Objective To construct the wt-p53's eukaryotic expression vector pE6/p53/GFP that was controlled by the radiation induced promoter and research its functions.Methods Radiation response element E6 was synthesized by gene synthesis.The wt-p53 cDNA sequence was prepared from pcDNA3.1(+)/p53 plasmid by PCR.IRES2-EGFP report gene segment was prepared from double cistron expression vector IRES2-EGFP by enzyme digestion.After sequenced and identified,the recombinant plasmid was transformed into H1299(p53-/-)cell with Lipofectamine 2000,and the cell lines in stable expression was screened by G418.In the H1299(p53-/-)cell transfected with the recombinant plasmid or without,wt-p53 mRNA expression was analyzed by RT-PCR,the p53 expression by Western blot when exposed to 4 Gy 8 MV X ray for 0,3,8,12,24,36 h or when exposed to 0,1,2,4,8 Gy 8 MV X ray for 12 h.The cell cycle of H1299(p53-/-)cell transfected stably with the recombinant vector was analyzed by flow cytometry after exposed to 4 Gy 8 MV X ray.Results The recombinant pE6-p53/GFP plasmid had been constructed correctly and the expression of p53 gene in the transfected H1299 cell lines had been determined.After 4 Gy X ray radiation,the expression of wt-p53 protein had a significant rise.The transfected H1299 cell lines stopped in G1 stage after radiation and their cloning efficiency decreased notably.Conclusion We had constructed successfully the recombinant pE6/p53/GFP plasmid that was regulated by radiation induced response element E6.This provides experimental data for radiation-gene therapy of non-small lung cancer.

Objective To study the effects of irradiation-induced wt-p53 positive feedback circuit on cell life cycle,apoptosis rate and irradiation sensitivity of adenocarcinoma of lung in vitro.Methods The therapeutic group pE6R4-p53-EGFP/H1299 cells and the other 3 control groups H1299(p53-/-) ,pE6-p53EGFP/H1299 and pR4-p53-EGFP/H1299 cells were irradiated with 8-Mev electron beam generated by a linear accelerator at a dose rate of 400 cGy/min and a source-skin distance(SSD) of 100 cm,12 h exposure to irradiation.The cell life cycle and the rate of apoptosis were analyzed by FCM.Mean lethal dose(Do) and sensitive enhancement ratio(SER) index were calculated from the irradiation dose-survival curve.Results In 12 h post 4-Gy 8MV-X-ray irradiation,FCM analysis showed that most cells in therapeutic group were arrested at G0/G1 stage(75.13?1.42) %,compared with the 3 control groups(38.47?0.87) %,(62.57? 0.76) %and(51.23?2.41) %,respectively.After irradiation,the cell apoptotic rate in each group was higher than that in the cells without irradiation.The apoptotic rate in the therapeutic group was(23.73? 0.21) %,which was 5.69,1.51 and 2.57 folds respectively higher in the 3 control groups(4.17?0.12) %,(15.67?0.32) %and(9.23?0.15) %.Do values were 0.91,1.073 and 1.413 Gy respectively in pE6R4-p53-EGFP/H1299,pE6-p53-EGFP/H1299 and H1299 cells.The SER,derived from Do values,was 2.63 and 1.34,respectively.Conclusion Irradiation up-regulates wt-p53 gene expression,regulates cell cycle and induces cell apoptosis.Thus this positive feedback circuit increases the sensitivity of lung adenocarcinoma to irradiation.

Objective To explore the safety and efficacy of resistance training (RT) and aerobic exercise (AT) for low back pain during pregnancy. Methods From June to October, 2015, 34 women gestated 20-23 weeks with low back pain accepted resistance training and aero-bic gymnastics exercises twice a week for 12 weeks. They were measured with blood pressure and body mass index (BMI), and assessed with Visual Analogue Scale (VAS) of pain, Self-rating Anxiety Scale (SAS) and Oswestry Disability Index (ODI) before and after treatment. They were investigated the injury and symptoms with questionnaire. Results No musculoskeletal injuries and groin bleeding occurred. Symptoms, such as dizziness and pelvic pain were infrequent (1.5%). The BMI increased (t=-5.791, P<0.001) and the blood pressure did not obviously change (t=1.441, P=0.159). The muscle strength (t=-5.081, P<0.001) and endurance (t=-5.019, P<0.001) increased, and the scores of VAS (t=-5.179, P<0.001), ODI (t=-5.206, P<0.001) and SAS (t=-5.024, P<0.001) significantly improved after treatment. Con-clusion The resistance training combined with aerobic training program during pregnancy is safe and efficacious for pregnant women with low back pain.

Objective To clone mcp1, mcp2 and mop3 genes that encoding methyl-accepting chemotaxis proteins(MCP) of Campy/obacter jejuni for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of the microbe for determining chemotaxis-inducing substances and to understand relationship among MCPs and chemotactic inducers. Methods The segments of mep1, mcp2 and mcp3 genes were amplified by PCR and then sequenced after T-A cloning. Prokaryofic expression systems of the genes were subsequently constructed. SDS-PAGE pins Bin-Rod Gel Image Analyzer were used to examine the expression of target recombinant proteins rMCP1, rMCP2 and rMCP3, and Ni-NTA affinity chromatography was performed to purify the rMCPs. Rabbits were immunized with each of the three rMCPs to obtain antisera. Immunodiffusion assay was performed to measure the titers of antisera. IgG in each of the antisera were extracted by ammonium sulfate precipitation and DEAE-32 ion exchange chromatography, and IgG F(ab')2 were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography. Chemotactic model in vitro of C. jejuni based on HAP( hard-agar plus) method was established to determine the chemo-taxis-inducing effect of eight candidate substances. Chemotaxis inhibition test based on IgG F(ab')2 bloc-king was applied to determine the function and diversity of MCPs. Results The segments with expected si-zes amplified from mcp1, racp2 and mcp3 genes were obtained by PCRs, and their nucleotide and putative amino acid sequences were completely same as the reported. The constructed prokaryotic systems could effi-ciently express rMCPs with the yields about 10% of the total bacterial proteins. Immunization with rMCP1, MCP2 and rMCP3 enables the rabbits to produce specific antibody. All the antisera had 1: 4 immunodiffusion titers. Both bovine bile and sodium deoxycholate(DOC) were able to induce ehemotactie movement of C. je-juni in a dosage-dependent manner ( P < 0.05 ). When MCP1 and MCP2 were blocked with their IgG F(ab')2, the ehemotaetic ability of C. jejurd were remarkably decreased (P <0. 05). However, MCP3 blocking did not affect the chemotaxis ( P > 0.05 ). Conclusion The C. jejuni MCPs are successfully ex-pressed in this study. Bovine bile and DOC are the inducers for chemotaxis of C. jejuni, and MCP1 and MCP2 are involved in the process of ehemotaxis iadueed by DOC.

Objective To clone fliH, fliⅠ, fliY and fliN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the loca- tions of Flirt, FliⅠ, FIiY and FIiN. Methods The fliH, fliⅠ, fliY andfliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotie expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression prod- ucts. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain anti- sera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH, FliⅠ, FliY and FIiN. Results Segments of fliH, fliⅠ, fliY andfliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and puta- tive amino acid sequences from the four genes were 100% compared with the reported sequences. The con- structed prokaryotic systems efficiently expressed rFliH, rFliⅠ, rFliY and rFliN with the outputs of approxi- mate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliⅠ, rFliY or rFliN could pro- duce antibody. The antisera had the titers above 1:100 000, and could recognize the corresponding recombi- nant proteins and membrane proteins of L interrogans to display positive Western hybridization bands. Flirt, FliⅠ, FliY and FliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The pro- karyotic expression systems was successfully constructed in this study, which could efficiently express flagel-lum-associated proteins FliH, FliⅠ, FliY and FliN of L. interrogans. The antisera with high titers to recognize their protein antigens were also obtained. Flagellum-associated proteins Flirt, FliⅠ, FIiY and FIiN are the inner envelope proteins and/or outer envelope proteins of L. interrogans.

Objective To screen the efficient antigenic epitopes in genus-specific envelope proteins OmpL1 and LipL21 of Leptospira interrogans for further development of multiple antigenic peptide (MAP)vaccine.Methods Based on bioinformatic technique,the combined epitopes of T and B lymphcytes in OmpL1 and LipL21 molecules were screened.Nucleotide fragments of each epitopes were amplified by PCR and then constructed their phage display systems.Using antisera against rOmpL1,rLipL21,L.interrogans serogroup Icterohaemorrhagiae strain Lai and leptospirosis patients' sera as the first antibodies.respectively,Western blot assays were performed to determine the immunoreaetivity and reactive ability of the epitopes with different antisera.Resuits Four combined epitopes of OmpL1 and two combined epitopes of LipL21 were selected out by the predicting procedure.All the amplified epitope fragments were accurately inserted into the region at N end of phage PⅢ protein and successfully expressed.All of the antisera could recognize each of the epitopes.Based on the results of Western blot,the two LipL21 epitopes at 97-112 and 176-184 showed similar strong hybridization signals with any of the antisera,and the hybridization signals of four OmpL1 epitopes with the three antisera were 173-191,87-98,297-320 and 59-78,from strong to weak.Conclusion The six combined epitopes in this study are efficiently antigenic.And the epitopes at positions 97-112 and 176-184 in LipL21 as well as the epitopes at position 87-98 and 173-191 in OmpL1 have a potential for developing leptospiral MAP vaccine.

Objective To determine the effect of Mycobacterium tuberculosis spp. inducing transformation of THP-1 cells to epithelioid cells (EC) and the involved signaling pathways and their regulations. Methods THP-1 cells infection models respectively infected with M. tuberculosis strains H37Rv, bovis and phlei were established. Indirect immunofluorescent staining assays were used to detect the expressions of monocyte/macrophage differentiation antigen CD115 and EC differentiation antigen CD82 of the THP-1 cells before or after infection. By Sandwich ELISA Kits, the phosphorylation levels of p38MAPK, Akt1 and STAT3 of the THP-1 cells before or after infection were measured. The alterations of CD115 and CD82 expression levels were examined when the associated signaling pathways were blocked with specific blocking agents. Results CD115 expression was weakened and CD82 expression was strongly increased in all the THP-1 cells infected with the three strains. A temporal up-regulation of the p38MAPK phoshporylation level but no obvious alteration of Akt and STAT3 phosphorylation levels after THP-1 cells infected by strain H37Rv or bovis. The THP-1 cells infected with anyone of the three strains continuously expressed CD115 after MAPK, PI3K/Akt or JAK/STAT of the cells was blocked. Although JAK/STAT was blocked, the THP-1 cells respectively infected with the three different strains still expressed CD82. However, CD82 expressed in THP-1 cells infected by the strain H37Rv or bovis was disappeared when p38MAPK and PI3K/Akt pathways of the cells were blocked. Conclusion Strain H37Rv and bovis can induce the infected THP-1 cells transforming to EC and p38MAPK and PI3K/Akt signaling pathways participate and regulate the transformation procedure. Of the two signaling pathways p38MAPK seems to be more important.

gluconate in both G1 phase and S phase cells.The permeability of G1 phase cells to I-was higher than that in S phase cells,but to gluconate was lower than that in S phase cells.CONCLUSIONS: The density of the volume-activated Cl-current,the anion permeability of the channel and the sensitivity of the current to tamoxifen were different between the CNE-2Z cells in G1 phase and those in S phase.The results suggest that the expression of tamoxifen-sensitive,volume-activated chloride channels is differentiated at different stages of the cell cycle.

AIM: The roles of Cl-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 ?mol/L arrested cells in G 1 phase (G 1 population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G 1 phase. The results suggest that activation of Cl-channels and RVD is necessary for facilitating cells to proceed to the S phase from G 1 phase and maintaining cell proliferation.

AIM:To investigate the type of chloride channel activated by cisplatin in poorly differentiated na -sopharyngeal carcinoma cells (CNE-2Z cells).METHODS:The technique of whole-cell patch-clamp was used to investi-gate the role of Ca 2+in the activation of cisplatin-activated chloride currents and to analyze the effect of hypertonic stress on these currents in CNE-2Z cells.RESULTS:Chloride currents were induced when the cells were exposed to the calcium -free cisplatin solution , showing the similar density to the currents induced by cisplatin with the presence of extracellular cal -cium.However , the latency and the peak time of cisplatin-activated currents in the absence of extracellular calcium were prolonged.The activation of cisplatin-activated chloride currents was insensitive to the depletion of intra-and extracellular calcium.Calcium channel antagonist nifedipine had no effect on the cisplatin -activated chloride currents , while hypertonic solution completely inhibited those currents .CONCLUSION:The cisplatin-activated chloride currents are independent on intra/extracellular calcium .The chloride channels activated by cisplatin are not calcium-activated chloride channels , but are probably volume-sensitive chloride channels .