Objective To investigate the variation of immune index in patients with systemic lupus erythematosus (SLE) treated with autologous purified CD+34 cells transplantation and to clarify the relationship with pathogenesis and prognosis. Methods Flow cytometry (FCM) and enzyme linked immunosorbent assay(ELISA) were used to test lymphocyte subsets, C3, C4, CH50, autoantibodies and immunoglobulin for 18 cases of SLE before and after transplantation. Results The results showed that the ratio of all the T cell subsets reduced obviously in early postgraft and recovered gradually in 1 to 3 months after transplantation except CD45 RO+CD+4 cells. The levels of serum C3, C4, CH50 increased significantly after transplantation. No case relapsed within one year after transplantation, but 2 patients relapsed one year after transplantation. The levels of the indexes in the patients with relapse were significantly lower than those in the patients with persistent remission, including C4 in the entire course, CH50 in the 3rd and 12th month after transplantation and CD45 RA+ CD+8 cells in the 6th month after transplantation. However, the ratio of CD45 RO+ CD+4 cells in the first month after transplantation in the patients with relapse was higher than that in the patients with persistent remission. Conclusion Autologous purified CD+34 cells transplantation is effective for treating SLE. Survey of immune indexes before and after transplantation is important to investigate the pathogenesis of SLE. Moreover, these immune indexes can be used to predict therapeutic efficacy of SLE.

Objective To identify the polyreactivity of a monoclonal natural anti-keratin autoantibody 3B4 and to analyze its possible molecular me chanism.Methods enzyme-linked immunosorbent assay (ELISA)and immunohistoche mistry were applied to test the binding reactivity of 3B4 against different anti gens and tissues.The variable region genes and their amino acid composition wer e sequenced.Results 3B4 could reacted with a range of antigens and tissues,i n addition to keratin and skin.The variable region genes of its light chain and heavy chain showed high homology with germline genes VK1 am4 and VH1 J558.42.H CDR3 region,which mainly composed of short side chain amino acids(from 294 to 324 nucleotides around the heavy chain),was the only motif that differs from ot her highly homologous immunoglobulin genes.Conclusions The monoclonal natural anti-keratin autoantibody 3B4,with its variable region genes highly homologo us to germline genes,is highly polyreactive.The flexibility of HCDR3 may contr ibute to the polyreactivity.

Background Reasearches showed that α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation and ameliorates the ocular surface abnormalities in a scopolamine-induced dry eye rat model,and the managing effect of sodium carboxymethylcellulose (CMC) on dry eyes also has been determined.However,whether α-MSH can enhance the therapeutic effects of CMC remains to be investigated.Objective This study was to investigate the protective effects of α-MSH combined with CMC on ocular surface in a scopolamine-induced dry eye rat model.Methods Sixty clean female Wistar rats were randomly divided into normal control group,model control group,NaCl group,CMC group,α-MSH group and α-MSH+CMC group,and 10 rats for each group.The dry eye models were established by subcutaneous injection of scopolamine hydrobromide at 9:00,12:00,15:00 and 18:00 per day for 28 days.0.9％ NaCl solution,1×10 3 mg/ml α-MSH solution,0.5％ CMC eye drop,and 1 ×10-3 mg/ml α-MSH+0.5％ CMC solution were topically administered twice a day (8:00,17:00) since the initial day of modeling according to grouping.Shirmer Ⅰ test (S Ⅰ t),breakup time of tear film (BUT) and corneal fluorescence staining were performed before and 7,14,21,28 days after the application of drugs.At 28 days following the administration of drugs,the eyeballs of the rats were collected.Hemotoxylin and eosin staining was employed to examine the morphology of corneas,and periodic acid schiff (PAS) staining was used to count the conjunctival goblet cells.This study protocol was approved by Experimental Animal Ethic Committee of Tianjin Medical University (SYXK 2009-0001),and the use and care of the rats complied with ARVO Statement.Results The S Ⅰ t and BUT values were significantly reduced,and the corneal fluorescence staining scores were significantly increased over time following modeling in the model control group (all at P＜0.01).No significant differences were found in the S Ⅰ t,BUT and corneal fluorescence staining scores between model control group and NaCl group at various time points (all at P＞0.05).At 7,14 and 21 days after intervention,the S Ⅰ t values were (4.800±0.789),(4.100±0.516) and (4.300±0.856) mm in the α-MSH+CMC group,which were considerably higher than (2.875 ±0.719),(2.375 ±0.619) and (2.532±0.957)mm in the NaCl group (all at P＜0.01).At 7 days after intervention,the BUT values were (4.938± 1.843) seconds and (5.000±1.491) seconds in the α-MSH group and α-MSH+CMC group,which were significantly higher than (3.250±1.000) seconds in the NaCl group (both at P＜0.01).The corneal fluorescence staining scores in the CMC group,α-MSH group and α-MSH+CMC group were significantly lower than that in the NaCl group,with the lowest score in the α-MSH +CMC group (all at P＜0.05).The thickening of corneal epithelial layer,corneal edema and arrangement disorder of corneal stroma were found in the model control group and NaCl group;while slight corneal edema and epithelial cell proliferation were exhibited in the α-MSH+CMC group by hemotoxylin and eosin staining.PAS staining showed that the number of goblet cells was much more in the CMC group,α-MSH group and α-MSH+ CMC group than that in the model control group and NaCl group (all at P ＜ 0.01).Conclusions The sole application of α-MSH or CMC alleviates ocular surface damage and morphological abnormality to certain extent,and the combination of α-MSH and CMC generates more effective protection in comparison with sole administration of α-MSH or CMC.The early application of the drugs plays an improvement role in tear secretion and tear film stability in dry eyes.

AIMUnderstanding the modes and activities of metal bacterial chlorophylls as PHD sensitizers with DNA.

METHODSThe modes and activities of the interaction of DNA and metal complexes of bacterial chlorophyll, which have been prepared by extraction and synthesis reaction, have been discussed according to the ultraviolet-visual spectrum and nucleic acid electrophoresis.

RESULTSIt indicates that the system of DNA and metal complexes have enchanced the interaction by the ultraviolet-visual spectrum. At the same time, it also indicates that metal complexes of bacterial chlorophyll and DNA have different combining way and have strong cutting effect in illumination by the nucleic acid electrophoresis.

CONCLUSIONThis paper proved that metal bacterial chlorophylls as PHD sensitizers have great advantage.

Bacteriochlorophylls ; chemical synthesis ; chemistry ; pharmacology ; Copper ; chemistry ; DNA ; metabolism ; Electrophoresis ; HL-60 Cells ; Humans ; K562 Cells ; Nickel ; chemistry ; Organometallic Compounds ; chemical synthesis ; chemistry ; pharmacology ; Protein Binding ; Spectrophotometry, Ultraviolet ; Zinc ; chemistry

AIMTo find a new PDT sensitizer.

METHODSThere were four complexes (Cu, Zn, Co, Ni) synthesized through reaction of metal and deprivating-Mg bacteriochlorophyll in the organic solvent. Their antitumor action was detected by MTT.

RESULTSThe ultraviolet-visual spectrum and the fluorescence spectrum of these complexes showed that synthesis of these four complexes was succeeded. And these metal complexes have potent antitumor action on two kinds of leukaemic cells.

CONCLUSIONMetal bacteriochlorophylls as PDT sensitizers have very good properties and this is a way to develop new PDT sensitizers.

Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Bacteriochlorophylls ; chemistry ; Cell Proliferation ; drug effects ; Cobalt ; chemistry ; Copper ; chemistry ; HL-60 Cells ; cytology ; Humans ; K562 Cells ; cytology ; Metals, Heavy ; chemistry ; Nickel ; chemistry ; Organometallic Compounds ; chemical synthesis ; chemistry ; pharmacology ; Photosensitizing Agents ; chemical synthesis ; chemistry ; pharmacology ; Zinc ; chemistry

OBJECTIVEIsobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrometry were used to screen differentially expressed plasma proteins in cold stress rats.

METHODSThirty health SPF Wistar rats were randomly divided into cold stress group A and control group B, then A and B were randomly divided into 3 groups (n = 5): A1, A2, A3 and B1, B2, B3. The temperature of room raising was (24.0 +/- 0.1) degrees C, and the cold stress temperature was (4.0 +/- 0.1) degrees C. The rats were treated with different temperatures until 12 h. The abdominal aortic blood was collected with heparin anticoagulation suction tube. Then, the plasma was separated for protein extraction, quantitative, enzymolysis, iTHAQ labeling, scx fractionation and mass spectrometry analysis.

RESULTSTotally, 1085 proteins were identified in the test, 39 differentially expressed proteins were screened, including 29 up-regulated proteins and 10 down-regulated proteins. Three important differentially expressed proteins related to cold stress were screened by bioinfonnatics analysis (Minor histocompatihility protein HA-1, Has-related protein Rap-1b, Integrin beta-1).

CONCLUSIONIn the experiment, the differentially expressed plasma proteins were successfully screened in cold stress rats. iTRAQ technology provided a good platform to screen protein diaguostic markers on cold stress rats, and laid a good foundation for further. study on animal cold stress mechanism.

Animals ; Blood Proteins ; chemistry ; Cold Temperature ; Mass Spectrometry ; Rats ; Rats, Wistar ; Stress, Physiological

OBJECTIVETo establish the patent ductus arteriosus model in preterm rats using the improved natural development method.

METHODSIn the light of the flaws of the natural development method in establishing the patent ductus arteriosus model, the experimental technology was modified to avoid the influence of fixation, dehydration, and section method on blood vessel diameter. Cesarean section was performed for a Wistar rat pregnant for 19 days, and 8 neonatal rats were obtained. After they were sacrificed by dislocation, they were embedded as a whole to avoid dehydration, and the microsection and horizontal section were made. After hematoxylin and eosin staining, a microscope was used to measure the inner diameters of the ductus arteriosus, the main pulmonary artery, and the descending aorta.

RESULTSAfter the cesarean section for the rat pregnant for 19 days, patent ductus arteriosus occurred in all the 8 neonatal rats. The measurements of the inner diameters of blood vessels were as follows: the long diameter and short diameter of the descending aorta were 354±106 and 182±140 μm, respectively; the short diameter of the ductus arteriosus was 155±122 μm, and its area was 36,847±42,582 μm(2); the long axis and short axis of the main pulmonary artery were 589±150 and 174±170 μm, respectively.

CONCLUSIONSThe improved natural development method can help to successfully establish the patent ductus arteriosus model in preterm rats.

Animals ; Animals, Newborn ; Disease Models, Animal ; Ductus Arteriosus, Patent ; etiology ; Female ; Male ; Rats ; Rats, Wistar

Abstract?AIM: To investigate mechanism of bradykinin ( BK) on inflammations of retinal pigment epithelium ( RPE) cells.?METHODS: ARPE -19 cells were cultured in vitro, stimulated by 100nM BK for 24h. Cell morphology changes were observed by microscope, and BK receptor localization was detected through cell immunofluorescence. Changes of Ca2+in BK and BR antagonist stimuli were detected by laser scanning confocal microscopy.The expressions of COX-1, COX-2, eNOS and iNOS protein in control group and BK group were detected by Western Blot.?RESULTS: After the stimulation of BK, there was no significant changes of ARPE-19 cells in morphology.Kinin B1 receptors ( B1R ) and B2 receptors ( B2R ) could be detected in ARPE-19 cells.Compared with control group, Ca2+concentrations significantly increased in BK group; in B1R antagonist group and B2R antagonist group Ca2+concentrations increased less than BK group; B1R and B2R antagonist group showed no obvious changes in Ca2+concentrations.Compared with control group, COX-2 and iNOS protein concentrations were significantly increased in BK group (P<0.001).?CONCLUSION:BK induces the increasing expression of COX-2 and iNOS in the cultured ARPE cells through binding with either B1R or B2R.

Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

A full-length cDNA of GGPPS gene from Tripterygium wilfordii suspension cells was obtained by use of RACE strategy (GeneBank: KM978333), and then analyzed by bioinformatics approaches. TwGGPPS cDNA has 1857 nucleotides and an open reading frame (ORF) encoding a protein of 514 amino acid residues. The deduced protein has isoelectric point (pI) of 7.85, a calculated molecular weight about 57.13 kD, 5 conserved domains and 2 functional domains. PSORT Prediction showed it was located at plasma membrane. Phylogenetic analysis demonstrated that TwGGPPS1 was similar to GGPPS from other species of plants. For the first time the cloning of geranylgeranyl diphosphate synthase gene from T. wilfordii was reported, it lays the foundation for further research of diterpenoids biosynthetic pathway.
Amino Acid Sequence ; Cloning, Molecular ; Farnesyltranstransferase ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Tripterygium ; chemistry ; enzymology ; genetics