Objective To study the application effect of feedback training mode on new neonatal recovery technology.Methods 101 midwives,obstetric doctors and pediatricians who participated the training were tested about the professional knowledge and skills of new neonatal recovery technology according to the teaching material of neonatal asphyxia recovery and neonatal resuscitation guidelines (2007 revision in Beijing).The results showed no statistical significance.And they were randomly divided into the control group (51 cases) and the observation group(50 cases).The control group adopted traditional method,that was,the teacher taught first and then the students took practice.The observation group used feedback training method.Results The theoretical and technical results of the observation group were significantly higher than those of the control group.The learning motivation,learning initiative,cooperation degree,ability of observation and solving problem and operation proficiency were also significantly higher than those of the control group.Conclusions Feedback training methods can significantly increase the learning effect of perinatal personnel,and make popularized the new neonatal recovery technology.

Objective To investigate the early diagnostic value of cornel confocal microscopy for the screening of small neuropathy in elderly patients with type 2 diabetic mellitus.Methods In the prospective study,96 elderly patients with diabetes as study group and 46 patients with non-diabetes as the control group were continuously collected from our hospital endocrinology and ophthalmology out patients during May 2014 to February 2016.The 96 cases of type 2 diabetes were subdivided into 47 patients with diabetic peripheral neuropathy (DPN)and 47 patients with nowdiabetic peripheral neuropathy(non DPN).Results The diabetes course was shorter in non-DPN group than in DPN group(P=0.000).The levels of glycosylated hemoglobin and urine albumin were lower in the nonDPN than in the DPN(P =0.072,0.007,respectively).The corneal nerve fiber density was lower in the DPN group than in NDPN group (P =0.000).Corneal nerve fiber density was higher in control group than in DPN and NDPN group.The differences in number of corneal nerve fibers showed no statistical significance between DPN and NDPN group (x2 =2.391,P =0.314).But the number of corneal nerve fibers was significant less in DPN and NDPN group than in control group(x2 =16.014,P =0.000).The negative correlation was found between the course of disease and corneal fibrous density by using single factor linear regression analysis.The number of corneal nerve fibers was lower in smoking group than in non-smoking group(P=0.003).The multiple linear regression analysis showed that duration of diabetes was a risk factor for diabetic neuropathy.Conclusions In some elderly diabetic patients with non-neuropathy,corneal nerve fiber density and number have been significantly decreased before nerve conductive velocity is reduced.Therefore,corneal confocal microscopy can be used to detect and diagnose small diabetic neuropathy in elderly patients with diabetes mellitus.

Objective To research the homology and cross reaction characteristics of human papillomavirus(HPV)16 type L2 N-terminal(1-200)protein in clinical common HPV infection types.Methods The amino acid sequences of the common HPV infection types(6,11,16,18 ,etc.)were blasted and it was found that 1-200 N-terminal sequence of L2 protein was highly homologous.The gene of HPV16 L2(1-200)was amplificated from tissue sample of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2(1-200).After sequencing identification,the recombinant plasmid was tranformed into E.coli BL21(DE3).Induced by IPTG,the fusion protein containing HPV16 L2(1-200)was expressed and analyzed by both SDS-PAGE and Western blot.Furthermore,the specific binding capacity of the fusion protein to the HPV 6,11,16 and 18 DNA positive patient serums were analyzed by Western blot.The fusion protein was purified with Ni-NTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG of 98 condyloma acuminatum patients,135 cervix cancer patients and 96 healthy control subjects were detected respectively by indirect ELISA.Results After comparing the amino acid sequences of the common HPV infection types(HPV6,11,16,18,etc.),We found that the homology of HPV L2(1-200)reached 52.7%-74.3%.The recombinant plasmid PGEX-4T-1-HPV 16 L2(1-200)was constructed successfully.Highly expressed HPV16 L2(1-200)fusion protein was obtained and the expression level was account for up to about 22.6% of total bacterial protein.The relative molecular mass(Mr)of the fusion protein is about 49×103,which matches up to the expected Mr Meanwhile,the serums of HPV 6,11,16,18 DNA positive patients were used as the first antibody and the specific band was detected respectively at about 49 × 103 by Western blot.Indirect ELISA showed that the A490 values of the specific IgG of condyloma acuminatum group,cervical cancer group and healthy control subjects were 0.753 ± 0.262,0.756 ± 0.274 and 0.178 ± 0.157 with the positive rate were 89.8%,88.9% and 9.4% respectively.There was no significance of the specific IgG between condyloma acuminatum group and cervical cancer group(P＞0.05),but it was significant among the three groups(P＜0.001).Conclusion The N-terminal 1-200 amino acids of HPV L2 has high homology and there exits cross reaction among the most common HPV infection types.

Objective To express in prokaryotic system and to analyze the antigenic specificity of EB virus(EBV) latent membrane protein 2(LMP2) multi-epitopes gene rich of T cell and B cell epitopes.Methods Using on-line prediction service, T cell epitopes and B cell epitopes of EB virus latent membrane protein 2 were predicted. The genes rich of CTL and th cell epitopes were selected as the candidate gene sequences, while B cell epitopes around them were taken into account. The finial selected multi-epitope gene was synthesized after being optimized according to prokaryotic codon bias and inserted into prokaryotic expression vector pET32a( + ) to get the recombinant plasmid: pET32a( + )/EBV-LMP2 multi-epitopes. After transformed into E. coli BL21 (DE3) and induced by IPTG, the target multi-epitopes gene can be expressed as Trx-His fusion protein. The expression products can be identified by SDS-PAGE and Western blot. Moreover, rabbit serum antibody to EBV membrane protein and nasopharyngeal carcinoma(NPC) patient serum were used respectively to detect the antigenic specificity of the multi-epitopes. Meanwhile, 6-8 weeks female BALB/c mice were immunized with EBV-LMP2 multi-epitope at 2 week intervals, three times in all, Trx-His protein and PBS were set as the control groups. At the second week after the last immunization, the mice were sacrificed. LDH and indirect ELISA were taken to detect the specific spleen CTL activityand specific IgG in serum, which reflected the immunogenicity of the EBV-LMP2 multi-epitope. Results Two amino acid sequences which locate at the LMP2 (aa195 -232 ) and LMP2 (aa419-436 ) were selected and connected in series to be the target gene. The recombinant plasmid containing EBV-LMP2 multi-epitope gene successfully constructed and the target protein was expressed in E. coli BL21 ( DE3 ). The relative molecular mass(Mr) of The expression products is about 27 × 103 , which matches up to the expected Mr. The antigenic specificity of the multi-epitopes protein was identified by Western blot and the multi-epitopes protein also can be detected by rabbit serum antibody to EBV membrane protein and NPC patient serum respectively. In the result of the animal experiment, EBV-LMP2 multi-epitope was able to induce the specific CTL activity in BALB/c mice. With the increasing of the effector: target ( E: T) 1: 5,1: 10, 1: 25, the CTL activity was also increased wih( 12.52% + 2.59% ), (21.80% + 1.08% ), (23.68% + 3.74% ) respectively; EBV-LMP2 multi-epitope was able to induce LMP2-specific antibody response(A490 =0.258 +0.040) as compared with Trx-His protein(A490 =0.095 +0.011) and PBS(A490 =0.068 +0.014,P＜0.05＝. Conclusion The EBV-LMP2 multi-epitopes gene was designed successfully and expressed precisely in prokaryotic expression system with good antigenicity and immunogenicity.

Objective This study was designed to compare the mRNA expression of Toll-like receptors 2 (TLR2) and Toll-like receptors 4 (TLR4) in monocytes between brucellosis cases and healthy controls,to explore the role and value of TLR2 and TLR4 in the pathogenesis of brucellosis.Methods From March to June in 2015,a total of 155 brucellosis cases at different clinical stages were diagnosed in Ulanqab Center for Endemic Disease Prevention and Control and 50 healthy controls were selected as research subjects.Subjective clinical symptoms of all patients were observed.Serological detection of all cases was done using serum tube agglutination test (SAT).The mRNA expression of TLR2 and TLR4 in peripheral blood cell was detected by real-time PCR technique.Results Fever,hyperhidrosis,weak,bone ache and hepatosplenomegaly were found in patients of acute (69 people),subacute (34 people) and chronic (52 people) periods.The incidences of fever and hyperhidrosis in acute and subacute periods were higher than that in chronic period,and the differences were statistically significant (x2 =58.427,26.190,all P ＜ 0.01).The incidence of bone ache in chronic period was higher than that in acute period,and the difference was statistically significant (x2 =9.264,P ＜ 0.01).In the agglutination titer of 1:800 and higher,the positive rate of chronic period was lower than those in acute and subacute periods,and the differences were statistically significant (x2 =14.302,8.682,all P ＜ 0.01).The mRNA expression levels of TLR2 and TLR4 were different among different clinical periods of brucellosis patients,and the differences were statistically significant (F =17.502,24.931,all P ＜ 0.01).The expression levels of TLR2 mRNA (8.67 ± 2.39) and TLR4 mRNA (12.38 ± 3.87) in acute period of brucellosis patients were higher than those of subacute period (5.21 ± 1.76,7.62 ± 2.21),chronic period (1.25 ± 0.47,1.72 ± 0.55) and healthy control (1.17 ± 0.23,1.43 ± 0.62),and the differences were statistically significant (all P ＜ 0.01).The expression levels of TLR2 and TLR4 mRNA in subacute period of brucellosis patients were higher than those of chronic period and healthy controls,and the differences were statistically significant (all P ＜ 0.01).The expression levels of TLR2 and TLR4 mRNA were not significantly different (all P ＞ 0.05) between chronic period and healthy controls.Conclusion TLR2 and TLR4 may be the specific recognizing receptors participated in the immune response in brucellosis and associated with the occurrence and development of brucellosis.

AIM: To observe the effect of osthole on tricalcium phosphate (TCP) particles-induced calvarial osteolysis in vivo.METHODS:Male ICR mice were randomly divided into sham group , TCP group and osthole group .A mouse calvarial model of osteolysis was established by TCP particles .On the second postoperative day , osthole (20 mg/kg) was locally injected into the calvarium under the periosteum 3 times a week.Two weeks after osthole treatment , blood and calvaria were collected to determine the level of bone turnover markers such as alkaline phosphatase ( ALP) , osteocalcin and tartrate-resistant acid phosphatase ( TRACP) .The periosteum was performed to examine the release of tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6) and IL-1βby ELISA.The calvaria was obtained for histological and molecular analyses.RESULTS:Data from HE and TRACP staining revealed that osthole prevented TCP particles-induced obvious increase in osteoclastogenesis and resorption area in the metaphysis of mouse calvaria .Osthole treatment increased ALP ac-tivity and osteocalcin level , and dncreased the activity of TRACP in the mouse serum compared with TCP group .Further-more, TCP particles-induced the releases of TNF-α, IL-6 and IL-1βwere significantly suppressed by osthole treatment .In addition, Western blot demonstrated that endoplasmic reticulum ( ER) stress markers such as glucose-regulated protein 78 (GRP78) and CAAT/enhancer binding protein homologous protein (CHOP) were significantly up-regulated in TCP parti-cles-implanted calvarial mice , indicating that TCP particles triggered an ER stress response in the mouse calvarial osteolysis model , which obviously attenuated by osthole .CONCLUSION:Osthole inhibits TCP particles-induced calvarial osteolysis in mice, which is mediated by inhibition of ER stress signaling pathway .

Aim To investigate the effect of MTX(included(±)MTX,(+)MTX and(-)MTX)on the proliferation of ECV304 cells and to explore its mechanisms.Methods ECV304 cells were cultured.The cell proliferation was determined by MTT.The morphological changes were inspected by inverted microscope.Cell cycle phases were assayed by propidium iodide staining flow cytometry.Results ECV304 cells were treated with(+)MTX,(-)MTX and(±)MTX at 1～150 μmol·L~(-1) for 24,48,72 h.The results showed that the proliferation of ECV304 cells was significantly inhibited under different conditions.The order of the inhibited efficacy was(+)MTX>(±)MTX>(-)MTX.The morphology of ECV304 cells were changed by(+)MTX,(-)MTX and(±)MTX treatment,which included the cell shrinkage,chromatin condensation.After administration of 10 μmol·L~(-1) of(+)MTX,(-)MTX and(±)MTX for 48 h,the cell cycle phases were assayed by propidium iodide staining flow cytometry.The result showed DNA replication was interfered by(+)MTX,(-)MTX and(±)MTX treatment.Conclusions The proliferation of ECV304 cells has the chiral selective effects by(+)MTX and(-)MTX treatment,and the inhibition on ECV304 cells proliferation of(+)MTX is significantly stronger than that of (-)MTX.

Objective To explore the alteration of brain pain functional areas in patients with functional dyspepsia (FD) irritable bowel syndrome (IBS) overlap by rectal balloon volume stimulation and magnetic resonance imaging (MRI) and the differences with IBS alone patients and healthy individuals were compared.Methods A total of 11 IBS alone patients,16 IBS overlapped with FD patients (IBS-FD) and 10 healthy controls were recruited.Sensory thresholds and visual analogue scale (VAS) were recorded during the rectal balloon air injection process. The changes of brain activated areas were analyzed by functional MRI (fMRI) when the rectum was stimulated at the volume of 50,100 and 150 ml.The data were analyzed by least significant difference (LSD) test.Results Under rectal volumetric stimulation,the sensory thresholds of IBS-FD group and IBS alone group were (53.14 ± 16.05) ml and (59.20 ± 20.55) ml and the difference was not statistically significant (LSD test,P＞0.05).There was no significant difference in VAS score between IBS alone group and IBS-FD group (LSD test,P＞0.05).Rectal stimulated under different volume,the results of fMRI indicated the activation of anterior cingulated cortex, dorsolateral prefrontal cortex,postporietal cortex,thalamus and insular cortex in both IBS alone group and IBS-FD group.And there was no significant difference in activated areas and intensity between IBS alone group and IBS-FD group (LSD test,P＞0.05).Conclusions There was no significant difference in activations of brain areas between IBS alone and IBS-FD patients under rectal volumetric stimulation. Under rectal volumetric stimulation,although symptoms overlapped,there was no evidence of the overlap of braingut axis and visceral hypersensitivity between IBS alone and IBS-FD.

Objective To study serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and its correlation with accumulation degree of 99Tcm-MDP on bone and arthritis and to study the role of sICAM-1 in the diagnosis of patients with early RA. Methods Serum levels of sICAM-1 were measured by immunoenzymetic assay (ELISA). 99mTc-MDP scintigraphy was carried out for the same patients. The significant test between serum levels of sICAM-1 of three groups was performed with analysis of variance; Correlations between serum levels of sICAM-1 and index of semiquatitive 99Tcm-MDP were obtained using sperman's ranks correlation method. Results Compared with control group,serum levels of sICAM-1 were significantly higher in standard group (group A) (P<0.01) and non-standard group (group B) (P<0.05). No significant difference was found between group A and B(P>0.05). Serum levels of sICAM-1 were positively correlated with 99Tcm-MDP scintigraphy. Most patients of non-standard group and standard group showed visible medium-high uptake of 99Tcm-MDP. While control group had not obviously uptake of 99Tcm-MDP. Conclusion Serum levels of sICAM-1, semiquatitive 99Tcm-MDP scintigraphy, accumulation degree of radiopharmuceuutial on bone and arthritis are helpful for diagnosis of early rheumatoid arthritis and judge-ment of the clinical status and improvement of the prognosis.

Objective To clone and express cathepsin B gene of Echinococcus granulosus(EgCatB)and analyze EgCatB protein by using bioinformatics tools and online databases. Methods The total RNA of E. granulosus was extracted and reverse?ly transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In?Fusion PCR cloning method and expressed by a wheat germ cell?free system,and then the recombinant protein was identified by Western blotting. The signal peptide,transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online soft?ware SignalP 4.1,TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently,the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally,the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam,SMART,Predictprotein,Swiss?model,NetOGlyc 4.0 and NetNGlyc 1.0 approaches. Results The EgCatB gene was successfully amplified from cDNA of E. granulosus and ex?pressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a cata?lytic active center was formed through Gln106,Cys112,His282 and Asn302. It was found that there were nine O?glycosylation sites in the EgCatB sequence,but no N?glycosylation sites. Conclusions The EgCatB gene is cloned and expressed successfully,and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful informa? tion for further functional study of the EgCatB protein.