Objective:Generation and functional analysis of EBV LMP2A specific CTL elicited by DC transfected with recombinant adenovirus in vitro .Methods:PBMC were isolated from healthy EBV carriers and NPC patients, and then cocultured with autologous mature Ad5 LMP2A transfected DC at the ratios of 20∶1. Cytotoxicity of LMP2A specific CTL was determined with LDH release assay, the populations of CTL were performed by FACS,the IFN ? secretion and FasL mRNA expression of the CTL were detected by biological activity assays and RT PCR, respectively.Results:The results showed that high cytotoxicity of LMP2A specific CTL could be elicited by autologous transfected DC. The cytotoxicity boosted with the increase of transfected DC stimulation times, but there were no significant changes between two and three stimulations.The phenotypic analysis demonstrated that the LMP2A specific CTL induced at day 14 consisted of a majority of CD4 + and CD8 +T cells, and only a small percentage of CD56 + cells. The IFN ? secreted in the supernatants of cell culture also increased with the stimulation times. In addition, the specific CTL at day 14 from EBV healthy carriers could express FasL mRNA.Conclusion:Strong and functional EBV specific CTL could be generated by autologous mature DC transfected with adenovirus encoding LMP2A, which could provide a rationale for the immunotherapy of EBV associated NPC. [

Objective To analyze the expression profile of long non-coding RNA (lncRNA) in the lipopolysaecharide (LPS)-induced inflammation of monocyte-derived macrophages.Methods Peripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. The macrophages were divided into blank control group and LPS (1 mg/L) stimulated 12 hours group. Culture supernatants and cell pellets were harvested in each group, enzyme linked immunosorbent assay (ELISA) was used to assay the production changes of interleukins (IL-1β and IL-6), and tumor necrosis factor-α (TNF-α) in the supernatant. The technique of lncRNA microarray was used to test the lncRNA expression profile in LPS-induced inflammation of macrophages and control macrophages. The raw data of lncRNA were pretreated for normalization. Five lncRNA expressions were validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Furthermore, qRT-PCR was used to detect the expression of NR_028034 in macrophages after LPS-induced inflammation.Results ① The contents of IL-1β (ng/L:562.93±61.17 vs. 59.74±15.68), IL-6 (ng/L: 702.46±92.31 vs. 71.66±18.25) and TNF-α (ng/L: 794.50±63.89 vs. 85.12±22.07) in the LPS group were significantly higher than those in the blank control group (allP < 0.01). These results indicated that the inflammatory model of human macrophages was constructed successfully. ② Compared with blank control group, and 1479 lncRNA which have more than 2 folds variation and significant difference (P < 0.05) by statistical analysis was defined as lncRNA with differential expression. Among these lncRNA, LPS group showed 953 up- regulated and 526 down- regulated genes by 2 folds and 49 up- regulated and 35 down- regulated genes by 5 folds. ③ qRT-PCR results were generally consistent with the microarray data. ④ The expression of NR_028034 was increased by (4.41±0.65), (11.56±2.04), (18.58±1.36) folds compared with blank control group at 3, 6, 12 hours after LPS stimulation (allP < 0.01).Conclusions These data show a significantly altered lncRNA expression profile in the LPS-induced inflammation of monocyte-derived macrophages, suggesting that lncRNA may be involved in regulation of macrophages inflammatory response.

Objective To investigate the effects of lincRNA-cox2 on the polarization of murine RAW264. 7 macrophages by analyzing the expression of lincRNA-cox2 in RAW264. 7 macrophages of M1 and M2 phenotypes. Methods Murine RAW264. 7 cells were induced by IFN-γand LPS to polarize to M1 phenotype, and were induced by IL-4 to polarize to M2 phenotype. The expression of lincRNA-cox2 in M1 and M2 macrophages were analyzed by real-time quantitative PCR ( RT-PCR) . We designed and synthesized siRNA oligo for lincRNA-cox2 and unrelated sequences. Then the siRNA oligo and NC oligo were transfected into RAW264. 7 cells by LipofectmineTM 2000. The transfected RAW264. 7 cells were induced by IFN-γand LPS or by IL-4 to polarize to M1 or M2 macrophages. Enzyme linked immunosorbent assay ( ELISA) was performed to measure the secretion of IL-10 and IL-12 induced in different conditions. The expression of in-ducible nitric oxide synthase ( iNOS ) , TNF-α, arginase 1 ( Arg-1 ) and found in inflammatory zone 1 (Fizz1) at mRNA level were detected by RT-PCR. The M1 macrophages were transfected with siRNAs to knock down the expression of lincRNA-cox2 for analyzing the biological effects of lincRNA-cox2 on the polar-ization of macrophages. Results The relative expression of lincRNA-cox2 in M1 macrophages was signifi-cantly higher than that in RAW264. 7 cells and M2 macrophages. Compared with the control group, the RAW264. 7 cells transfected with lincRNA-cox2-siRNA showed decreased secretion of IL-12 and inhibited expression of iNOS and TNF-αat mRNA level after IFN-γand LPS induction, but increased secretion of IL-10 and enhanced expression of Arg1 and Fizz1 at mRNA level after IL-4 induction. Transfecting the M1 mac-rophages with lincRNA-cox2-siRNA inhibited the secretion of IL-12, but promoted the secretion of IL-10. Conclusion This study indicated that lincRNA-cox2 was involved in the regulation of macrophage pheno-types by promoting the polarization to M1 macrophages and inhibiting the polarization to M2 macrophages.

Objective To analyze the expression profile of circular RNA (circRNA) in LPS-treated murine peritoneal macrophages (MPMs) and to investigate the effects of mmu_circ_0000790 (circ790) on the secretion of pro-inflammatory cytokines in MPMs following LPS stimulation.Methods MPMs were isolated from C57BL/6 male mice and then stimulated with (LPS treatment group) or without (blank control group) 1 mg/L of LPS for 12 hours.Supernatants of cell culture and cell pellets were collected from each group.Enzyme linked immunosorbent assay (ELISA) was used to measure the changes in the secretion of IL-1β,IL-6 and TNF-α in the supernatants.Expression profile of circRNA in macrophages from the two groups (n=3) was analyze by circRNA microarray.Some differentially expressed circRNAs were validated by real-time PCR (RT-PCR).Moreover,RT-PCR was also performed to detect the expression of circ790 in MPMs after LPS stimulation.Small interfering RNA (siRNA) oligo specific for circ790 was designed and synthesized.Then the synthesized siRNA oligo and normal control (NC) oligo were transfected into MPMs by LipofectamineTM2000.The transfected MPMs were treated with LPS.ELISA was used to detect the levels of IL-1β,IL-6 and TNF-α in the supernatants.Arraystar's home-made microRNA target prediction software was used to predict circ790/microRNA.Results The concentrations of IL-1β,IL-6 and TNF-α in the LPS treatment group were significantly higher than those in the blank control group (P<0.01).These results indicated that the inflammatory model of MPMs was successfully constructed.Statistical differences in 34 differentially expressed circRNAs were found between the two groups (P<0.05).Among them,12 circRNAs were up-regulated and the other 22 circRNAs were down-regulated in the LPS treatment group.RT-PCR results were generally consistent with the microarray data.The expression of circ790 was increased by (1.94±0.15),(3.18±0.13) and (4.21±0.22) folds as compared with that of the blank control group at 3,6 and 12 h after LPS stimulation (P<0.05).After transfecting the MPMs with circ790-siRNA,the secretion of IL-6 was significantly decreased (P<0.05) without notable influence on the secretion of IL-1β and TNF-α.circ790 could function as a microRNA sponge to regulate the gene expression.Conclusion These data show a significantly altered circRNA expression profile in the LPS-treated MPMs.circ790 may be involved in the regulation of IL-6 secreted by macrophages.

Objective To detect the serum level of long non-coding RNA (lncRNA ) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in pulmonary tuberculosis (TB) patients ,and to evaluate its diagnostic value .Methods The expression of serum MALAT1 in 56 hospitalized TB patients , 35 latent TB infection (LTBI) individuals and 40 healthy controls were detected by real-time quantitative PCR .Serum levels of MALAT1 before and 3 ,6 months after anti-TB therapy in 16 TB patients were determined .Receiver operating characteristic (ROC) curve analysis was used to evaluate the sensitivity and specificity of serum MALAT 1 .The comparison between two groups was performed by Student t-test , and the comparison among three groups was performed with one-way analysis of variance test .Results Serum level of MALAT1 in TB patients was (2 .10 ± 1 .05) ,which was significantly higher than those in LTBI individuals (1 .16 ± 0 .51) and healthy controls (1 .02 ± 0 .44 ,F= 28 .53 ,P< 0 .01) .The MALAT1 level in TB patients with positive sputum smear was significantly higher than that in patients with negative sputum smear (2 .42 ± 1 .03 vs 1 .43 ± 0 .74 ,t= 2 .66 ,P< 0 .01) .Compared with pre-treatment (2 .28 ± 0 .79) ,the serum MALAT 1 levels decreased significantly in 3 months (1 .35 ± 0 .39) and 6 months (1 .05 ± 0 .30) after anti-TB therapy (t= 4 .33 ,6 .05 ;both P< 0 .01) .The area under the curve (AUC) of serum MALAT1 was 0 .821 , with sensitivity and specificity of 0 .732 and 0 .850 , respectively .Conclusion The expression of MALAT1 is up-regulated in TB patients , and could be used as potential novel biomarkers for the diagnosis of TB .

Objective To screen stable express A30P a-synuclein PC12 cell as the research object,and to preliminarily investigate the activity of different glycogen systhesis kinase 3β (GSK3β) for the regulation of autophagy pathway causes a-synuclein autophagy level change and its effects on cell growth.Methods The vector of A30P a-synuclein was used to transfect to PC12 cells.G418 was used to screen stable expressed cells.Liposome method was used to transfect GSK3β-expressed plasmid,GSK3β silence plasmid,and blank plasmid into these cell lines,and stable expressed activity of GSK3β cell lines were screened.Western blot was used to detect the expression of GSK3 β after transfection and verify the transfection efficiency.Western blot was used to test groups of cells in LC-3 Ⅱ,Beclin1,and a-synuclein expressions.Methyl thiazolyl tetrazolium (MTF) method was used to detect the change of cell proliferation.Terminal dexynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) method was used to detect apoptosis.Results (1) When GSK3β expressed group was compared to GSK3β silence group,blank plasmidgroup,and blank control group,the expressions of LC-3 Ⅱ and Beclin1 were significantly increased (P ＜ 0.05),the expression of a-synuclein was significantly reduced (P ＜ 0.05),the results of MTT and TUNEL showed that the cells were the decrease of apoptosis and increase of cell proliferation,with a statistically significant difference (P ＜ 0.05).(2) When GSK3β silence group was compared to GSK3β express group,blank plasmidgroup,and blank control group,the expressions of LC-3 Ⅱ and Beclin1 were significantly reduced (P ＜ 0.05),the expression of a-synuclein was significantly increased (P ＜ 0.05),the results of MTT and TUNEL showed that the cells were the increase of apoptosis and decrease of cell proliferation,with a statistically significant difference (P ＜0.05).Conclusions (1) Activation of GSK3β activity can improve the level of cell autophagy,enhance the ability of alpha-synuclein degradation,and promote cell survival.(2) Inhibition of GSK3β activity can reduce the level of cell autophagy,weaken the ability of alpha-synucleindegradation,and reduce cell survival.(3) Autophagy is closely related to the pathogenesis of Parkinson's disease (PD).The improvement of the level of autophagy and enhancing of the degradation of asynuclein is a new way of the future treatment of PD.

In order to investigate the effects of electric pulses on cancer cells, we carried out the experiments with exposing HepG2 and L02 to electric pulses (1 kV/cm, l00 micros, 1 Hz) for different lengths of time (8 s, 15 s, 30 s, 60 s). Annexin V-FITC Kit and Flow cytometry were used to study the apoptosis of treated cells. The results showed that the electric pulses of 1 kV/cm, l00 micros, 1 Hz for 8 s could not induce tumor cells apoptosis. Apoptosis was observed when tumor cells were stimulated for 15 s and longer, and the apoptosis percentage increased with the increase of stimulation time. Furthermore, tumor cells were more sensitive than normal cells in response to electrical pulses. Rhodamine 123 and Laser Scanning Confocal Microscope (LSCM) were used to make a real-time study of mitochondrial transmembrane potential (Deltapsim) when the tumor cells were exposed to electric pulses for 60 s. No significant change of Deltapsim was observed within 30 s stimulation. After that, the Deltapsim increased sharply and declined later, suggesting that the mitochondrial pathway may be one of the apoptosis mechanism induced by electric pulses.
Apoptosis ; radiation effects ; Electromagnetic Fields ; Hep G2 Cells ; Humans ; Membrane Potential, Mitochondrial ; physiology ; radiation effects ; Time Factors

Objective Detecting plasma level of circular RNA(circRNA)hsa_circ_0009024 in pulmonary tuberculosis(TB)patients,and evaluating its diagnostic value for TB.Methods From January 2016 to December 2016, a hosptial-based, case-control study was performed, which include 90 untreated active pulmonary tuberculosis patients(TB group),75 healthy people(healthy control)and 84 patient with other diseases(other disease group).Plasma level of circRNA hsa_circ_0009024 was detected by real-time quantitative polymerase chain reaction.Furthermore, the 90 patients with TB were divided into different subgroups according to cavity formation and the lung fields involvement: patients without lung cavity(55 cases)vs those with lung cavity(35 cases),patients with involvement of <2 lung fields(49 cases)vs≥2 lung fields(41 cases).Plasma levels of hsa _circ_0009024 of 41 TB patientswere monitored andcomparedbefore and after 3 months anti-TB therapy.The sensitivity and specificity of plasma hsa_circ_0009024 were analyzed by using the receiver operating characteristic(ROC)curve.The comparison between two groups was performed with Mann-Whitney U test and the comparison among multigroupswas conducted with Kruskal-Wallis H test.Results Plasma levels of hsa_circ_0009024 in TB patients[1.98(1.42, 2.71)]were significantly higher than healthy controls[1.03(0.78,1.33)]and other disease groups[1. 13(0.77,1.51)](H=76.58,P<0.0001).Plasma levels of hsa_circ_0009024 in cavity pulmonary TB patients were higher than pulmonary TB patients without cavity(U=392.50,P<0.0001).Plasma levels of hsa_circ_0009024 in TB patients with involvement of ≥2 lung fields were higher than <2 lung fields(U=590.50,P=0.0008).As compared to pre -treatment[2.01(1.41, 2.71)], the plasma hsa_circ_0009024 levels decreased significantly in 3 months[1.22(0.85,1.47)](U=292.50,P<0.0001)after anti-TB therapy.The area under the receiver operating characteristics curve(AUC)of plasma hsa_circ_0009024 in discriminating the patients with TB from normal controls, pneumonia patients and lung cancer patients were 0.841 and 0.811, respectively.Conclusion The hsa_circ_0009024 can be used as a potential biomarker in TB diagnosis and monitoring.

Although immune checkpoint inhibitors(ICIs)are widely used in cancer therapy,showing great advantages and development potential,it is accompanied by a series of immune-related adverse reactions,of which myositis is a potentially fatal adverse event,which has attracted great attention.Herein,we reported a case of advanced esophageal cancer with myositis after treatment with camrelizumab,which was characterized by myasthenia gravis(MG)with myasthenic crisis,and recovered after active rescue by multidisciplinary cooperation.