Objective To investigate the feasibility of labeling iris pigment epithelial(IPE)cells of rabbits with 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester(CFSE). Methods Enzyme-assisted microdissection was used to isolate the cultured rabbit′s IPE cells.The third or forth subcultured IPE cells were incubated with 2.5,5,10,20,and 40 ?mol/L of CFSE for 1,5,and10min respectively.The fluorescence intensity was detected by flow cytometry,and the leakage of CFSE and its dyeing were observed by fluorescence antibody labeling. Results Incubation with 20 ?mol/L CFSE under 37℃for1minute was the most optimal condition for IPE cells labeling.The coloration of IPE cells stained by CFSE lasted 4 weeks.There was no leakage of dye from labeled rabbit IPE cells to non-labeled human IPE cells in mixed culture process. Conclusion With the advantages of high rate of dyeing,long time of tracing,safety and convenience,CFSE can be used as a new method to label the rabbit′s IPE cells.

Forty-five healthy male dogs were wounded with high or middle-velocity steel ball on the lower part of the face.and the findings were as follows:(1)Craniocerebral injuries concomitant with maxillofacial gunshot wounds were charcterized by brain contusion in the entry side of the temporal lobe and extradural hemorrhage in the entry side of the middle cranial fossa,and their highest incidence and severity were found in those cases with mandible fracture due to high-velocity missiles.(2)The larger the amount of the absorbed energy from the bullet,the higher the incidence and severity of the craniocerebral injury.(3)The incidence and severity of the craniocerebral injury were positively correlated to the value of vibrational acceleration measured on the pareital bones,which suggests that vibrational acceleration plays an important role in the precipitation of craniocerebral injury secondary to maxillofacial gunshot wounds.

To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase II (CaMK II), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was detected in neurons. The results showed that the expression of CaMK II, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (P<0.05), and that of iNOS and AC peaked at 8 h and 12 h respectively. It was suggested that there might be some epileptogenic factors in the ACM and such signal pathways as NOS-NO-cGMP, Ca2+/CaM-CaMK II and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.

Objective To evaluate the effect of dexmedetomidine on cognitive dysfunction after thoracic surgery in patients undergoing one-lung ventilation.Methods Sixty-two patients,aged 45-64 yr,of ASA physical status Ⅰ or Ⅱ,with body mass index ranged from 17.5 to 25.5 kg/m2,scheduled for elective thoracic surgery,were randomly allocated into 2 groups (n =31 each) using a random number table:dexmedetomidine group (Dex group) and control group (C group).Dexmedetomidine 0.5 μg/kg was infused for 10 min starting from the time point before induction of anesthesia,followed by continuous infusion at a rate of 0.5 μg · kg-1 · h-1 until 30 min before the end of surgery in Dex group.The equal volume of normal saline was administered instead in group C.Before induction and at 24,48 and 72 h after surgery,venous blood samples were collected for determination of levels of S-100 beta protein and neuronspecific enolase in serum by ELISA.Cognitive function was assessed by Mini-Mental State Examination at 72 h after surgery.Results The levels of S-100 beta protein and neuron-specific enolase in serum were significantly increased after surgery than before induction in the two groups.Compared to C group,the levels of S-100 beta protein and neuron-specific enolase in serum were significantly decreased after surgery,and the incidence of postoperative cognitive dysfunction was decreased in Dex group (26％ vs 6％).Conclusion Dexmedetomidine can effectively reduce the nerve damage during one-lung ventilation and significantly inhibit the development of postoperative cognitive dysfunction in patients undergoing thoracic surgery,indicating that dexmedetomidine is suitable for thoracic surgery.

Objective:To investigate the immunogenicity of pORF5 plasmid protein,and further to screen and identify its im-munodominant domian.Methods: 10 different fragments of pORF5 gene including full length were amplified from the DNA of Chlamydia trachomatis serovar D by PCR and cloned into appropriate site of pGEX-6p vector to construct recombinant vectors after digested with BamHⅠand NotⅠrestriction endonucleases.After identification by PCR and sequencing,the recombinant plasmids were transformed into XL1 Blue E.coli to express the GST fusion proteins.ELISA and Western blot were carried out to identify the immunogenicity and immunoreaction of pORF5 plasmid protein.10 different fragments were reacted with sera from patients urogenitally infected with Chlamydia trachomatis, mouse polyclonal antibodies and mouse monoclonal antibodies of pORF5 plasmid protein with ELISA method.Results: pORF5 plasmid protein displayed strong immunogenicity and could induce a strong antibody response in human.The reactivity of human antibodies almost completely disappeared,when the native structure of pORF5 plasmid protein was de-stroyed.F6 that only lacked the N-terminal 66 amino acids was recognized by antibodies in ELISA as strongly as the whole pORF5 plasmid protein was.However,no other fragments were significantly recognized although there was a minimal reactivity of F2 and F3 with antibodies.Conclusion:pORF5 plasmid protein was an immunodominant antigen containing conformation-dependent epitope,and the C-terminal three quarters of pORF5 amino acid sequence was required for maintaining its immune dominance and conformation.The significance of the above findings lay a foundation for the further study on pORF5 protein function and vaccine development.

Objective To construct DNA vaccine containing MOMP gene of Chlamydia trachomatis and to observe immune response in mice. Methods Mice of 4 - 6 weeks old were immunized with pcDNA3.1-MOMP or pcDNA3.1 intramuscularly at a dose of 100 μg. Booster immunizations were employed at 2-week interval for two times. Specific antibody in the sera of mice and the level of IFN-γ in murine spleen lymphocyte supernatant were detected by ELISA. The proliferation response of spleen cells was detected by MTT assay. Results Significant specific antibody titers were observed and the highest titer was 1: 1 024 in mice after three times immunization with pcDNA3.1-MOMP. The proli-feration response of spleen cells were significantly higher than that of mice injected with pc DNA3.1. IFN-γ reached(532.0 + 45.4)pg/mL in immunized mice. Conclusion Strong responses of humoral and cellular immunity can be evoked by DNA vaccine of pcDNA3.1-MOMP in mice.

Whether transforming growth factor-beta2 (TGF-beta2) induces apoptosis of human trabecular meshwork cells was investigated in vitro. Cultured 3-5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF-beta2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmission electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79 +/- 0.44)%, (4.43 +/- 1.17)% and (9.60 +/- 2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-beta2 with the difference being significant between experimental group and control group [(1.41 +/- 0.34)%]. It was concluded that TGF-beta2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.
Apoptosis/*drug effects ; Cells, Cultured ; Trabecular Meshwork/*cytology ; Transforming Growth Factor beta/*pharmacology ; Transforming Growth Factor beta2

To explore the effect of coriaria lactone (CL)-activated astrocyte-conditioned medium on the cerebral TNF-alpha of normal rats, the CL-activated astrocyte-conditioned medium (ACM) was injected into the lateral ventricle of SD rats. The rats were observed for behavioral changes, and the changes of the expression of TNF-alpha in the cerebral cortex and hippocampus were immunohistochemically examined by employing SP method. TNF-alpha level was assessed by means of radioimmunoassay in homogenate of cerebral cortex and hippocampus as well as cerebrospinal fluid. Seizure episodes were observed in ACM group 30 min after the ACM injection, but they were not observed in the control group. Immunohistochemical detection showed that the immunoreaction of TNF-alpha in hippocampus and cerebral cortex of rats were stronger than that of the control group 4 h after the ACM injection (P<0. 05). In this group, the concentrations of TNF-alpha in homogenate of cerebral cortex and hippocampus and cerebrospinal fluid were higher than those of the control group (P<0.05). It is suggested that the ACM activated by CL can enhance the expression of TNF-alpha in normal rats, and is related to epileptogenesis.

To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells. CT358 gene from the Chlamydia trachomatis (C. trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedCI vectors. The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins. The GST-CT358 fusion protein was used to immunize mice to raise the antibodies, which specifically recognized CT358 without eross-reacting with other unrelated proteins. The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay (IFA). Meanwhile, pDSRedC 1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection. The hypothetical protein CT358 was identified in the inclusion membrane of C. trachomatis-infected cells for the first time,and it was detected as early as 12 h after C. trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of CT358 via a transgene failed to affect the subsequent ehlamydial infection. These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein, giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.

Objective To localize and characterize the plasmid protein pORF5 in the Chlamydia trachomatis(Ct) infected cells. Methods The open reading frame encoding for pORF5 protein from the Ct plasmid was amplified and cloned into the pGEX-6p vector. The recombinant plasmid pGEX-pORF5 was transformed into XL1-blue E. coli to express fusion protein with the glutathione-s-transferase (GST). After purified with Glutathione Sepharose 4B beads, the pORF5 fusion protein was used to immunize mice to make monoclonal and polyclonal antibody. The antibodies were used to localize the endogenous pORF5 protein and detect the expression pattern in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). At the same time, ELISA was used to determine whether pORF5 plasmid protein was expressed and immunogenic during Ct infection in humans. Results pORF5 was detected a dominant signal in the cytosol of the Chlamydia-infected cells with a pattern similar to that of anti-CPAF. pORF5 also appeared in the RBs and EBs in small quantity. Athough pattern was similarly, pORF5 did not overlap with CPAF. pORF5 protein was strongly recognized antiserum in an ELISA. Conclusion The pORF5 plasmid protein was identified as a secreted protein with good immunogenicity, pORF5 gene was to express the endogenous target protein during human infection.