Objective To construct DNA vaccine expressing Mycobacterium tuberculosis(Mtb) immunodominant antigen Ag85A and analyze its anti-tuberculosis T cell responses in BCG primed-mice after DNA vaccination boosting.Methods The coding gene of Ag85A mature fragment was amplified by PCR with H37Rv genomic DNA as template,and then cloned into the eukaryotic expression vector pVAX1 to construct Ag85A DNA vaccine.After purification,Ag85A DNA vaccine was injected intramuscularly twice in BCG primed-mice with BCG vaccination and DNA vaccination alone as control.Eight weeks post-vaccination,spleen lymphocytes were separated and were then used to analyze Mtb antigen specific effector T cell response and polyfuntional IFN-γ/TNF-α/IL-2 secreting CD4+ T cell frequencies and intensities,and CD8+T cell responses by IFN-γ ELISPOT assay and intracellular staining,respectively.Results Compared to BCG vaccinated-and DNA vaccinated-mice,Ag85A DNA boosting not only enhanced significantly BCG primed-mice IFN-γ+TNF-α+IL-2+,IFN-γ+ IL-2+,TNF-α+IL-2+ and IL-2+ CD4+ T cell frequencies and IL-2 secretion,but also improved significantly IFN-γ-secreting and IL-2-secreting CD8+ T cell frequencies.Condusion Ag85A DNA vaccine was constructed successfully and was demonstrated to enhance significantly BCG primed-mice Mtb antigen specific CD4+ and CD8+ T cell responses when boosting,which is beneficial to improve BCG immunogenicity and its waning immune protection against Mtb.

In order to synthetically train students to do scientific researches independently,inspire their enthusiasm and go-aheadism about study,and improve the quality of experimental teaching,we have been exploring to update experimental content and reform experimental system for many years,and have commenced a number of "Series Experiments".The setup of"Series Experiments" which means several separate experiments are organized together by their internal relations has already showed us a favorable effect.

Objective To explore the effect of ? - aminobutyric acid B receptor(GABABR)on brain damage induced by recurrent febrile seizures (FS). Methods Rats were randomly divided into four groups: control group (37. 0 ℃ water, n = 8), FS group (45.2 ℃ water,n=8), FS + baclofen group (45.2 ℃ water,77 = 8), FS + phaclofen group (45. 2 ℃ water,n=8). FS in rats were induced for ten times in a bath of warm water, once every 2 days. The intensity, latency and duration of the seizure in rats were recorded. The expression of c - fos gene and Fos protein were examined by in situ hybridization and immunohistochemistry, respectively. Results Compared with those of FS group, the seizure latency gradually prolonged, and the seizure duration was shortened in FS + baclofen group. In FS+ phaclofen group, the seizure latency was shorter and the seizure duration was longer than those of FS group. The seizure intensity was lessened in FS + baclofen group while aggravated in FS + phaclofen group compared with that of FS group. The expression of c - fos gene and Fos protein increased significantly after recurrent FS. Baclofen down regulated the expression of c -fos gene and Fos protein, while phaclofen enhanced the expression of them. Conclusion The study by using the agonist and the inhibitor of GABABR showed that GABABR might play a crucial role in the development of FS- induced brain damage.

Objective To evaluate the changes of left ventricular (LV) bulk rotation and untwisting in transplanted hearts using 2-dimensional speckle tracking imaging(STI).Methods Basal and apical LV short-axis images were acquired in 15 heart transplant recipients 3 months post surgery(HT group) and 56 healthy control subjects.Basal and apical rotation versus time profiles were drawn using 2-dimensional STI software.Appropriate values were chosen from the dataset obtained and compared between two groups.Results ①Compared with the control group,the heart rate,anterior-posterior diameter of left atrium,enddiastolic interventricular septum thickness,left ventricular posterior wall thickness,isovolumic relaxation time and E/e ratio were significantly increased,e and a values were decreased significantly in HT group (P ＜ 0.05).② No significant difference was noticed in the peak degrees of LV bulk rotation,the degrees of LV bulk rotation at the time of aortic valve closure and mitral valve opening (P =0.700,0.984,0.495,respectively) between 2 groups.In both groups,systolic rotation reached its maximum at end-systole [(96.1 ± 8.4) ％ in HT group vs (100.5 ± 6.3) ％ in control group,P =0.065].③Significant decreases in untwisting rate and trend untwisting variables were observed in the HT group(P ＜0.001).Conclusions 3 months after transplanted,left ventricular bulk rotation of cardiac allografts remained normal,and significant decreases in both untwisting rate and trend untwisting variables showed that the diastolic function of cardiac allografts was impaired.

Objective To explore the effect of nitric oxide (NO) on ?-aminobutyric acid B receptor (GABA_BR) subunits during recurrent febrile seizures (FS).Methods Twenty-four Sprague-Dawley rats aged 21 days were randomly divided into 4 groups: control group (37.0 ℃ water,n=8), FS group (45.2 ℃ water,n=8), FS + SNP group (45.2 ℃ water,n=8), FS+L-NMMA group (45.2 ℃ water,n=8). FS rats were induced 10 times in a warm-water bath, once every 2 days. The plasma level of NO was detected by the spectrophotometer. The expressions of GABA_BR subunit mRNA and c-fos gene were examined by in situ hybridization. The expressions of GABA_BR subunit and Fos protein were observed by immunohistochemistry. Results The plasma level of NO increased in FS + SNP group while decreased in FS+L-NMMA group compared with that in FS group. The expressions of GABA_BR_2 were down-regulated in FS+SNP group, while GABA_BR_1 hardly changed compared with those in FS group. In FS+L-NMMA group, both the expression of GABA_BR_2 and GABA_BR_1 up regulated compared with those in FS group. The expressions of c-fos gene and Fos protein were significantly enhanced after recurrent FS. SNP elevated the expressions of c-fos gene and Fos protein, while L-NMMA down regulated the expressions of them.Conclusion NO may play a regulatory role through modulating GABA_BR function in the pathogenesis of recurrent FS.

Objective To explore the influence of ?-aminobutyric acid B receptor(GABA_BR)on carbon monoxide (CO)/heme oxygenase(HO-1)system during recurrent febrile seizures (FS).Methods Sprague-Dawley rats aged 21 days were randomly divi- ded into 4 groups:control group and FS group,FS+baclofen group,FS+phaclofen group.FS in rats were induced 10 times in a bath of warm water, once every 2 days.The plasma level of CO was detected by the dual wave lengh spectrophotometer;the expressions of GABA_BR and HO-1 mRNA were examined by insitu hybridization;the expressions of GABA_BR and HO-1 protein were observed by immunohistochemistry.Results The plasma level of CO increased in FS+baclofen group,while decreased in FS+phaclofen group compared with FS group.The expressions of GABA_BR and HO-1 upregulated in FS+baclofen group,while decreased in FS+phaclofen group compared with FS group.There were significant difference (All P

Objective To study the distribution of apolipoprotein E(apoE) genotypes and alleles in children with primary nephrotic syndrome(PNS),and to investigate the relationship between apoE gene polymorphism and disturbance of serum lipid metabolism.Methods Forty-six children with PNS were compared with 39 age-sex-matched healthy children.Serum levels of total cholesterol(TC),triglyeride(TG),high density lipoprotein-cholesterol(HDL-C),low density lipoprotein-cholesterol(LDL-C),apoA_1,apoB and apoA_1/B were detected,and polychain reaction single-strand conformation polymorphism(PCR-SSCP) combined with gene sequence determination was used to confirm the apoE genotypes in two groups.Results 1.Serum TC,TG,HDL-C,LDL-C,apoB and apoA_1/B in PNS group were higher than those of control group,respectively(P

Objective To investigate the growth and exocrine function of BM-MSC derived from PBC patients.Methods To compare the growth patterns and cytokines secretions between PBC patients and healthy controls by student's t test.Results ① There was no difference in growth profile and speed between PBC patients and healthy controls.② The level of TGF-β1 was much lower in the supernatant of BM-MS from OBC patients than health controls [(2.6±1.9)vs (8.2±6.7)ng/ml,t=-3.641,P=0.001].There were no other differences between two groups' BM-MSC.③ The super natant concentration of interlukin-10 of the third BM-MSC subculture from healthy controls was lower than that of the primary subculture [(18.5±5.0) vs (12.4±3.1) pg/ml,t=2.368,P=0.045],and that of hepatic growth factor from the second subcuhure was higher than the primary subculture [(0.21±0.07) vs (0.35±0.08) ng/ml,t=-2.874,P=0.021].There were seldom discrepancies in other cytokines between different generations of BM-MSC.Conclusion BM-MSC from PBC patients may have almost the similar characters in growth pattern and cytokines secretion as,except the TGF-β1,which was much lower than those from healthy controls.The second subculture of BM-MSC might be more suitable for the treatment to patients with PBC.

AIM: To study the role of TGF-?/Smad pathway in the development of renal fibrosis in diabetic nephropathy.METHODS: Rats were induced to diabetic nephropathy by using tail intravenous injection of STZ.The expression of TGF-?_1,Smad2/3 protein and mRNA in kidney were examined at 2,4,8 and 16 weeks after STZ induction.CTGF,collagen-Ⅲ,PAI-1 mRNA expression in kidney at 16 weeks of STZ-induced diabetic nephropathy and normal rats were studied by RT-PCR.RESULTS: Weak TGF-?_1,Smad2/3 protein were detected in normal renal tissues while strong TGF-?_1,Smad2/3 staining were observed in renal tissues of diabetic nephropathy(0.057?0.030/0.223?0.040;0.017?0.010/0.153?0.010,respectively,P

AIM: To explore the relationship between various mitochondrial (mt) DNA tRNA Leu (UUR) and ND1 gene mutations and type 2 diabetes mellitus (T2DM) among Chinese in Hubei Province. METHODS: PCR restriction fragment length polymorphism (PCR-RFLP) analysis was used to screen point mutations of mtDNA ( 3 243, 3 256, 3 290, 3 316, 3 394, 3 421, 3 426, 3 460, 3 593) in 174 T2DM and 207 healthy controls. Then, DNA sequencing, reverse dot blot hybridization and Genchip were used to compare and confirm mutations. All mutations were analyzed by DNASTAR and Antherprot softwares. RESULTS: In diabetic group, there were 5 carriers (2.9%) of 3 316 G→A (Ala→Thr) mutation, 4 (2.3%) of 3 394 T→C (Tyr→His) mutation, 1 (0.6%) of 3 593 T→C(Val→Ala) mutation, and 1 (0.6%) of 3 618 T→C(Phe→Phe) mutation. Among 3 316 (G→A) mutations , there were more than 1 point mutations in 2 cases, one accompanied with 3 256 C→T(Arg→Arg) and 3 688 G→C (Ala→Pro) mutations, another accompanied with 3 606 A→G(Leu→Leu) mutation. 3 606 (A→G), 3 618 (T→C) and 3 688 (G→C) were novel mutations, GenBank accession number is DQ092356. In controls, only 3 316 (G→A) mutation was found in 1 subject (0.5%). There was significant difference between two groups for 3 394 (T→C) mutation frequencies (P