Objective To observe the influence of coal burning fluorosis on learning and memory ability in rats and reveal its possible mechanisms. Methods Healthy 48 SD rats were divided into control, low-fluoride and high-fluoride group. All rats in fluoride exposed groups were fed with the eom polluted by drying processes with burning coal containing high level of fluoride obtained from the endemic fluorosis area to produce the animal model of fluorosis. The experiment period were 3,6 mouths, respectively. The ability of leaning and memory was measured by Morris test and cholinesterase activity detected by photometric method at 3 or 6 month after experiment, respectively. Results Fluoride contents signifieantlly influenced the escape latency, the numbers of crossing the platforms and the time of staying the platforms(the value of F was 29.29,6.47,6.50, respectively, P<0.01).In addition, the numbers of crossing the platforms and the time of staying the platforms were influenced by the exposed time(the value of F was 16.11,45.59, P<0.01). Furthermore, the fluoride contents and the exposed time had an interaction between the numbers of crossing the platforms and the time of staying the platforms (the value of F was 4.67,5.68, P<0.05 or<0.01). Three months after the experiment, the mean values of escape latency [(14.71± 4.85)s] of rats in highly fluoride exposed group were significantly prolonged as compared with controls [(9.28±4.22)s]; 6 month after the experiment, the mean values of escape latency[(12.42±8.03)s, (17.48± 8.05)s] of rats in both groups exposed to fluoride were significantly prolonged as compared to controls [(7.04± 3.29)s, P<0.05]. The decreased numbers of crossing the platforms[(1.62±0.87)number] and the declined time of staying the platforms[(16.70±5.02)s] were found in the rats exposed to high fluoride as compared to controls [(3.53±1.67 )number, (23.33±5.35)s, P<0.05]. The fluoride contents obviously influenced the activities of acetylcholinesterase and butylcolinesterase (the value of F was 12.83,13.27, P<0.01). On the other hand, the times of breeding also influnced the activities of butylcolinesterase (the value of F was 16.26, P<0.01). In 3 months of the experiment, the activities of butylcolinesterase [(0.55±0.12)kU/g] in low fluoride exposed group were significantly decreased in comparison with controls[(0.73±0.10)kU/g, P<0.05]. The activities of acetylcholinesterase[(0.62±0.42)kU/g] and butylcolioesterase[(0.58±0.10)kU/g] in high fluoride group were significantly decreased as compared to eontrois[(1.41±0.52), (0.73±0.10)kU/g, P<0.05]. The correlation analysis showed that there was a negative correlation between the cholinesterase and the escape latency(r=-0.68, P< 0.01), and a positive correlation between the cholinesterase and the time of staying the platforms(r=0.57, P< 0.01). Conclusions The ability of learning and memory in rats with coal buring fluorosis was decreased, which might be connected to the decreased activity of cholinesterase in a dose-effect correlation.

Objective To construct lentivirus containing CXCR4 gene and transfect MCF-7 cells , and obtain CXCR4 high-expressing MCF-7 cells. Methods CXCR4 gene was amplified by RT-PCR to construct CXCR4/pSin-EF2, which was transfected into HEK293T cells with psPAX2 and pMD2G vector for lentivirus packing. Packaged lentivirus was used to transfect human breast cancer cells MCF-7, with empty lentivirus as control. CXCR4 mRNA and protein expression levels were detected by RT-PCR and Western blot before and after transfection. And flow cytometry was used to detecte cell surface CXCR4 expression. Results The recombinant plasmid CXCR4/pSin-EF2 was constructed successfully,identified by double digestion and sequencing, and transfected into HEK293T cells to obtain high-titer lentivirus. RT-PCR and Western blot confirmed that the expression of CXCR4 in MCF-7 cells increased significantly after CXCR4 lentivirus transfection. Flow cytometry results showed that the CXCR4 positive rate increased from 26.78% to 99.29%, while there is no significant difference in CXCR4 expression between vector-transfected MCF-7 cells and non-transfected MCF-7 cells. Conclusion CXCR4 lentivirus and the breast cancer cell line with high and stable expression of CXCR4 (MCF-7CXCR4) were successfully constructed.

Objective To investigate the expression of extraeellular signal-regulated protein kinase (ERK1/2)pathway in rat brains with fluorosis and the effects of fluoride on learning and memory of the rats,and to reveal the mechanisms of damaged nervous system resulted from the toxicity of the ion.Methods Seventy-two SD rats were divided into 3 groups and 24 rats were in each group.Three groups were fed respectively with different concentrations of fluoride(NaF)for 6 months to establish rat models with fluorosis.Controls were fed with tap water (NaF＜0.5 mg/L):lower and higher concentration group were fed with water containing NaF(5,50 ms/L).Animals are sacrificed after 6 months of treatment with fluoride and the dissected brains were kept for analysis.The protein levels of ERK1/2 in rat brains were detected by Western-blotting and the mRNA level by RT-PCR. The spatial learning and memorizing ability was measured by Morris water maze test. Results The ERK1/2 protein in control group,lower and higher concentration group was 0.944±0.10,1.253±0.02,1.953±0.07,the differece being statistieally sighificant between any two groups (P ＜ 0.05). The phospho-ERKl/2 protein in control group,lower and higher concentration group was 0.73±0.08,0.77±0.07,1.28±0.11,the differece being statistieally sighificant between any two groups(P ＜ 0.05);the activation rate of phospho-ERK1/2 in lower and higher concentration group [(68.4± 3.8)%,(64.1±3.2)%] was decreased compared to control group[ (82.3±10.7)%],the differece being significant(P ＜ 0.05). In the navigation trial,longer escape latencies of lower concentration group on the second, the third,the fifth and the sixth day were observed[ (46.0±8.0),(24.0±2.7),(8.9±5.3),(7.4±4.1 )s] compared to the control[ (39.3±6.9),(19.1±9.1 ),(8.3±3.4),(4.8±2.7)s],the differece being significant (P ＜ 0.05 or ＜ 0.01 );the similar results were also observed in the higher concentration group[ (36.9±16.8),(37.7±12.9), (19.7±7.6),(12.2±5.7 )s],and the escape latencies of the higher concentration group on the third,the fifth and the sixth day were longer than that in lower concentration group. In the probe test,the rats took more time to reach the first cross in lower and higher concentration group[(1.17±0.75),(4.18±1.10)s] than control group[ (5.89± 0.56 ) s ],the differece being significant (P ＜ 0.05 or ＜ 0.01 ) ;stayed shorter [ ( 17.05±4.25 ),(18.20±4.57 ) s ] than control [(25.37±5.65 )s ] in platform area (P ＜ 0.01 );the activation rates of ERK1/2 were directly correlated with the time taken to reach the first cross platform located in the probe test(r = 0.364,P ＜ 0.05) and the activation rates were also directly correlated with the escape latencies on the sixth day(r = 0.497,P ＜ 0.05). Conclusion Long-term exposure of excessive fluoride induces the change of expression and activating rate of the ERK1/2 in rat brains,leading to the decreased capacity of learning and memory.

Objective To investigate the effects of sepia on stem cells,granulocyte and monocyte progentior cells and peripheral WBC in mice.Methods Different dosages of sepia were given to normal and model mice with hematopoietic system impairment respectively.The numbers of CFU-S,CFU-GM and peripheral WBC in normal and model mice were measured respectively with the method of hematopoietic progenitor cells cultured in vitro and the technique of experimental hematology.Results Sepia could enhance the number of CFU-S,CFU-GM and peripheral WBC in normal mice significantly,resist the decrease of CFU-S,CFU-GM,and peripheral WBC in model mice of hemapoiecsis impaired effectively and promote the restoration of those indices mentioned above in model mice significantly.Conclusion Sepia has significant effects on stimulating granulopoiesis in bone marrow in mice.The mechanism may be related to regulating immunological function and inducement GM-CSF and other sorts of cellular factors,which in turn promote the multiplication differentiation of CFU-S and CFU-GM.

Objective To evaluate the roles of magnetic resonance imaging and proton magnetic resonance spectroscopy(1H-MRS) in the diagnosis of meningiomas. Methods 98 patients with meningiomas underwent conventional pre-contrast MR and contrast MR. Among them, 28 cases had two dimensional single voxel or multi voxel 1 H-MRS simultaneously both in the lesion's region and the contralateral side. Results On precontrast MR images of 98 cases, T1 WI showed 58.1% (61/105) isointensities, 31.4% (33/105) faintly low intensities and 10. 5% (11/105) mixed intensities; T2WI showed 40. 0% (42/105) isointensities, 41.0%(43/105) hyperintensities, 10.5% (11/105) faintly low intensities and 8.5% (9/105) mixed intensities. After administration of Gd-DTPA, the solid part of the tumors exhibited various enhancement in all the 98 cases.28 cases of MRS exhibited specific different spectral peaks, including increased of choline-containing compounds(Cho), absent or decreased of acetylaspartate(NAA), and the unchanged of creatine(Cr). The value of NAA, Cr, Cho, NAA/Cr, Cho/Cr, NAA/Cho in the tumor center of meningioma were 0. 09 ± 0.06,0.31 ± 0. 22, 0.46 ± 0. 16, 0.33 ± 0. 42, 1.50 ± 0. 68, 0. 15 ± 0.08, compared with the contralateral normal region, Cr has no significant difference (P ＞ 0. 05), NAA, Cho, NAA/Cr, Cho/Cr, NAA/Cho had significantly differences(P ＜ 0.05). Conclusion Conventional pre-contrast MR and contrast MR is the most important dignostic means for meningiomas, 1H-MRS combined with MRI can improve the diagnostic accuracy of meningiomas.

Objective To investigate the changes of oxidative stress level in brain tissues and serum, and learning and memory in rats with oxidative stress level in nerve damage in chronic fluorosis. Methods The rats were randomly divided into 3 groups according to the body weight, eight rats in each group, i.e., control group, drinking water containing less than 0.5 mg/L of fluoride; lower fluoride exposure group, drinking water containing 5 mg/L of fluoride; higher fluoride exposure group, drinking water containing 50 mg/L of fluoride. The animals were examined six months after initiating the experiment. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA), as well as learning and memory, were measured. Results Escape latency in higher fluoride exposed group[ (14.37±3.48)s] was significantly higher than that of controls[ (5.84±1.87)s] and exposed te lower fluoride [ (7.18±1.42)s], the difference being statistically signifieant(P<0.05). As compared with controls[ (2.17±0.11)× 103 U/L , (0.79±0.11)×103 U/g Pr] ,the rats exposed to higher fluoride and lower fluoride exhibited lower levels of T-AOC [(1.37±0.27)×103 U/L,(0.24±0.06)×103 U/g Prand (1.20±0.14) x 103 U/L,(0.41 ~ 0.10)×103 U/g Pr], the difference being statistically signifieant(P<0.05). As compared with controls[ (2.34±0.16) mmoL/L, (2.97±0.11)mmol/g Pr] and low fluoride exposed group[ (2.68±0.33)mmoL/L, (3.38±0.21)mmol/g Pr], higher level of MDA were observed in higher fluoride exposed group[ (3.72±0.59)retool/L, (4.01±0.21)mmol/g Pr], the difference being statistically significant(P<0.05). Conclusion The results indicated that higher amount of fluoride induced an increased level of oxidation, which might result in the decreased capacity of intelligence of rats with fluorosis.

OBJECTIVETo construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.

METHODSWe firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.

RESULTSOur results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.

CONCLUSIONOur protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Histones ; genetics ; Humans ; Polymerase Chain Reaction ; methods

OBJECTIVETo investigate the effects of Newcastle disease virus (NDV) infection on the expression of survivin and cell cycle in human tongue squamous carcinoma TSCCa cells.

METHODSThe proliferation of TSCCa cells infected with NDV in vitro was evaluated by means of MTT assay, and survivin expression in the infected cells was detected using RT-PCR and Western blotting. Flow cytometry was performed to assess the changes in the cell apoptosis, cell cycle and cell proliferation index (PI) of the cells.

RESULTSNDV infection resulted in decreased survivin expression and increased apoptosis of TSCCa cells, with reduced cell percentage in G2/M and S phases and lowered PI of the cells, showing significant differences from those of the negative control cells (P<0.05).

CONCLUSIONNDV infection can inhibit survivin expression, affect the cell cycle of TSCCa cells and induce their apoptosis.

Apoptosis ; physiology ; Blotting, Western ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cell Cycle ; physiology ; Cell Line, Tumor ; Host-Pathogen Interactions ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Newcastle disease virus ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Tongue Neoplasms ; metabolism ; pathology ; virology

Adult ; Anti-HIV Agents ; therapeutic use ; Familial Primary Pulmonary Hypertension ; HIV ; pathogenicity ; Hepacivirus ; pathogenicity ; Hepatitis B virus ; pathogenicity ; Humans ; Hypertension, Pulmonary ; drug therapy ; virology ; Male ; Sulfonamides ; therapeutic use