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Precancerous lesions of gastric cancer (PLGC) is a pathological change accompanied by intestinal metaplasia and dysplasia on the basis of chronic atrophic gastritis. It is also an important stage of "inflammation-cancer transformation" on gastric mucosa. Paying attention to the intervention of PLGC has important value and significance for the secondary prevention of gastric cancer. PLGC has the characteristics of occult onset, toxin damaging collaterals, and long course of disease, which is highly consistent to the pathogenesis characteristics of incubative pathogenic factors. Based on the relevance of incubative pathogenic factors and PLGC, treatment of PLGC from the perspective of incubative pathogenic factors should be mainly strengthening the spleen and stomach, and combined with the methods of regulating qi and dissipating dampness, and removing blood stasis and detoxification. It should also pay attention to the prognosis.Paying attention to the body-mind treatment can reduce the re-occurrence , so as to provide a new way of thinking for treating PLGC from incubative pathogenic factors.
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Objective:The mechanism of Indigo Naturalis in the treatment of ulcerative colitis (UC) was predicted by GEO chip combined with network pharmacology, and preliminarily verified by molecular docking. Methods:The main active components and targets of Indigo Naturalis were obtained by searching TCMSP, SymMap database, SwissTargetPrediction and Pharmmapper. The UC disease targets were obtained from DrugBank database, OMIM database, TTD database, Disgenet database and GEO gene chips. Venn diagram was used to show drug-disease common targets. The protein-protein interaction (PPI) network of intersection targets was analyzed by String platform, and Cytoscape 3.8.2 software was used to construct the PPI network of components and disease targets. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on the core targets. AutoDock Vina 1.2.1 was used for molecular docking, and the results were visualized by Discovery studio Visualizer. Results:A total of 10 active components and 184 compound targets of IN-UC, of which 42 were core targets, were enriched and analyzed by GO and KEGG. The therapeutic effect of Indigo Naturalis on UC may involve activation of various process, such as reactive oxygen species metabolism, heme binding, protein phosphatase binding, secretory granule exocytosis, cytoplasmic vesicles, cell focal adhesion and cell substrate connection, and regulates PI3K/Akt signal pathway, human cytomegalovirus infection signal pathway, EB virus infection signal pathway HF1 signaling pathway and insulin resistance signaling pathway to treat UC. Conlusion:Indigo Naturalis has the characteristics of multi-component, multi target and multi pathway in the treatment of ulcerative colitis. Through PTGS2, CAT and other core targets, it can regulate PI3K/Akt signal pathway, human giant cell signal pathway, HIF-1 signal pathway to play a therapeutic role.
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Objective:To analyze the changes in biological characteristics including infectivity, growth and pathogenicity of Chlamydia muridarum ( Cm) after serial passage in vitro in special conditions in order to provide reference for screening attenuated live vaccines and virulence-related genes. Methods:Wild-type Cm strain (G0) was cultured for several passages using conventional cell culture method under alternate unassisted and assisted culture conditions. Then, the 28th generation (G28) of Cm was selected and compared with the parental G0 strain in terms of centrifugation dependence, attaching ability, intracellular growth curve, plaque size and fallopian tube lesions after genital tract infection in a mouse model. Results:Compared with the parental G0 strain, the G28 strain showed significantly decreased dependence on centrifugation during cell infection ( P<0.05) and increased attachment capacity to cells ( P<0.05). No significant differences were observed in the growth curves 32 h after cell infection or in the plaque sizes between the parental G0 and G28 strains. In the in vivo virulence test, fallopian tube lesions were observed in 87.5% of G0-infected mice and 37.5% of G28-infected mice ( P<0.05). Conclusions:Compared with the parental G0 strain, the G28 strain showed significantly enhanced in vitro infection ability, but decreased in vivo pathogenicity, which brought hope for further identification of virulence genes, isolation of attenuated strains with single genotype and development of live attenuated Chlamydia vaccines.
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First-class education for postgraduate is the foundation for the construction of "double first-class" education. Universities play an important role in postgraduate education. This paper explored the measures for the reform and innovation of the construction of "double first-class" education for postgraduate students in our university, which include perfecting the supervisor's responsibility and authority mechanism, deepening the reform on curriculum system, strengthening the construction of sharing platform, and improving the evaluation mechanism of training quality and so on. In conclusion, initial achievement from the reform and innovation of training mode was observed, which provides a useful reference for the construction of "double first-class" education for local universities.
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Objective To explore the molecular mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein of Chlamydia trachomatis,so as to provide an experimental basis for further clarifying the pathogenesis of Chlamydia trachomatis.Methods HeLa cells were divided into two groups:carbonyl cyanide m-chlorophenyl hydrazone (CCCP,an apoptosis inducer) group was stimulated by CCCP for 30 minutes,and pORF5 + CCCP group was pretreated with pORF5 plasmid protein for 18 hours followed by CCCP for 30 minutes.Then,Western blot analysis was performed to determine the expression of apoptosisrelated proteins Bcl-2,Bax and caspase-3,JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential in HeLa cells,and cytochrome c release from mitochondria was analyzed by indirect immunofluorescence assay.To analyze whether high-mobility group box 1 (HMGB1) protein participated in the anti-apoptotic role of pORF5 plasmid protein,HMGB 1 shRNA and control RNA were separately transfected into the HeLa cells,which were then stimulated by pORF5 plasmid protein and CCCP.Then,the protein expression of Bcl-2,Bax,activated caspase-3 was determined,and cytochrome c release was analyzed.Data were compared between two groups by using paired t test.Results pORF5 plasmid protein could antagonize the CCCP-induced decrease of mitochondrial membrane potential,and the red/green fluorescence intensity ratio was significantly lower in the CCCP group (0.4 ± 0.1) than in the pORF5 + CCCP group (1.7 ± 0.3;t =6.95,P ＜ 0.01).The protein expression of Bcl-2 in the HeLa cells in the pORF5 + CCCP group was 5.3 ± 0.6 times more than that in the CCCP group (t =8.62,P ＜ 0.01),while the protein expression of Bax and activated caspase-3 in the pORF5 + CCCP group significantly decreased by 79％ ± 10％ (t =9.23,P ＜ 0.01) and 75％ ± 8％ (t =4.26,P ＜ 0.05) respectively compared with the CCCP group.Compared with the control RNA transfection group,the HMGB1 shRNA transfection group showed significantly decreased mitochondrial membrane potential in the HeLa cells (t =11.23,P ＜ 0.01),increased cytochrome c release,decreased Bcl-2 expresson (t =7.19,P ＜ 0.05) and increased Bax expression (t =13.06,P ＜ 0.01) after stimulation with pORF5 and CCCP.Conclusion Chlamydia trachomatis plasmid protein pORF5 plays an anti-apoptosis role by blocking the mitochondrial apoptotic pathway through HMGB1 protein.
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Objective To analyze the protein expression profile of HeLa cells transfected with pORF5 gene of Chlamydia trachomatis. Methods A lentiviral expression vector containing pORF5 gene was constructed. The lentiviral expression vector and helper plasmids were co-transfected into 293T cells to construct the recombinant lentivirus, which was used to infect HeLa cells. HeLa cells transfected with pORF5 gene and control HeLa cells were sorted out by flow cytometry. The isobaric tags for relative and absolute quantitation ( iTRAQ) approach combined with nano-liquid chromatography-tandem mass spec-trometry ( NanoLC-MS/MS) analysis was performed to understand protein expression profiles and to iden-tify and quantify the differentially expressed proteins in the pORF5-transfected HeLa cells ( pORF5-Hela) and the control HeLa cells. Quantitative real-time PCR ( qRT-PCR ) and Western blot analysis were performed to detect the expression of some proteins at mRNA and protein levels, respectively. Results HeLa cell line stably transfected with pORF5 gene and control HeLa cell line were constructed successful-ly. Totally 314 proteins were differentially expressed between the pORF5-HeLa and control HeLa cells, 159 of which showed increased expression and the other 155 showed decreased expression in pORF5-HeLa cells. The differentially expressed proteins were involved in many processes, such as metabolic process, immune response, biological adhesion and so on. Results of qRT-PCR showed that the expression of HIST1H1C(histone H1. 2C), HBA1(hemoglobin subunit alpha), PARK7(parkinson disease protein 7), HMGB1(high mobility group protein B1) and HMGB2 at mRNA level in pORF5-HeLa cells were up-regulated, while the expression of CLIC1 ( chloride intracellular channel protein 1 ) , KRT7 ( typeⅡ cy-toskeletal 7), SFN(14-3-3 protein sigma) and CDKN2A(cyclin-dependent kinase inhibitor 2A) were down-regulated. Western blot analysis confirmed the enhanced expression of HMGB1 and PRAK7 at pro-tein level. The results of qRT-PCR and Western blot analysis were consistent with proteomic data. Con-clusion Expression profiles for differentially expressed proteins between pORF5-HeLa and control HeLa cells were established successfully. The differentially expressed proteins regulated by pORF5 gene were found to be related to cell metabolism, proliferation, adhesion and so on, suggesting that pORF5 might promote the growth and proliferation of Ct by regulating protein expression and biological behavior of host cells.
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Objective To evaluate inhibitory effect of Chlamydia trachomatis plasmid-encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor-alpha(TNF-α). Methods The recombinant lentiviral expression vector containing pORF5 gene and helper plasmids were co-transfected into 293T cells to prepare the recombinant lentivirus. Then, the lentivirus particles were collected and concentrated, and used to infect HeLa cells. Flow cytometric screening identified stable pORF5-expressing HeLa (pORF5-HeLa) cells. Meanwhile, the empty plasmid was transfected into HeLa cells to prepare control HeLa cells. The two cell lines were both divided into two subgroups to be treated with 20μg/L TNF-αand fresh culture medium respectively for 6 hours. Then, Hoechst 33258 staining was performed to observe morphological changes of apoptotic cells, flow cytometry to detect cell apoptosis, real-time PCR to measure the mRNA expression of Caspase3, Bax and Bcl-2, and Western blot analysis to determine the protein expression of Bax and Bcl-2. Results After 6-hour treatment with TNF-α, Hoechst 33258 staining showed variable degrees of karyopyknosis and karyorrhexis, and highly-refractive blue apoptotic bodies in the pORF5-HeLa cells and control HeLa cells. The pORF5-HeLa cells and control HeLa cells both showed significantly higher apoptosis rate in the treated subgroup than in the untreated subgroup (pORF5-HeLa cells:35.5%± 4.5%vs. 9.5%± 1.5%, t=13.53, P<0.01;control HeLa cells:63.6%± 5.8%vs. 7.9%± 0.9%, t=32.36, P<0.01). Compared with treated control HeLa cells, treated pORF5-HeLa cells showed significant decreases in mRNA expression of Bax(72.8%)and Caspase 3(84.5%)(t = 35.29, 42.25, respectively, both P < 0.01), as well as in Bax protein expression(t = 17.58,P < 0.01), but significant increases in Bcl-2 mRNA and protein(6.8 times)expression(t = 87.12, 18.93, respectively, both P <0.01). Conclusion pORF5 plasmid protein can inhibit TNF-α-induced HeLa cell apoptosis likely by increasing the expression of anti-apoptotic protein bcl-2 and decreasing the expression of pro-apoptotic proteins Caspase-3 and Bax.
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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.
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Objective:To investigate whether pORF5 plasmid protein of Chlamydia trachomatis(Ct) induces 1L-1βand 1L-18 production in THP-1 cells,and its potential molecular mechanism.Methods:pORF5 plasmid protein was used to stimulate THP-1 cells at different concentrations(0,3,6,12,24,36 μg/ml),then the inflammatory cytokines IL-18 and IL-1βwere detected by ELISA at the time of 0,8,16,24,36 h;The mRNA expression of NALP3 inflammasome were detected by Realtime-PCR,and Caspase-1 activity was determined by Western blot analysis.THP-1 cells were transfected with siRNA targeting NALP3 and ASC gene for 24 h or pretreated with Caspase-1 inhibitor(Z-YVAD-FMK) for 30 min,and subsequently stimulated with pORF5(24 μg/ml) for 24 h,then secretion of IL-1βand IL-18 were analyzed by ELISA.Results: The pORF5 plasmid protein induced THP-1 cells to secrete IL-1βand IL-18 by dose-and time-dependent manners,production of IL-1βand IL-18 reached their peaks(491 pg/ml and 186 pg/ml) at concentration of 24 μg/ml,and the peak amount of IL-1βand IL-18 occurred at 24 h and 16 h post-stimulation respectively.pORF5 plasmid protein in-creased mRNA expression of NALP3 inflammasome and activated Caspase-1 in THP-1 cells.NALP3 siRNA,ASC siRNA and Z-YVAD-FMK reduced pORF5-induced IL-1βand IL-18 production when compared with control groups(P<0.05).Conclusion:pORF5 plasmid protein could induce THP-1 cells to produce IL-1βand IL-18 through NALP3 inflammasome activation,which may play an important role in the pathogenesis in Ct infection.
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Objective To investigate the immune injury in genital tract of BALB/c mice induced by plasmid protein pORF5 of Chlamydia trachomatis and its possible mechanism.Methods GST(glutathione-S-transferases)-pORF5 fusion protein was expressed and digested with PreScission Protease to obtain the target protein without GST tag.After further purification and endotoxin removal,pORF5 protein was injected into the posterior fornix of BALB/c mice on day 1,3 and 6,while the control groups were injected with PBS or GST protein respectively,and then all the mice were sacrificed on day 7 to evaluate genital tract gross pathology and histopathological characterization.The levels of TNF-α,IL-1β and IL-6 in serum,splenocytes culture supernatant and vaginal douche were detected by ELISA.Results Mice in pORF5 group developed different degrees of swelling in isthmic portion and ampulla of uterine tube,connective tissue adhesion and hydrosalpinx in the genital tract tissues,while the PBS group and the GST group did not show any obvious change.The inflammatory score showed that the genital tract pathology in pORF5 group was much more severe than PBS and GST control groups (P＜0.O1).The levels of TNF-α,IL-1β and IL-6 in vaginal douche and splenocytes culture supernatants in pORF5 group were obviously higher than those of PBS and GST groups (P＜0.05).The levels of TNF-α and IL-1β in serum were also higher than those of GST and PBS groups (P＜0.01).Conclusion pORF5 plasmid protein could induce pathological immune response in the genital tract of BALB/c mice,which may be associated with the increase of the production of the inflammatory cytokines TNF-α,IL-1β and IL-6 in BALB/c mice.
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Objective To investigate the effect of heat shock protein 10 (HSP1O) of Chlamydophila pneumoniae in inducing TNF-α on THP-1 cells and the roles of TLR4 and TLR2 involved in it.Methods Purified native recombinant HSP10 from Cpn(CHSP10) were produced and inactivated the endotoxin contamination,then different concentration (0.5,1,5,10,20,30 μg/ml) of CHSP10 were used to stimulate THP-1 for different time (0,6,12,24,36,48,60 h).TNF-α were measured by using human TNF-α ELISA kit and compared among different groups.THP-1 were collected and analyzed for TLR2 and TLR4 mRNA levels and protein expression by RT-PCR and immunofluorescence.Peritoneal macrophages isolated from wide-type (C3 H/HeN) and TLR4-deficient mice (C3H/HeJ) were stimulated with endotoxin-free proteins respectively,and the TNF-α were measured.Furthermore,neutralizing anti-human TLR2/TLR4 McAb as a blocking Ab was preincubated with THP-1,after stimulation with CHSP10,ELISA was used to detect the concentration of TNF-α.Results TNF-α can be induced with CHSP10 in THP-1,while it significantly decreased with heated or deproteinized CHSP10.Both TLR2 and TLR4 mRNA and protein were detected in THP-1.Macrophages from C3H/HeN mice displayed higher TNF-α compared with it from C3H/HeJ mice after stimulation with CHSP10.The CHSP10-induced TNF-α would obviously decline when treated with antiTLR2/TLR4 McAb.Conclusion As a potential inflammation related protein,CHSP10 are involved in the pathogenesis of Cpn inducing inflammation cytokine TNF-α.TLR2 and TLR4 appear to be involved in CHSP10-mediated expression of TNF-α.
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ObjectiveTo construct a mouse model for studying pathophysiology and mechanism of human Chlamydia trachomatis genital infection.MethodsInnate immunity-deficient C3H/HeJ female mice were infected intravaginally with human C.trachomatis serovar D urogenital isolates for screening the highest violent clinical strain.The clinical strain UT0603 as well as standard strain D/UW-3/CX were then used to reinfect na(i)ve mice,the lower genital tract shedding were monitored by swabbing every 3-7 day over the entire infection period by culture.Some mice were sacrificed at early infection stage to detection of in site Chlamydia growth by immunofluorescence assay,then all the mice were sacrificed at later infection stage to evaluate upper genital tract gross pathology and histopathological characterization.ResuIts In the lower genital tract,Chlamydia shedding time course were significantly prolonged in clinical strain infected mice.Chlamydia not only growth in the lower genital tract,the live organism also ascending and growth in the upper genital tissue.The gross appearance under naked eyes and dilation and inflammation scores under microscope all showed that the genital tract pathology from the clinical strain infected mice were much more severe than standard strain infected control mice.Conclusion Together,all these results demonstrated that a mouse model for Chlamydia genital infection was constructed.
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Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.
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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.
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Quality is the lifeline of graduate education and the tutor team plays an important role in postgraduate training. To control the selection of tutors strictly, to implement the combination of openness and stability in tutor team, and to carry out academic exchanges actively are the keys to construct an excellent tutors staff and to ensure the quality of graduate student training.
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ObjectiveTo purify and characterize the monoclonal antibody (McAb) against Chlamydia trachomatis pORF5 plasmid protein.Methods The hybridoma cells stably secreting specific McAb against pORF5 were cultured in a large scale,and protein G purification by affinity chromatography was used to purify 2H4 McAb.ELISA was used to determine the antibody titer,and identify McAb isotype.Immunofluorescence assay (IFA) and Western blot were performed to detect McAb specificity.Results The purity of 2H4 antibody was 93％,the titer reached 1:1024,and 2H4 McAb was identified to belong to IgG2a isotype,2H4 McAb reacted strongly with the GST-pORF5 fusion protein and endogenous pORF5 protein expressed by Chlamydia trachomatis serovar A,D,L2,Chlamydia muridarum ( MoPn ),Chlamydia psittaci 6BC,but not other chlamydial plasmid proteins and Chlamydia pneumoniae(Cpn) AR39 strain.Conclusion2H4 McAb against pORF5 protein was successfully purified with a high titer and specificity which lay a foundation for further study on pORF5 protein structure and function.
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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.
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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.
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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.
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To explore the effect of coriaria lactone (CL)-activated astrocyte-conditioned medium on the cerebral TNF-α 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-α in the cerebral cortex and hippocampus were immunohistochemically examined by employing SP method. TNF-α 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-α 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-α in homogenate of cerebral cortex and hippocampus and cerebrospinal fluid were higher than those of the control group (P＜0.05). Itis suggested that the ACM activated by CL can enhance the expression of TNF-α in normal rats,and is related to epileptogenesis.