Objective To explore the effects of murinecytomegalovirus(MCMV)infected mouse embryonic fibroblasts(MEF)on the proliferation and activation of regulatory T cell in vitro. Methods A co-culture system of T cell and MCMV infected MEF(T-MEF MCMV)was established.The viral load of supernatant was determined by plaque assay.The proliferation of T cells was observed with cell counting.The proportion of CD4+ CD25+ cells was measured by flow cytometry.The levels of Foxp3 protein were measured by Western blot.Reverse transcription polymerase chain reaction(RT-PCR)assay was utilized tO determine whether MCMV infection of MEF influenced the level of transforming growth factor(TGF)-β mRNA.The level of TGF-β protein in supernatant was measured by double-antibody sandwich enzyme linked immunosorbent assay(ELISA).The difference between T-MEF MCMV group and control group was assessed by one-way ANOVA.Results When T cells were co-cultured with MCMV infected MEF for 1 day and 3 days,the viral load in supernatant decreased.But when co-culture lasted for 6 days,the antiviral effect obviously diminished,as the viral load[(5.58±0.67)×105 PFU/mL]of the experimental group showed no statistic difference with MEF MCMV control group[(6.05±0.34)×105PFU/mL].When co-cultured with MCMV infected MEF for 3 days,T cell increased from pre-culture level of[(2.02±0.05)×106/mL]to(2.25± 0.13)×106/mL(P＜0.05).But when co-culture lasted for 6 days,the number of T eelI returned to (2.08±0.14)×106/mL,which had no statistic difference with that of co-culture for 3 days system. Both the expressions of Foxp3 protein and the proportion of CD4+ CD25+ Foxp3+ Treg cells in T-MEF MCMV group were up-regulated as the infection time extended,which were two times and three times of the control group level,respectively.The mRNA level(A value)of TGF-β in MCMV infected MEF increased from baseline of 1.09±0.13 to 3.15±0.54 on day 3 after infection.The expression of TGF-β in supernatant 3 days after infection was(3.85±0.32) μg/L,which was significantly higher than that before infection[(1.74±0.14)μg/L,P＜0.05].Conclusions Activated T cells have antiviral effect.However,the function of T cells is rapidly inhibited after activation,which may be due to the expression of Foxp3 mRNA induced by MCMV infected MEF and increased CD4+ CD25+ Treg proportion of the co-cultured T cells.TGF-β level is significantly increased after CMV infection,which may be an important mechanism of Treg proliferation.MCMV may manipulate Treg to evade specific immune elimination and,as a result,to cause CMV replication.

Objective To investigate the effect of jejunal infusion of amino acids on secretion of gastrointestinal hormone in healthy dogs.Methods Six healthy adult dogs were treated with jejunal fistulas and femoral vein intubation.Twenty-four hours after the operation,solution of 8 different amino acid monomers (experimental group) or normal saline (control group) were infused into the jejunum of the dogs every 24hours.The levels of cholecystokinin (CCK),motilin,and gastrin in the peripheral plasma were measured using radioimmunoassay at the start of infusion (0 minute),and 30,60,90,and 120 minutes after infusion.Results Compared with the control group,the serum CCK level in the phenylalanine group was significantly higher 30 and 60 minutes after infusion [(1.25 ±0.19) ng/L vs.(0.66 ±0.14) ng/L,(1.23 ±0.12) ng/L vs.(0.80 ± 0.03) ng/L,both P ＜ 0.01],while that in the tryptophan group was significantly higher 30 minutes after infusion [(1.08 ±0.26) ng/L vs.(0.66 ±0.14) rig/L,P ＜0.01].The other measurement results showed no statistically significant differences.Conclusions Jejunal infusion of phenylalanine or tryptophan may stimulate the secretion of gastrointestinal hormone to some extent.Aromatic amino acids (phenylalanine and tryptophan) is more potent in triggering the release of CCK than aliphatic (leucine,isoleucine,and methionine) and charged amino acids (aspartic acid,arginine,and glutamate).The mechanism may be related to the properties of the amino acids.

Female ; Genetic Predisposition to Disease ; HSP70 Heat-Shock Proteins ; genetics ; Humans ; Lung Neoplasms ; genetics ; Male ; Polymorphism, Genetic

Objective To investigate the anti-oxidant effect and the possible mechanisms ofpicrodide II in cerebral ischemia/reperfusion injuries in rats. Methods A total of 90 adult, healthy, mmale Wistar rats were used to established the middle cerebral artery occlusion reperfusion (MCAO/R) models by intraluminal monofilament suture on the left external-internal carotid artery. The treatment group and the positive control group were respectively injected with 1.0％ picroside II (10 mg/kg, 250 μl) and salvianic acid A sodium (10 mg/kg, 250 μl) via the tail vein, and the negative control group and sham-surgery group were injected with 0.1mol/L phosphate buffer saline (PBS) 250 μl. The neurological deficit scores were evaluated with Bederson's test. The cerebral infarction volume was observed with tetrazolium (TTC) staining. The apoptosis positive cells were counted by terminal deoxynucleotidyl transferase dUTP nick-end labeling and the expressions of inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) were detected with immunohistochemical assay.The concentration of iNOS and SOD proteins in brain tissue was detected by enzyme linked immunosorbent assay.Results Neurological behavioral malfunction appeared in all the rats with MCAO/R. The infarction focuses emerged in the ischemic hemisphere following the MCAO/R injuries. The number of apoptotic cells and the expression of iNOS increased while the SOD reduced after MCAO/R. After the treatment of picrodide Ⅱ, the nervous behavioral function (1.28±0.38)improved, the infarction volume(68.73±4.46)％ reduced, the number of apoptosis positive cells(6.10± 1.26), the expressions and the concentrations in brain tissue of iNOS(4.67+0.51)decressed while those of SOD (0.53 ±0.14) increased significantly compared with the negative control groups(t=3.16、 2.51、 4.15、3.12、 3.25, P＜0.05). Conclusion PicrodideⅡ might play a neuroprotective effect by inhibiting the neuronal apoptosis and the expressions of iNOS and SOD after cerebral ischemia/reperfusion injuries.

Objective:To screen the early biomarkers associated with lung carcinoma by using proteomic method. Methods:The proteins of early lung squamous cell carcinoma tissues and adjacent normal tissues were separated by two-dimensional gel electrophoresis (2-DE) and differentially expressed proteins were screened by mass spectrometry (MS). The differential expressions of part proteins were verified by immunohistochemical method. Results:The proteomic profiles of lung squamous cell carcinoma and the adjacent normal tissues were established and the average matched protein spots in the two group were 626 and 602, respectively. Ten protein spots with higher expression in lung squamous cell carcinoma tissues were identified as Annexin 1(Anx-A1),heat shock proteins (HSP27), etc. The proteins were mainly related with cell cycle and signal transduction. The results of immunohistochemistry showed that the positive expression rates of Anx-A1 and HSP27 in lung squamous cell carcinoma tissues were significantly increased (P<0.05). Conclusion:Proteomic technology was an effective method for preliminary identification of the proteins associated with early lung carcinoma. The identified proteins laid a solid foundation for further screening the molecular markers for early diagnosis and treatment of lung cancer.

Objective We examined the in vivo effect of the antisense or/and decoy oligonucleotide of nuclear factor-?appa B (NF-?B) on balloon-injured smooth muscle cell proliferation and MMP-9 in the carotid artery of rats. Methods Sprague-Dawley rats underwent balloon-dilation injury of the left carotid artery. Rats were divided into 7 groups(n=18) and each group included 6 time points (6 h and 1,3,5,7,14 d)(n=3). Results The mean intima/media ratio increased significantly in sense group、scramble group and model group and reached the maximum at 7 d after rat injured carotid artery, compared with normal group, antisense group, decoy group and decoy plus antisense group(P

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
Astrocytes/*cytology ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; Neurons/*cytology ; Stem Cells/*cytology ; Tretinoin/pharmacology

Objective:To investigate the clinical feature, diagnosis and treatment of autoimmune pancreatitis(AIP).Methods:Clinical data of 20 AIP patients admitted to the First Affiliated Hospital of University of Science and Technology of China from Jan 2014 to Dec 2019 were retrospectively analyzed.Results:Nineteen patients were diagnosed with type 1 AIP and 1 patient was with type 2 AIP. Fifteen patients were diagnosed by imaging, serology and other organ involvement, and 5 patients were confirmed by postoperative histopathology. Thirteen patients received glucocorticoid therapy. Five patients have not received glucocorticoid therapy after surgery.One patient refused treatment, and 1 patient is currently under clinical observation. Seventeen of the 20 patients were followed up, 11 patients were on glucocorticoid therapy with related clinical symptoms being gradually improved, serum IgG4 decreased and imaging findings improved. Five patients did not relapse after drug withdrawal. Three patients had recurrence of jaundice after drug withdrawal. One patient had recurrence of pancreatic lesions after drug withdrawal. Two patients had recurrence of high serum IgG4 after tapering the doses, these 6 patients were treated with steroid maintenance therapy. One patient died of repeated gastrointestinal bleeding 2 months later, and another 4 surgical patients and 1 patient under clinical observation are in good condition.Conclusions:AIP should be diagnosed in combination with clinical manifestations, serological examination, imaging examination and histopathology, especially focal lesions should be differentiated from pancreatic cancer, so as to avoid missed diagnosis and unnecessary surgical intervention.

Objective To explore the role of CD4+CD25+regulatory T cells(Treg)in the co-culture svstem consisting of T cells and mouse embryo fibroblasts(MEF)infected with murine cytomegalovirus (MCMV)in vitro.Methods A co-culture system of T cells and MCMV infected MEF(MEFMCMV)was established.The viral load in the supernatants was determined by plaque assay.TH1/TH2 differentiation-specific transcription factors T-bet/GATA-3 were assayed by Western blot.The levels of cytokines IL-4,IL-10and IFN-γ in the co-culture supenatants were measured by double-antibody sandwich ELISA.Results After 3 d incubation,the viral load in the supernatants of TdepTreg-MEFMCMC group,in which the T cells depleted Treg(TdepTreg)were co-cultured together with MEFMVMV,was significantly lower than that in MEFMCMV group.And in the co-cultivation of MEFMCMV and T cells without Treg the expressions of T-bet/GATA-3,IL-4,IL-10 and IFN-γ incteased significantly.Compared with the virus content in the TdepTreg-MEFMCMV group,the MCMV load in the"add-on Treg group"increased and the levels of T-bet/GATA-3 and IFN-γ were lower,and the expression of IL-4 didn't show any significant difference. Compared with the level of IL-10 in the TdepTreg-MEFMCMV group,the IL-10 level in the"add-on TREG group"didn't show any significant differece when the Treg ratio among total T cells was 1%～2%,and increased significantly when the Treg ratio was more than 5%.These eflfects were all correlated with Treg ratios in a dose-dependent manner.Conclusion MCMV infected MEF can induce the proliferation and activation of effector T cells,but Treg can inhibit the T cell-mediated protective effect on MCMV infection.

BACKGROUND:IPS e. max Press porcelain crown has been widely used in clinical restoration because of its aesthetic effect and biological properties. OBJECTIVE:To explore the effect of IPS e. max Press porcelain crown in esthetic restoration of anterior teeth in terms of marginal adaptation and color match. METHODS:Total y 52 patients with 138 defective anterior teeth (20 males and 32 females, 18-45 years old, including 62 anterior tooth defects, 46 tooth traumas, 30 denticles) were divided into two groups randomly:treatment group (26 cases) and control group (26 cases) were given IPS e.max Press porcelain crown and conditional Co-Cr al oy handcuffed ceramic crowns, respectively. Al the cases were asked for a second visit in 1 week after restoration, and the integrity of ceramic veneer, marginal adaptation, gingival health and color match were evaluated during 6-month and 2-year fol ow-ups. RESULTS AND CONCLUSION:The degrees of satisfaction on the color, shape and adaptation in the treatment group were al higher than those in the control group (P<0.05). At 6 months after restoration, the marginal adaptation, gingival health and color match in the treatment group were al better than those in the control group (P<0.05). At 2 years after restoration, there was also a significant difference in the marginal adaptation, gingival health and color match between the two groups (P<0.05). IPS e.max Press porcelain crown has the advantages of aesthetics and biological characteristics, so it can be used as an ideal restoration method for esthetic restoration of anterior teeth.