Objective:To investigate the peripheral mechanism by studying the histological changes of masseter muscles using HE stains and substance P(SP) and protein gene product 9.5(PGP9.5) immunohistochemical stains.Methods: Fifteen male Sprague-Dawley were randomly assigned into occlusal interference group(n=12) and control group(n=3).In occlusal interference group,0.4 mm thick crowns were bonded to the rats'first molar of the maxillary.In the control group,rats were anesthetized and mouths were forced open for about 5 min but restorations were not applied.1,5,10,and 21 d after 0.4 mm occlusal alteration treatment,mechanical pain thresholds of bilateral masseter muscles were quantitatively measured by modified electronic anesthesiometer in control group and occlusal interference group.The rats were euthanized by transcardiac perfusion after deep anesthetization at different time points.The paraffin sections of masseter muscles were made and processed for HE,SP,and PGP9.5 immunohistochemical staining.Results: Decreased head withdrawal threshold to mechanical pressure was detected in masseter muscles on both sides following occlusal interference.Histological stains of masseter muscles presented intact following occlusal interference,and no inflammatory cells were observed in both sides.Intensely stained PGP9.5 was observed at 1 d in occlusal interference groups and maintained until the end of the experiment.SP expression was the most obviously increased at 5 d in both sides and gradually decreased to the level of control.Conclusion: Experimental occlusal interference-induced masticatory muscle pain is associated with peripheral sensitization of nociceptive neurons rather than muscle damage and inflammation.

AIM: To establish human lymphocyte cell line secreting monoclonal antibody against Rhesus(D) antigen and analyse the nucleotide and deduced amino acid sequences of a human monoclonal anti-D Fab fragment. METHODS: By using PCR method, the cDNA of human anti-(Rhesus D) antibody(lgM ?)Fab fragment was amplified from an Epstein- Barr-virus-transformed cell line. Cloning and subsequent sequence analysis of the Fab fragment was performed. The deduced amino acid sequence was compared and analysed with previously published sequences. RESULTS : A band of approximate 700 and 650 base pairs was amplified using lgM heavy chain primers and ? light chain primers, respectively. Sequence analysis indicated that the deduced amino acid sequences was in agreement with the characterization of the amino acid present in the human lg Fab fragment. CONCLUSION: The cloning and sequencing of a human anti-Rhesus (D) antibody Fab fragment cDNA will make benefits for production of recombinant anti-Rhesus (D) antibody and prevention of Rh haemolytic disease in the newborn.

Objective Study on producing human monoclonal antibodies against Rhesus (D) antigen that was suitable for use as blood group typing reagent. Methods B lymphocytes from a Rh negative woman, which can produce anti D antibodies were transformed by Epstein Barr virus(EBV). Antibody secreting cells were enriched by RhD + group O erythrocytes and cloned by limited dilute method. By using one step emzymatic method on microplates, one thousand normal blood donors with a common Rh phenotype were tested with the supernatant of cell culture medium as well as a polyclonal human anti D and a commercial monoclonal anti D serum. Results Three human B lymphocyte lines secreting monoclonal antibodies to Rh (D) were established. One of them produced lgM antibody. The titer of the monoclonal antibodies was 64～128. Study on 1000 blood donors, the results did not show any discrepancy among the three different anti Rh(D) serum. Conclusion These monoclonal antibodies against D antigen could be used in Rh(D) typing.

AIM: To investigate the effect of norcantharidin(NCTD)on proliferation and invasion of human breast cancer cell line SKBR3 in vitro and its anticancer mechanisms.METHODS: MTT assay was used to determine SKBR3 cell proliferation. Light and FACScan were used to detect apoptosis and cell cycle. The invasiveness of SKBR3 was evaluated by the adhesion test,Matrigel experiment and the crossing-river test.RESULTS: NCTD had inhibitive effects on growth of SKBR3 cells in a dose and time-dependent manner, with the IC50 value of 12.5 mg/L at 24 h.The cells treated with 10 mg/L NCTD for 24 h and 48 h showed typical apoptotic morphology and hypodiploid peak before G1 phase. The cell cycle was arrested at G2/M phase. The apoptosis percentage was up to 3.44% and 6.17%, and the G2/M percentage was up to 35.82% and 38.70%. NCTD also could inhibit obviously the adhesion, movement and invasive capability simulating human basement membrane of SKBR3. Its effect was also in a dose-dependent manner. In the NCTD-treated group, crossing-river time was prolonged significantly and passing-membrane cells markedly decreased. CONCLUSION: NCTD in vitro inhibits not only the proliferation and growth of human breast cancer cells but also invasion and metastasis of the cells at relatively low concentration. NCTD shows prominent anti-tumor effects.

AIM:To study the blocking effect of shRNA on the expression of PSMA gene in LNCaP cell line by using shRNA eukaryotic expression vector.METHODS:Three pairs of DNA templates coding shRNA,synthesized against PSMA and cloned into the vector pSilencer 2.1-U6-neo,which was named pSilencer 2.1-U6-neo-shRNA,were identified by restriction endonuclease digestion analysis and DNA sequencing.LNCaP cells were then transfected with these three pSilencer 2.1-U6-neo-shRNAs and the negative control pSilencer 2.1-U6-neo-NC.After G418 selection,the cells were selected and the interfering effect was detected by RT-PCR and Western blotting.The biological behaviours of the transfected LNCaP cells were also tested.RESULTS:Restriction endonuclease digestion analysis and DNA sequencing results all showed that the 3 target segments were cloned into pSilencer 2.1-U6-neo vector respectively.After transfected into LNCaP cells,the inhibitory ratio of PSMA mRNA was 33.15%,9.26% and 41.97% respectively,and that of PSMA protein was 26.26%,6.47%,40.69% respectively.The p-shRNA3 was chosen to test the cell growth and its invasive power in vitro.The results showed that after interfering,the invasiveness of LNCaP cells were enhanced.CONCLUSION:The vector-based shRNA on PSMA gene effectively knocks down the PSMA gene expression.The successful construction of PSMA shRNA makes it possible for further study of the interaction between PSMA and prostate cancer.

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α(+) dendritic cells (DCs) in vitro was investigated in this study. Immature CD8α(+) DCs were prepared from C57BL/6 (H-2(b)) bone marrow cells by using a cytokine cocktail. On the 3rd day of culture, CD8α(+) DCs were pulsed by allogeneic (Balb/c, H-2(d)) EL9611 leukemia antigen, or RM-1 syngeneic prostate cancer antigen, with the concentration series of 0, 2.5, 5.0, 10.0, 20.0 μg/mL, respectively, then antigen-loaded immature CD8α(+) DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1:1, 2:1 and 4:1. T cell proliferation was measured by MTT assay. Cytokines including interferon gamma (IFN-γ) and interleukin-10 (IL-10) in CD8α(+) DCs and T co-culture supernatant were detected by using ELISA. Cytotoxic effect of antigen-specific T cells was tested by LDH release assay. Conventional mature DCs (mDCs) induced from C57BL/6 (H-2(b)) bone marrow cells by using granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) served as a control. The results showed that the proliferative activity of T cells stimulated by CD8α(+) DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8α(+) DC/T ratio increased (P<0.05). When antigen concentration ≤ 5 μg/mL and CD8α(+) DC/T ratio ≤ 2:1, the ability of CD8α(+) DCs to stimulate T cell proliferation was higher than mDC control in allogeneic tumor antigen-pulsed groups (P<0.05), but not in syngeneic tumor antigen-pulsed groups (P>0.05). The level of IFN-γ and IL-10 in CD8α(+) DCs and T cell co-culture supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups (P<0.05), and the cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when the CD8α(+) DC/T was 1:1 or 2:1 (P<0.05). There existed a negative correlation between the level of IL-10 and T cell proliferation. T cell cytotoxicity assay showed that when CD8α(+) DCs were pulsed with allogeneic tumor antigen, the maximal T cell killing efficiency could reach (100±7.7)%, whereas syngeneic tumor antigen-pulsed group had only (65.0±3.4)%. It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α(+) DCs could stimulate T cells to exert the GVT effect in vitro, and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen. The optimal condition was low allogeneic tumor antigen pulsation (≤ 5 μg/mL) and low CD8α(+) DC/T ratio (1:1 and 2:1).

Objective To construct the murine allogeneic acute GVHD model.Methods C57BL/6 (H-2b) mice were used as the donors and Balb/c (H-2d) mice as the recipients in allogeneic bone marrow transplantation (BMT).Groups were set as total body radiation (TBI) control group (n =4),GVHD group (n =10),simple BM transplantation group (n =10) and normal control group (n =4).For TBI control group,mice were subjected to TBI but did not receive BMT after radiation.For GVHD group,5 days before TBI,gentamycin (320 mg/L) and erythromycin (250 mg/L) were added into the drinking water,and on the day of transplantation,mice received one total dose of 8.0 Gy 60Coγ TBI,and within 5 h,2 × 106 C57BL/6 BM cells and 1 × 107 C57BL/6 spleen cells were transfused per mouse via the tail vein.For simple BMT group,the pretreatment was the same as GVHD group,and mice received only 2 × 106 C57BL/6 BM cells per mouse via the tail vein.The mental status,activity,posture,fur,weight,and stool were observed after transplantation.Survival time of each mouse was recorded,survival rate was calculated,and survival curve was drawn.Pathological examination was done for the liver,skin,small intestine and BM on the brink of death.Results The median survival time (MST) in TBI control group,GVHD group and BMT group was (9.0 ± 0.7),(32.0 ± 3.2) and ( 17.5 ± 1.6) days respectively,and there was significant difference between every two groups (P ＜ 0.01 ).Pathological examination in TBI control group showedhematopoiesis exhaustion.GVHD group showed acute GVHD symptoms 10-13 days after allo-BMT,and the pathological changes of the skin,liver and small intestine corresponded to those of Ⅰ to Ⅱ degree of GVHD.Simple BMT group also showed acute GVHD symptoms 10-13 days after alloBMT,but their GVHD manifestation and histological changes were less serious and only 0 to Ⅰ degree of GVHD could be seen.ConclusionStable acute GVHD model can be constructed by transfusion of allogeneic BM cells and spleen cells into Balb/c mice after lethal TBI.

Objective:To study the possible immune escape mechanisms of HCoV-OC43 from human dendritic cells(DC).Methods:HCoV-OC43 was isolated from clinical specimen using BSC-1 cells and identified by Real-time PCR,and the cytopathic effect was observed by phase contrast microscope.DCs were induced in vivo using hu-GM-CSF and IL-4 cytokines,and after 7 days of differentiation,DCs were infected by HCoV-OC43.The morphology of HCoV-OC43 infected DC was observed by transmission electron microscope,and the cytokines related to DC functions were detected by Real-time PCR after infection.DC proportion and function related co-stimulatory molecules were analyzed by flow cytometry.Results:In vitro HCoV-OC43 infected human DC model was successfully built.HCoV-OC43 can infect DC and generate immune response of DC in vitro,but no virus nucleonic acid could be detected in culture supernatant.The DC expression of IFN-α,IFN-β,CCL3 and CCL5 were significant decreased when infected with HCoV-OC43,but the expression of costimulatory molecules including HLA-DR,CD1c and CD86 were not affected by HCoV-OC43 infection.Conclusion:Human DC could be infected by HCoV-OC43 and generate immune response,but could not produce progeny virus.HCoV-OC43 may escape from immune response by suppressing the expression of IFN-α and other inflammatory cytokines and chemokines in DC.

AIM: To clone and express a human monoclonal anti-D Fab fragment in E. coli and make benefits for the expression of the whole immunoglobulin molecules of anti-D. METHODS: The gene of anti-D Fab fragment was cloned into the phagemid vector pComb3. After analyzing by PCR and restriction site analysis, the recombinant was expressed in E. coli and the expressed protein was analyzed by SDS-PAGE and ELISA. RESULTS: The result of SDS-PAGE confirmed that E.coli expressed a 48 kD protein. The ELISA result demonstrated that the cell culture supernatant reacted with Rh+ group O human erythrocytes, but was not recognized by Rh-group O human erythrocytes. CONCLUSION: Expressed Fab fragment has the antigenic specificity for human erythrocytes.

AIM: To discuss the relationship between prostate specific membrane antigen(PSMA) and prostate cancer and to seek a target for diagnosis and therapy of prostate cancer.METHODS: A pair of primers was designed according to the published PSMA mRNA sequence.Total RNA was extracted from prostate cancer tissues and was reversely transcribed into cDNA,which was used as a template for PCR to amplify the PSMA gene.The recombinant was sequenced and the result was analyzed by BLAST.The PSMA5 gene specific primers were designed to identify its expression in different cells and prostate tissues.RESULTS: A new alternatively spliced variant of PSMA named PSMA5 was discovered when sequencing the recombinant.PSMA5 showed well pathological tissue-specificity,and its expression rate in prostate cancer,benign prostatic hyperplasia of prostate,and normal prostate tissue were 92.6%,78.8% and 10.0%,respectively.It expressed specifically in Pca cell line LNCaP,not in cell lines of PC3,bladder carcinoma,renal carcinoma,or hepatoma.CONCLUSION: A new alternative spliced variant of PSMA named PSMA5 was discovered,which was well correlated with prostate cancer and benign prostatic hyperplasia.This finding may give a new clue to the evolution of prostate cancer and may provide a target for the diagnosis and therapy of prostate cancer.