Objective To analyze changes of DNA demethylation analysis of FOXP3 TSDR at the beginning of the secondary pulmonary tuberculosis patientsby utilizing real-time PCR technology.Methods To select 47 patients of secondary pulmona-ry tuberculosis as a research group from June 2014 to May 2015 and 40 healthy donors as a control group.The peripheral blood mononuclear cells (PBMC)of research group and control group were isolated.CD4+CD25+T cells were isolated from PBMC.Genomic DNA was isolated from CD4+CD25+T cells.PCR was performed in a final reaction volume containing dem-ethylation-specific primers.Plasmid standard was generated by PCR products were enzyme digestion,TOPO TA cloning,and recycling and purification.A real-time PCR system was established by quantitatively analyzing the specificity of FOXP3 TS-DR demethylation to treg (regulatory T-cell).Treg numbers of control group at week 0 and research group treated at week 0,week 2,week 4 and week 8 by using real-time PCR assay of the FOXP3 TSDR demethylation.The experimental data was analyzed by using SPSS 1 6.0 software.Results The M.tuberculosis in sputum of research group were positive by smear mi-croscopy,however the results of control group were negative.The treg frequency of control group,2+ group and 3+ group respectively was 1.63%±0.70%,1.96%±0.10% and 0.86%±0.21%,respectively.The difference between the treg fre-quency of control group and that of 2+ group by smear microscopy had not statistical significance,however which of 3+group was opposite.The average treg frequency of research group treated at week 0,2,4 and 8 respectively was at 1.05%, 2.04%,3.44% and 2.79%,range of which respectively was 0.32%~2.03%,0.95%~3.95%,2.35%~4.95% and 1.02%~4.27%,95% confidence interval of which respectively was (0.93%,1.18%),(1.85%,2.24%),(3.27%,3.61%) and (2.60%,2.98%).The treg frequency of difference between control group and research group at week 0 had statistical significance (t=4.669,P<0.05).The treg frequency was influenced by time of therapy,using One-Way ANOVA analysis (F=347.2,P<0.001,df=3,within-subjects Contrasts:F=407.4,P<0.001,df=3).Test of the treatment time and group interaction effect was linear (F=678.2,P<0.001,df=1).Conclusion DNA demethylation analysis of FOXP3 TSDR was high sensitivity and specificity in monitoring changes of treg at the beginning of the secondary pulmonary tuberculosis pa-tients.

Objective To investigate the role of bone marrow mesenchymal stem cells (MSCs) transplantation in repairing injured intestinal mucosa of acute pancreatitis.Methods MSCs were harvested and cultured from femurs of male SD rats.Twenty female SD rats were divided into three groups,and serve acute pancratitis (SAP) model was induced by intraperitoneal injection of L-arginine (2 g/kg) twice.Twelve hours after SAP model established,MSC transplantation group (n=8) were injected MSCs (5 × 106 cell/rat) through tail vein for three days,and SAP group (n=6) were injected the same volume of saline through tail vein as control.Control group (n=6) were only injected the same volume of saline without any treatment.All the rats were sacrificed at 72 hours after model established.The small intestinal tissues were taken for HE staining and pathological score,the TNF-α mRNA and IL-1β mRNA expression level in small intestine and pancreas were tested by RT-PCR.Y chromo-some (Sry) gene in pancreatic and intestinal tissue was examined by polymerase chain reaction (PCR).Results The relative expression quantity of TNF-a mRNA and IL-1β mRNA in pancreas was significant higher in SAP group and MSC transplantation group than in control group (7.22 ± 1.99,3.46± 1.75 vs 1.32 ± 1.04 ; 2.71 ± 0.56,1.92 ± 0.28 vs 0.61 ± 0.45 ),the difference was statistically significant (F=18.375,F=22.701; P＜0.05).Compared with SAP group,the expression quantity of TNF-α mRNA and IL-1β mRNA in pancreas was significantly decreased in MSC transplantation group,the difference was statistically significant (P＜0.05).The relative expression quantity of TNF-α mRNA and IL-1β mRNA in small intestine was significantly higher in SAP group and MSC transplantation group than in control group (3.93 ± 1.08,2.13 ± 0.53 vs 0.68 ± 0.42 ; 2.44 ± 1.54,1.02±0.44 vs 0.60±0.14),the difference was statistically significant (F=21.772,F=6.132; P＜0.05).The expression of TNF-αmRNA and IL-1β mRNA in MSC transplantation group was lower than that in SAP group,the difference was statistically significant (P＜0.05).Compared with SAP group,pathological score indicated that small intestine injure was slighter in MSC transplantation group (3.83±0.28 vs 2.83±0.56),the difference was statistically significant (F=12.013,P＜0.05).Sry gene could be detected in the pancreatic and intestinal tissue of MSC transplantation group.Conclusion Allogeneic MSC transplantation group can inhibit Pro-inflammatory cytokines expression in acute pancreatitis,relieve the intestinal mucosa injury and may involve in the intestinal tissue repair.

In order to express the gene of LEN-5 β-lactamase from a Klebsiella pneumoniae strain,plasmids in the strain were extracted and an 879bp product of LEN-5 gene was obtained with PCR.After being digested with Nde I and Xho I,LEN-5 gene was cloned into pET-26b (+) vector.Then it was confirmed by digestion and DNA sequencing in recombinant plasmid before transformed into E.coli BL21 (DE3).After inducing by IPTG,LEN-5 β-lactamase was expressed.Protein extraction was processed by ultrasonic and protein activity was detected by nitrocefin.The isoelectric focusing electrophoresis showed a pI of 7.6.These results indicated that the LEN-5 gene has been cloned and expressed in prokaryote cell successfully.

Objective To investigate the expression of Smac/DIABLO, XIAP mRNA in acute pancreatitis (AP) and the relationship with the severity in rats.Methods Fifty-four SD rats were randomly divided into three groups:sham-operation (SO) group, acute edematous pancreatitis (AEP) group and acute necrotizing pancreatitis (ANP) group.The models of AEP and ANP were induced by retrograde injection of 1％ and 3.5％ sodium deoxycholate into the pancreaticobiliary duct respectively.The specimens of pancreatic tissue at 3 h, 6 h, 12 h were collected, pathological changes of the pancreas were observed, apeptosis in pancreas were detected by TUNEL method and the expression of Smac/DIABLO, XIAP mRNA were analyzed by real-time PCR.Results Pathological changes of the pancreas confirmed the establishment of AEP and ANP.Apeptosis indexes in SO group, AEP group and ANP group were 0.67±0.82, 6.62 ±0.78 and 4.70 ±0.82, and the differences were significant (P< 0.05).The expression of Smac/DIABLO mRNA of AEP group increased with time, while the expression of ANP group decreased with time.Compared with SO group, Smac/DIABLO mRNA expressions at 6 h in AEP and ANP group were 2.41 ± 0.92 and 1.47± 0.53, and the differences were significant (P<0.05).By contrast, the expressions of XIAP mRNA in AEP group decreased with time,while the expressions in ANP group increased with time.The expressionsof XIAP mRNA at 6 h in AEP and ANP group were 5.51 ± 1.07 and 6.99 ± 1.00, and the differences were significant (P<0.05).Conclusions In acute pancreatitis, the expression of Smac/DIABLO mRNA was consistent with the apoptosis of pancreatic acinar cells, but not consistent with the severity of pancreatitis.The expression of XIAP mRNA was consistent with the severity of pancreatitis.Smac/DIABLO, XIAP mRNA is associated with regulation of apoptosis.

Objective To reproduce an SD rat model of prostatic calculus by using nanobacteria (NB), and explore the role of NB in contributing to prostatitis and prostatic calculus. Methods Twenty adult male SD rats were randomized to the control group and 20 to the model group. Rat prostate infection models were reproduced by infusing 0. 2 ml (Concentration, 1 Mai unit) NB suspension transurethrally. 0.2 ml physiological saline was infused transurethrally in the rat control group. The rats were sacrificed 4 and 8 weeks later and prostatic pathology were viewed by hematoxylin and eosin (HE) staining. Lithogenesis was observed by scanning electron microscope (SEM) or Transmission electron microscopy (TEM). Re-isolation, culture and identification of nanobacteria were also done in rat prostatic tissues. Results Chronic inflammatory changes of prostates were shown in the model group at both 4 weeks and 8 weeks after infusing NB suspension. Prostatic calculi were detected by SEM and TEM at 8 weeks in the prostates of the rat model group (7/10). Neither chronic inflammatory changes nor prostatic calculus was found in the control group. NB was positive in the model group, but negative in the control group. Conclusions NB infection could cause chronic prostatitis and prostatic calculus in rats.

Objective To investigate the expression of melatonin MT1 receptor in rats with acute necrotizing pancreatitis (ANP) and the protective effects of melatonin (MT) pre-intervention for the pancreas. Methods Fifty-four male Sprague-Dawley (SD) rats were randomly divided into three groups:sham-operation group, ANP group and MT-pretreated group. The models of ANP were induced by retrograde injection sodium taurocholate into the bili-pancreatic duct. MT group undergoing intraperitoneal injection 50 mg/kg 30 minutes before the establishment of ANP models. Four, 8 and 12 hours after the onset of operation, the levels of serum amylase and pathological changes of the pancreas were observed. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) and tumor necrosis factor-alpha (TNFα) in the pancreas were measured. The expression of MT1 protein and MT1 mRNA in pancreas were separately analyzed by immunohistochemistry and real-time PCR. Results (1) Pancreatic pathological damage in ANP groups was progressive exacerbated. It was obviously ameliorated in MT group as compared with ANP group ( P ＜ 0.05 ); (2) Compared with SO group, the levels of serum amylase, MDA and TNFα in the pancreas were significantly increased in ANP group (P ＜0.05 or P ＜0.01 ). They were markedly decreased in MT group as compared with ANP group [ 12 h, (2348.00 ±278.90)U/L vs (3194. 83 ±538.10)U/L,(2.255 ± 0.472 ) μmol/L vs ( 2.960 ± 0.722 ) μ mol/L, ( 102.929 ± 29.399 ) ng/L vs ( 378. 544 ±183.454)ng/L, P ＜ 0.05 ]. The level of SOD was decreased in ANP group compared with SO group (P ＜0.05) and increased in MT group[ 12h, (11.448 ± 1.594)U/L vs (8.427 ± 1.950)U/L, P＜0.05] ;(3)Compared with SO group, the expression of MT1 protein and MT1 mRNA in ANP group were down-regulated as the severity of the disease increased ( P ＜ 0.05 ). They were significantly higher in MT group than ANP group. Conclusions Melatonin pre-intervention is able to increase SOD level and decrease MDA, TNFα levels, thereby reducing pancreatic injury. The MT1 might play an important role in the pathogenesis of ANP. MT might exert protective effects for the pancreas in ANP rats through increase the expression of MT1.

Objective To analyze the value of serum Treg cell related factors and chemokines in patients with tuberculous pleurisy .Methods From July 2015 to December 2016 ,92 cases of tuberculous pleurisy in our hospital were selected as the observation group ,and 92 healthy persons at the same time were selected as the control group .The levels of Treg cell related factors[monocyte chemoattractant protein (MCP)-1 ,IP-10 ,CCL-3 and CCL-16] and IL-10 ,TGF-βand IL-35] were detected and compared in the two groups ,and the levels of these indexes were compared in different classifications and stages of tuberculous pleuritis .Results The ser-um Treg cell related factors and chemokine levels in the observation group were significantly higher than those in the control group (P<0 .05) .The expression level of tuberculous empyema was higher than that of dry pleuritis and exudative pleuritis ,the patients with exudative pleuritis were higher than those of dry pleuritis , and the patients with multiple pleuritis were higher than those with idiopathic and concomitant pleuritis ,the difference was statistically significant (P<0 .05) .Conclusion The serum Treg cell related factors and chemo-kines in patients with tuberculous pleurisy are highly expressed ,and the classification and staging of the dis-ease have great influence on the expression ,and the above indexes have high detection value in the patients with tuberculous pleurisy .

OBJECTIVETo study micro morphology and element-mixing distribution of different alloys welded in laser and analyze the feasibility of laser welding different alloys.

METHODSAlloys and titanium were matched into 4 groups: Au-Pt with Ni-Cr; Au-Pt with pure Ti; pure Ti with Ni-Cr; Ni-Cr with Co-Cr. They were welded in laser. Changes in metallography after hybridization of crystalline grain, ranges of heat-affected zone and pores were observed through SEM with ultra-thin windowed X-ray energy atlas. Meanwhile 10 testing points were chosen with area of 300 micro m x 900 micro m along the welding surface from the side A alloy to the side B alloy, than the element mixing distribution and tendency were analyzed with X-ray energy atlas.

RESULTS1. Hybridization of different alloys: (l) in the group of Au-Pt with Ti, there was titanium element mixing into Au-Pt tissue gradually and evenly on the Au-Pt side of the interface without clear boundary and increasing in size of crystalline grain. However, there was titanium crystalline grain increasing in size, irregular morphology and small sacks on the titanium side with clear boundary. (2) in the group of Ni-Cr with Ti, there was mixing regularly, slow transition and interlocks between crystalline grains on the Ni-Cr side of the in terface. Poor transition, clear boundary and small cracks were observed on titanium side. (3) in the group of Co-Cr with Ni-Cr, there was good transition, obscure boundary on both sides resulting from network, cylinder and branch structure growing. 2. Element-mixing distribution of different alloys. In fusion zone, the metal elements in matched groups mixed well and hybridized into new alloys except titanium blocks. The location of wave peak depended on the composition of alloys. Most of elements were from the alloy far from the fusion zone.

CONCLUSIONThe hybridization between pure titanium and any other alloys is not good The effect of laser welding different alloys is ideal except with pure titanium.

Objective To report the clinical management of the first case of human infection with influenza A (H7N4) in the world ,and to explore the clinical characteristics and therapeutic regimen of H7N4 infection in humans .Methods A case of confirmed human infection with influenza A (H7N4) virus was hospitalized in the Third People′s Hospital of Changzhou in Jiangsu Province on January 1st ,2018 . The clinical characteristics and treatment were analyzed by reviewing the clinical data and management of the patient ,and compared with the clinical characteristics of human infection with H 7N9 cases .Results The patient was an elderly woman with hypertension ,who had an exposure history of live poultry before onset .The patient had flu-like symptoms including fever , cough and expectoration . The symptoms worsened in the fifth day and presented as respiratory failure and circulatory disturbance .The count of white blood cells ,lymphocytes and platelets counts declined ,while the levels of aspartate aminotransferase and c-reactive protein slightly increased .The levels of creatine kinase and lactate dehydrogenase deformity increased .The pulmonary lesions manifested as exudation and consolidation of both side in a short period of time after the onset . Treatment was initiated according to the epidemiology history , clinical manifestations ,laboratory and imaging characteristics . The primary diagnosis was human infection of avian influenza virus .The patient was treated with oxygen therapy ,antivirus ,anti-infection and short-duration corticosteroids treatment . Early and timely throat swab specimens were tested . The patient recovered and discharged from hospital after active treatment .It was confirmed by the Chinese Center for Disease Control and prevention that the patient was infected with avian influenza A (H7N4) virus ,and no close contact was infected .Conclusions There are many similarities of clinical features between case of human infection with H7N4 and that of H7N9 . The early treatment of neuraminidase inhibitors is recommended according to the diagnosis and treatment of human infection with H 7N9 .In the clinical practice ,the avian influenza nucleic acid should be tested in time to determine the cause of the disease in those patients with suspected viral pneumonia ,especially in those who has an avian contact history .

Objective To explore the molecular mechanism of Atractylodes macrocephala in the treatment of Ulcerative colitis(UC)based on network pharmacology,and verify it with animal experiments.Methods The active components of Atractylodes macrocephala was screened from the TCMSP database,the TCM-ID database,and in combination with relevant references,and the corresponding targets were obtained through Swiss database.The relevant targets of UC were obtained from GeneCards database,construct the"drug-component-target-disease"network diagram and"pathway-active ingredient-target"network diagram and draw PPI network diagram;GO function enrichment analysis and KEGG signal pathway annotation analysis were carried out.Autodock software is used for molecular docking of active components and targets.Then,the experimental validation of the network pharmacology prediction was carried out.The mouse UC model was induced by dextran sodium sulfate(DSS).The pathological changes of the colon tissue,the number of goblet cells,and the positive expression of inflammatory factorswere detected by HE staining,AB-PAS staining and immunohistochemistry in colon tissue of UC mice.Results The results have shown 30 active ingredients including atractylolactone I,II and III were screened,and 591 corresponding targets were obtained,of which the key target was IL-1β、TNF-α and so on.Molecular docking show that the core components had good binding affinity with the key targets.And the results of animal experiments showed that the alcohol extract of Atractylodes macrocephala could significantly increase the colon length,reduce the DAI score,improve the pathological changes of colon tissue of UC mice,increase the number of goblet cells,and inhibit the expression of IL-1β,TNF-α in colon tissue.Conclusion This study indicated that Atractylodes macrocephala could regulate the release of inflammatory factors through multiple components,multi-target and multi-channel,which could inhibit inflammatory reaction and play a role in improving UC.