(control group), the other was fed with distilled water of 100ppm F-(experimental group). The incisors of these rats were extracted for immunohistochemical staining eight weeks later to investigate the expression of Cbfalpha1.The data was analyzed with Axioplan 2 imaging system and SPSS10.0 software package. RESULTS: Cbfalpha1 was expressed in the nuclear of ameloblasts at secretory stage, and the expression level in the experimental group was significantly higher than that in the control group(P<0.01). CONCLUSION: Overdose fluoride causes higher expression of Cbfalpha1 in the ameloblasts at secretory stage,which may effect the mineralization and development of enamel in a certain way and leads to the abnormal development of enamel.

[Filter coated with phenyl fluorone for enrichment and determination of indium by GFAAS].

The filter fiber column coated with phenyl fluorone was extended to pre-concentrate trace amount of indium determined by GFAAS. The optimized condition for filter fiber coated with phenyl fluorone was obtained. The enrichment condition was achieved when the pregnant solution at pH 5 flowed through the filter fiber column at the rate of less than 2.0 mL x min(-1), then 8mL HNO3 of 5.00 mol x L(-1) was used as an eluent. With a general graphite tube coated with tungsten and silver as matrix modifier, the sensitivity was enhanced with GFAAS. The method was validated by the determination of trace indium in water, artificial zinc, and aluminium samples. The detection limit of the method was 0.32 ng x mL(-1), the recovery was in the range of 95.0%-101%, and the RSD was between 1.8% and 7.0%.

[Effect of fluorosis on the expression of basic fibroblast growth factor in rat incisors].

OBJECTIVE: To investigate the effects of overdose fluoride on the expression of basic fibroblast growth factor (bFGF) in rat incisors. METHODS: Twenty Wistar rats were randomly divided into 2 groups: group I (control group) distilled water was given; group II (experimental group) 100 mg/L NaF was given. The rats were killed at the end of 8 th week. Immunohistochemical staining was used to study the expression of bFGF in rat incisors. RESULTS: Immunohistochemical results demonstrated the presence of bFGF in ameloblasts, odontoblasts of rat incisors. The expression of bFGF was reduced in group II (P < 0.01). CONCLUSIONS: Overdose fluoride inhibits the expression of bFGF and affects the interaction between dental epithelium and dental mesenchyme, which leads to the enamel demineralization.

[Precipitation trapping with phenylfluorone and determination of trace gallium, germanium, molybdenum and indium by GFAAS].

The preconcentration of trace gallium, germanium, molybdenum and indium by trapping with precipitation of phenylfluorone (PF), and the determination of the elements by GFAAS were developed. The effects such as those of acidity, amounts of PF, aging time, volume of test solution, and the coexistent ions on the preconcentration of the trace elements were examined in detail. The optimum conditions of preconcentration for Ga(III) were pH approximately 2 test solution 500 mL with added 10.00 mg x mL(-1) PF (2.00 mL) and aging for 4 h, those for Ge(IV) were pH approximately 2 test solution 500 mL with added 10.00 mg x mL(-1) PF (4.00 mL) and aging for 10 h, those for Mo(V) were pH approximately 3 test solution 1 000 mL with added 10.00 mg x mL(-1) PF (3.00 mL) and aging for 6 h, and those for In(III) were pH approximately 5 test solution 100 mL with added 10.00 mg x mL(-1) PF (3.00 mL) and aging for 10 h. The experiment results showed that the main contribution to trapping trace gallium, germanium, molybdenum and indium with PF precipitation was post-precipitation instead of coprecipitation. The detection limits (3s) were 0.12 ng x mL(-1) for gallium, 0.30 ng x mL(-1) for germanium, 0.046 ng x mL(-1) for molybdenum and 2.7 ng x mL(-1) for indium. The developed methods were successfully applied to the determination of trace amount of the elements in water samples, geological standard reference materials, and zinc concentrate samples by graphite furnace atomic absorption spectrometry.