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 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.

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.

This study was to investigate the permeability and absorbability of capsaicin cubosome across abdominal skin of the SD rats in vitro. Diffusion of capsaicin cubosome and cream was performed with the modified Franz diffusion cell technique. The capsaicin cubosome showed no enhancement of skin permeation within 24 hours. However, the deposition amounts of capsaicin in the rat skin in the cubosome group was markedly higher than those in the commercial cream group (P < 0.01). Cubosome showed excellent characetristic of skin-targed which could be a good carrier for the local transdermal drug delivery system.
Administration, Cutaneous ; Animals ; Capsaicin ; administration & dosage ; chemistry ; Kinetics ; Male ; Particle Size ; Permeability ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; metabolism ; Skin Absorption

OBJECTIVEThe research aimed to investigate the entophytic fungal community of Vitex negundo var. cannabifolia, including the biodiversity from different organs and the correlations with habitations.

METHODPDA medium was used to isolate and purify the endophytic fungi in V. negundo var. cannabifolia. The purified strains were identified by morphology together with similarity of 5.8S gene and internal transcribed spacer (ITS) sequence.

RESULTNinety-seven strains of endophytic fungi were isolated from V. negundo var. cannabifolia. They were identified belonging to 12 species, 10 genera, 8 families and 6 orders, while 60 isolations identified into 9 species, 9 genus ,7 families and 6 orders were obtained from the wild plants and 37 belonging to 7 species, 5 genus, 3 families and 3 orders were from the cultivated ones.

CONCLUSIONIt indicated that the entophytic fungi in V. negundo var. cannabifolia were diverse, and there were some differences at quantity and species in different sites and organs of V. negundo var. cannabifolia.

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 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.

Objective Pueraria extract puerarin,HPLC assay puerarin extract and compare different doses of correlation with the hang-over effect of puerarin evaluate different doses of puerarin liver hangover effect. Methods Extracted under optimal conditions obtained in the previous experiment puerarin spare,HPLC method for qualitative and quantitative detection of alcohol extract of kudzu root ( PRE) ,the male Kunming mice were randomly divided into control group,positive control group and puerarin group,each group of 10. Give mice fed pueraria extract,30 min after administration of liquor,drunk mice sobering observation time and the determination of mouse liver ADH,GOT,GPT con-tent in order to investigate the effect of puerarin on drunken mice. Results HPLC fraction was measured at 8 times the volume of 70% etha-nol,60 ℃ constant temperature water bath shaker at 30 min for optimal extraction conditions puerarin extraction. Compared with the positive control groups:low,medium and high doses of alcohol extract of pueraria can significantly shorten the time to sober up drunken mice,the dose of PRE could effectively inhibit the absorption of alcohol,reduce liver tissue ADH,GOT,GPT,the effects of high doses of PRE absorption of alcohol was small. Conclusion HPLC method capable of puerarin extract the qualitative and quantitative determination of puerarin on liver injury caused by acute alcoholism a protective regulatory role,and the hangover effect of puerarin dose showed a good positive correlation.

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

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