Objective:To investigate the immunogeneicity of a subunit vaccine of capsid protein precursor(P1) of swine vesicular diseas(SVD).Methods:In this study,the guinea pigs were immunized with the home-made antigen,T-lymphocyte proliferation response,blocking ELISA and micro-neutralization assay were used to detect the effect of the immunized responses in guinea pigs.Results:The results indicated that a retroviral-based vaccine carrying the capsid protein precursor(P1) of SVD was able to elicit strong SVDV-specific humoral immune responses in guinea pigs.Conclusion:It encourages further work towards the development of a vaccine against SVDV infection.

Objective To validate genetic suppression of metastastic capability of highly metastastic melanoma cells by endostatin transfection.Method pcDNA3.1-Endo eukaryotic expression vector contained insulin signal peptide sequence was transfected into highly metastatic mice melanoma cell strain B 16.The expression of endostain was detected by RT-PCR and Western blot experiment,melanoma cells were determined with adhere experiment,in vitro invasion and migration experiment and pulmonary metastasis experiment on C57BL/6 mice.Result Endostatin can obviously inhibit the capability of adherence,in vitro invasion and migration and pulmonary metastasis of melanoma cells.Among them,adhere inhibition ratio was 67.3%,in vitro invasion inhibition ratio was 48.4%,cell migration inhibition ratiowas 52.1%and pulmonary metastasis inhibition ratio was 67.3%.Conclusion Endostatin transfection can obviously inhibit the highly metastatie capability of melanoma cells.

The bacteriophage T7 RNAP gene was amplified via PCR from -lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.

Objective To acquire normative values of anorectal manometry and sensation in population of our country with different gender and age.Methods Healthy individuals from four medical centers were collected and divided into three group according to their age,group Ⅰ 18 - 39 years old, group Ⅱ 40-59 years old and group Ⅲ ≥60 years old.The parameters of anal of subjects at resting status was examined by pneumohydraulic capillary perfusion system and high resolution PC Polygraf HR desktop gastrointestinal dynamic monitoring system.Subjects were asked to simulate defecation and then the defecation related indexes were recorded.In the end rectoanal inhibitory reflexes (RAIR)and rectal sensation were assessed by aired balloon.One-way analysis of variance and independent sample test were performed to compare indexes among three groups with different age and between different genders. Results A total of 166 healthy subjects were enrolled,79 in group Ⅰ with 40 male,68 in group Ⅱ with 29 male and 19 in group Ⅲ with 11 male.There was no significant difference in anal sphincter length (ASL),valid anal sphincter length (VASL),resting anal sphincter pressure (RASP),squeeze anal sphincter pressure (SASP)and duration of valid squeeze anal sphincter pressure (VSASP)among three groups with different age (all P > 0.05 ).Compared between male and female,only SASP of male ((180.13±8.10)mmHg,1 mmHg=0.133 kPa)was significantly higher than that of female ((143.93± 6.59)mmHg,t = -3.489,P <0.001 ),no difference in other parameters was found (all P >0.05 ). There was no significant difference in rectal pressure (RP),rectoanal pressure gradient (RAPG),anal sphincter relaxation rate (ASRR),and rectoanal coordination (RAC)among three groups (all P >0.05). During simulated defecation,RP and RAPG of men ((61 .23±3.46)mmHg and (40.04±4.08)mmHg) were significantly higher than those of women ((44.47 ± 2.32)mmHg and (24.00 ± 2.59 )mmHg, t=-4.075 ,-3.367,both P <0.01 ).There was no significant difference in other parameters between men and women (all P >0.05).All participants had RAIR,and there was no significant difference neither among three groups nor between men and women (both P >0.05).There was no significant difference in first rectal sensation (FRS)and constant rectal sensation (CRS)among three groups with different age (all P >0.05).However,the maximum rectal tolerable sensation (MRTS)of group Ⅱ and group Ⅲ was significantly higher than that of group Ⅰ ((194.41 ±6.32)mL and (200.00±12.75)mL vs (167.80 ± 5 .00)mL,F = 6.698,P = 0.002).There was no significant difference in rectal sensation between different gender (all P >0.05 ).Conclusions In our country,SASP,RP and RAPG during simulated defecation of male are higher than those of female.The value of MRTS increases along with the age.

Objective To investigate the effect of acupuncture combined with antidepressant on behavioral changes and 5-hydroxytryptamine 1A and 2A receptor ( 5-HT1AR and 5-HT2AR) mRNA expression in nucleus raphes dorsalis (NRD) of sleep deprivation depression (SDD) rats. Methods Eight normal Sprague Dawley (SD) rats were caged together without any stimulus or treatment, serving as the normal group. Thirty-two SD rats were given solitary raise for 21 days, together with chronic unpredictable mild stress ( CUMS) and rapid eye movement ( REM) sleep deprivation to establish the model. Twenty-eight rats completed the modeling successfully, and were divided into model group, acupuncture group, medicine group, and combination group, 7 rats in each group. Rats in the model group were given modeling treatment but without any intervention. Rats in the acupuncture group were given acupuncture on acupoint Yintang, Shenting and bilateral Taichong, acupuncture stimulation lasting 15 s every 10 min and acupuncture retention for 20 min in each day. Rats in the medicine group were given gastric gavage of Zoloft solution (0.83 mg·kg-1·d-1) and Alprazolam solution (0.067 mg· kg-1·d-1) , and rats in the combination group were given both acupuncture and medicine intervention. The intervention for the three intervention groups lasted for 7 days. Results Compared with the normal group, body weight, Open-field scores and sucrose preference of the model rats were significantly decreased on modeling day 7, 14, 21 (P<0.05). On modeling day 28, body weight and Open-field scores of each intervention group were significantly increased compared with those of the model group (P<0.05), and Open-field scores of combination group differed from those of acupuncture group and medicine group ( P<0.05). Only sucrose preference of combination group was improved significantly compared with the model group (P<0.05). Compared with the model group, NRD 5-HT1AR mRNA expression level was increased in the combination group (P<0.05), and NRD 5-HT2AR mRNA expression level was down-regulated in all of the three intervention groups ( P<0.05). Conclusion Acupuncture and antidepressant treatment covering 7 days can evoke rapid effect on SDD rats, which can improve the slow effect of antidepressant.

ObjectiveTo establish normative values of esophageal manometry in different age groups and standard test method.Methods The values of esophageal manometry were tested in healthy subjects of 3 different age groups (Group Ⅰ: 18-39 years,Group Ⅱ: 40-59 years,Group Ⅲ:≥60 years) with pneumohydraulic capillary perfusion system.The repeatability and stability of this method in lower esophageal sphincter (LES) testing were observed. Results There were no significant differences of LES length (LESL),LES pressure (LESP) at the end of expiration,average LESP,residual pressure of LES and LES relaxation rate (LESRR) in three groups (P＞0.05).The LESP at the end of inspiration in Group Ⅰ (28.98± 1.11 ) mm Hg was significantly lower than that of Group Ⅲ (34.35±1.96) mm Hg (P＜0.05).Trandiaphragmatic pressure (Pdi) in Group Ⅰ (9.55±0.62) mm Hg was significantly lower than that of Group Ⅱ (13.05±0.76) mm Hg (P＜0.05).There were no significant differences in contraction amplitude and duration of proximal and distant esophagus in 3 groups (P＞0.05).UES pressure (UESP) of Group Ⅲ was significantly lower than that of Group Ⅰ and Ⅱ (P＜0.05).As for repeatability,the second recording of LESP in Group Ⅰ and Ⅱ were significantly higher than first recording (P＜0.05).Amplitude of distant esophageal peristaltic contraction (DEPC) of female was significantly higher than of male (P＜0.05).However,there was no significant difference in amplitude of proximal esophageal peristaltic contraction (PEPC)between femal and male (P＞0.05).ConclusionsThe normative values of esophageal manometry in different age groups have been obtained.The dynamic parameters of LES do not change with age.Between age 40 to 59 years old,the contraction of esophageal body is strongest.UESP decreases significantly in old people.If the subjects have enough time to adapt before test,it will help to get accurate and reliable dynamic parameters of LES.

Cardiovascular disease is the main threat to human health,which is one of the important causes of death in worldwide.N-methyl-D-aspartate receptor (NMDAR) is one of crucial ionic glutamate receptors.The physiology and pharmacology function of the composition of NMDAR are very complicated.Researches have shown that NMDAR with a high permeability to calcium and a unique feature of controlling numerous calcium-dependent processes.NMDAR affects the occurrence and development of cardiovascular disease such as hypertension,arrhythmia,myocardial infarction.

OBJECTIVESWe identified the long QT syndrome (LQTS) patients, and detected the potential risk of LQTS in family members by using genetic testing and electrophysiological analysis, which helped provide clinical evaluation and appropriate treatment.

METHODSDetailed clinical characteristics and familiar history were obtained from the whole family members of an idiopathic pediatric LQTS patient. Two hundred healthy subjects with the same ethnic background were recruited as controls. The entire coding sequences of three candidate genes including KCNQ1, KCNH2 and SCN5A were screened for mutations in the proband. The function of the mutation was then explored by whole-cell patch clamp techniques, and the genetic testing and risk assessment of the family members were performed.

RESULTSThe proband was clinically preliminary diagnosed as LQTS by 12-lead electrocardiogram. On the third day of metoprolol intake (25 mg, bid), she died suddenly at lunch. One heterozygous missense mutation (SCN5A-V411M) was identified in this proband, but the mutation was absent in 200 healthy subjects. The electrophysiological analysis indicated that SCN5A-V411M significantly increased the peak current density ((230.8 ± 27.6)pA/pF vs. (101.2 ± 10.9)pA/pF, n=10, P<0.01) and the late sodium current ((156.6 ± 13.6)pA/pF vs. (95.9 ± 7.9)pA/pF, n=12, P<0.01) of sodium channel compared to wide type. The enhanced sodium channel activation with a negative shift in the peak I-V relationship was significantly higher by -50 mV than wide type (85.0%± 7.4% vs. 41.5% ± 2.6%, P<0.01), while the steady-state inactivation curves remained unchanged. Additionally, mother and grandmother of the proband were the silent mutation carriers with no symptoms, who needed the appropriate clinical assessment and follow-up. The proband's twin sister and aunt died of sudden infant death syndrome.

CONCLUSIONSWe firstly reported a heterozygote missense mutation (SCN5A-V411M) in this Chinese family. V411M induced "gain of function" of sodium channel and formed the basis of type-3 LQTS. Genetic testing could help to increase the diagnostic accuracy, and facilitate clinical assessment and appropriate therapy to prevent sudden cardiac death of individuals with SCN5A-V411M mutation.

Objective: To explore the clinical and molecular genetic features of a Chinese patient with catecholaminergic polymorphic ventricular tachycardia (CPVT). Methods: Clinical data including resting electrocardiography, echocardiography and treadmill exercise testing of a patient with CPVT admitted to our department in March 2013 were analyzed, and the peripheral venous blood samples of the patient and his family members and 400 ethnicity-matched healthy controls were obtained. All exons and exon-intron boundaries of the six CPVT-related genes including RYR2, CASQ2, TRDN, CALM1, KCNJ2 and ANKB were sequenced to detect the variants related to CPVT. The relationship between the genotypes and phenotypes was analyzed to direct the target therapy. Results: Recurrent syncope induced either by exercise or extreme frightened fear was observed in this patient. There was no positive family history of syncope or sudden death. The resting electrocardiography and echocardiography of the patient were normal, while the exercise testing revealed bidirectional and polymorphic ventricular tachycardia. A cardiac ryanodine receptor gene mutation (R2401H) was identified in this patient, while this mutation was absent in his parents and sister and 400 controls. No variant was detected in the remaining five candidate genes. Treatment with high dose of metoprolol succinate (118.75 mg/d) was effective and patient was free of syncopal attack during the 2 years follow-up. Conclusion This is the first report on RyR2-R2401H mutation in Chinese patient with CPVT, and high dose of metoptolol is the effective therapy option for CPVT related to RyR2 mutation.

Objective To provide scientific evidence to identify and confirm severe acute respiratory syndrome (SARS) by laboratory detection.Methods Multiple clinical specimens were collected serially and systematically from the 4 suspected SARS patients, which occurred between Dec.2003 to Jan.2004 in Guangdong Province. The samples were tested by serologic and molecular methods.Results IgM or IgG antibodies against SARS-CoV were detectable after 6—8 days of the onset in four patients. The four-fold or greater rising in antibodies was clearly detected in three of the four patients, while the fourth patient’s seroconversion was from negative to positive. The results analysed by enzyme-linked immunosorbent assay( ELISA), immunoflourescence assay (IFA), and neutralization test were highly correlated. SARS-CoV RNA was just detected in 3 throat swab specimens from case 1 by real-time PCR. M, N and S genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from the positive samples. Sequencing results showed that they were SARS-CoV gene segments, and most closely matched SARS-CoV gene sequences were isolated from civet cats in Guangdong Province. Nevertheless, SARS-CoV was not isolated from any samples of the 4 patients.Conclusion Based on these results, the 4 reported cases were laboratorily confirmed as SARS cases.